Award Winners and Abstracts of the 31st Annual Symposium of The Protein Society, Montreal, Canada, July 24–27, 2017

Abstract

2016 Award Winners Montreal, Canada – The Protein Society, the premiere international society dedicated to supporting protein research, announces the winners of The 2017 Protein Society Awards. The awards were conferred at the 31st Annual Symposium of The Protein Society (July 24–27, 2017, Montreal, Canada). The Carl Brändén Award The Carl Brändén Award, sponsored by Rigaku Corporation, honors an outstanding protein scientist who has also made exceptional contributions in the areas of education and/or service to the field. The 2017 recipient of this award is Dr. Billy Hudson (Vanderbilt University). Dr. Hudson has worked tirelessly to develop the Aspirnaut K‐20 STEM Pipeline for Diversity Program that provides internships to an untapped pool of talented high‐school students to encourage them to work in the STEM fields and go on to college. Hudson's outstanding research accomplishments include seminal discoveries about the structure and chemistry of collagen IV scaffolds in extracellular basement membranes and have led to a potential treatment of diabetic kidney disease. The Dorothy Crowfoot Hodgkin Award The Dorothy Crowfoot Hodgkin Award, sponsored by Genentech, is granted in recognition of exceptional contributions in protein science which profoundly influence our understanding of biology. The 2017 award will be presented this year to two deserving nominees. The first is Dr. Juli Feigon (University of California, Los Angeles). Feigon's structural studies on proteins have largely evolved around proteins interacting with DNA or RNA, and has revealed interactions crucial to understanding DNA repair and regulation of gene expression. Feigon's recent accomplishment is structural analysis of the Tetrahymena telomerase complex, a multisubunit riboprotein complex responsible for the maintenance of telomeres. The structures provide new mechanistic knowledge of telomere function associated with aging and cancer. The co‐recipient of the Dorothy Crowfoot Hodgkin Award is Dr. Manajit Hayer‐Hartl (Max Planck Institute of Biochemistry). For the past 2 decades, Dr. Hayer‐Hartl has investigated the mechanism of GroEL and its co‐factor GroES. This work led to the insight that the chaperonin, in addition to preventing aggregation, profoundly influences the free‐energy landscapes for some proteins by accelerating folding through entropic destabilization of unfolded states in the confining environment of the folding cage, a mechanism that can be considered specific to chaperonin. The Hans Neurath Award The Hans Neurath Award, sponsored by The Neurath Foundation, seeks to honor individuals who have made a recent contribution of exceptional merit to basic protein research. In 2017, the Hans Neurath Awardee is Dr. Kazuhiro Nagata (Kyoto Sangyo University). Nagata has made fundamental discoveries advancing our understanding of protein quality control in the endoplasmic reticulum. Dr. Nagata's research focuses on functional analysis of collagen‐specific molecular chaperone, Hsp47; functional analysis of mammalian ER quality control and ER‐associated degradation; and ER‐associated degradation of misfolded proteins by the EDEM‐ERdj5 system. The Christian B. Anfinsen Award The Christian B. Anfinsen Award, sponsored by The Protein Society, recognizes technological achievement or significant methodological advances in the field of protein science. The recipient of this award in 2017 is Dr. Lewis Kay (University of Toronto). Dr. Lewis Kay has been involved in developing a large number of ground‐breaking tools and approaches that have revolutionized NMR spectroscopy and have rendered it one of the most powerful techniques in protein science. The research in Dr. Kay's laboratory focuses on the development of NMR techniques for studying macromolecular structure and dynamics and the application of NMR techniques to problems of biological and clinical importance. The Emil Thomas Kaiser Award The Emil Thomas Kaiser Award recognizes a recent, highly significant contribution to the application of chemistry in the study of proteins. The 2017 recipient is Dr. Thomas Muir (Princeton University). Muir is known for his innovative work to develop semisynthetic approaches, known as “expressed protein ligation,” to manipulate covalent structure of proteins. By combining tools of organic chemistry, biochemistry and cell biology, Muir has developed a suite of new technologies for making proteins with defined post‐translational modifications, enabling functional studies of how proteins work that would otherwise not be possible. The chemistry‐driven approaches pioneered by the Muir lab are now widely used by chemical biologists around the world. The Stein and Moore Award The Stein and Moore Award is named for Nobel laureates Dr. William Stein and Dr. Stanford Moore. The award venerates eminent leaders in protein science who have made sustained, high impact research contributions to the field. The 2017 recipient is Dr. John Kuriyan (University of California, Berkeley). Kuriyan' s major scientific contributions have been in understanding the regulation of eukaryotic cell signaling and the phenomenon of processivity in DNA repair. His contributions include seminal studies on the structural basis of regulating protein interactions and molecular mechanisms associated with cancer. These insights come from work on protein kinases such as the Src‐family kinases, Abelson tyrosine kinase, the epidermal growth factor receptor and Ca2+/calmodulin‐dependent kinase II. The Protein Science Young Investigator Award The Protein Science Young Investigator Award, named for the academic journal of the Society, Recognizes a scientist generally within the first 8 years of an independent career who has made an important contribution to the study of proteins. The 2017 recipient is Dr. David Pagliarini (University of Wisconsin, Madison). From the earliest point in his career, Pagliarini has made substantive and lasting contributions to our understanding of mitochondrial protein function. Taking an interdisciplinary approach, Pagliarini has revealed a large number of mitochondrial proteins have no established function, and many are associated with human disease. His goal is to use a range of techniques to connect “orphan” proteins with mitochondrial pathways and processes. The 2017 Protein Science Best Paper Award At the beginning of each year, two “best papers” are selected from articles published in “Protein Science” during the preceding 12 months. A junior author (typically the first author) is designated as the award winner and invited to give a talk at the following Annual Protein Society Symposium. The 2017 recipients are Charlotte Miton and Zach Schaefer. Charlotte Miton has already been something of a world traveler. Following completion of her Master's degree in France, she participated in research projects in Mexico and Italy before undertaking her Ph.D. in Cambridge with Drs. F. Hollfelder and M. Hyvonen. As Charlotte then narrates “Following my PhD work, I joined Dr. Nobuhiko Tokuriki at UBC in Vancouver, with whom I share a passion for tracking and elucidating the mechanisms behind functional transitions, mutational interactions and conformational changes that result from evolutionary selection. We had worked together during my time at Cambridge and felt that there were many potential pathways we could explore. This led to the core motivation behind the paper in Protein Science: based on the data being generated in the field on single evolutionary trajectories, how prevalent were the trends we were observing in the laboratory and to what degree could we use that information to make inferences from their protein structures? We felt that a general consensus on the role of mutational epistasis, i.e. non‐additive interactions between mutations, based on a quantitative survey of its type and prevalence, remained to be established. By 2017, we were able to gather, analyse and compare complete mutational data from nine evolutionary trajectories curated from the literature. This analysis revealed that epistasis plays a major, albeit hidden, role constraining evolutionary trajectories: about half of the mutations fixed during these nine trajectories were neutral or detrimental in the original WT background and only became positive at later rounds of evolution, following the prior fixation of permissive mutations.” The second Best Paper awardee, Zach Schaefer, worked with Tony Kossiakoff, who describes Zach “in a nutshell” as follows: “Zach was an undergrad at Reed. After graduation, he decided to take a year or two off to ‘find the meaning of life.’ That sounds very Reed‐like, you get the picture. He applied to my lab as a technician to see whether science was his thing. I usually don't like to be part of this type of experiment, but his mentor at Reed thought he was a good bet. So did I, and it was a good bet. He spent a couple of years with me and I put him on a number of hard projects. Interestingly, the work that is described in the Protein Science paper was completely initiated by Zach and a postdoc, Luke Bailey. They worked together on this, mainly in the background of other things. They had been working on projects that required Fabs to be coupled in a bivalent format and found that some of the best Fabs for an application actually became highly aggregated when put in this form. So, developing the polar ring was an important step leading to the ability to make bivalent Fab constructs. For more on the 2017 Protein Science Best Paper Award winners and their research, read the article by Protein Science Editor‐in‐Chief Brian W. Matthews “Protein Science best paper awards to Charlotte Miton and Zach Schaefer” here. Abstract Grouping by Topics Pages 1 – Amyloid and Aggregation 13 2 – Bioinformatics 24 3 – Chaperones 28 4 – Chemical Biology 35 5 – Computational Modeling/Simulation 46 6 – Design/Engineering 55 7 – Dynamics and Allostery 72 8 – Enzymology 83 9 – Evolution 97 10 – Folding 106 11 – Intrinsically Disordered Proteins 117 12 – Membrane Proteins 127 13 – Metabolic Engineering/Energy Applications 139 14 – Motors and Machines 139 15 – Peptides 141 16 – Protein Interactions and Assemblies 145 17 – Proteins in Cells 161 18 – Proteomics 167 19 – Proteostasis and Quality Control 175 20 – Single Molecule Studies 179 21 – Structure (X‐Ray/NMR/EM) 179 22 – Synthetic Biology 198 23 – Systems Biology 201 24 – Therapeutics and Antibodies 202 25 – Transcription/Translation/Post‐Translational Medications 207 01. AMYLOID AND AGGREGATION POS023 EGCG Disaggregates Human α‐Synuclein Fibrils and Modulates the Pathway to Form Stable and Distinct Oligomeric Species Sneha Roy1, Rajiv Bhat1 1Jawaharlal Nehru University The emerging evidence of toxic oligomeric species formed as intermediates during amyloidogenesis have led to the strategy of using small molecule modulators like polyphenols to inhibit the build‐up of‐ or ameliorate the toxicity of these species. We demonstrate that the flavonoid epigallocatechin gallate (EGCG) suppresses human α‐Synuclein fibrillation even at 5µM by affecting the oligomerization of α‐Syn. Additionally, EGCG also disaggregates the mature α‐Syn fibrils to disintegrated sheet like fragments as shown by Thioflavin T (ThT) and transmission electron microscopy (TEM). The effect of EGCG on α‐Syn structure and the dynamicity of the conformational transitions as investigated using ANS binding, circular dichroism (CD) spectroscopy and time‐resolved fluorescence measurements suggest that EGCG favours the natively unfolded structure of α‐Syn and significantly delays the structural transitions to characteristic ß‐sheet structure. The dynamic light scattering (DLS) and size‐exclusion chromatography (SEC) studies reveal that EGCG modulates the α‐Syn pathway towards two distinct types of oligomeric species which are also SDS‐resistant. By using fluorescence quenching and isothermal titration calorimetry (ITC), we also report that EGCG forms a weak binding complex with α‐Syn having a dissociation constant in the mM range. Interestingly, the cytotoxicity assays show a differential effect where EGCG generated oligomers completely rescue the breast cancer (MCF‐7) cells from α‐Syn toxicity but significantly reduce the viability of neuroblastoma (SH‐SY5Y) cells. Our findings suggest that even though EGCG modulates α‐Syn fibrillation, the differential toxicity of EGCG generated oligomers prompts further in‐depth investigation. POS074 Learning from the Amyloidogenic Peptides in Amyotrophic Lateral Sclerosis (ALS) Jen‐Tse Huang1 1Institute of Chemistry, Academia Sinica TAR DNA‐binding protein (TDP‐43) was identified as the main sediments in the histopathological inclusion bodies of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) in 2006. Our group have synthesized numerous TDP‐43 peptides and illustrated these fragments could form fibrillar aggregates, sharing similar morphology with neuronal cytoplasmic inclusions in ALS patients, and disrupt cellular membrane. Later on, we have further synthesized glutamine/asparagine (Q/N)‐rich and glycine (G)‐rich polypeptides from the TDP‐43 and characterized their structural/amyloidogenic properties with various biophysical methods. Through the microinjection and real‐time imaging, we have also proven that these aforementioned peptides were able to seed the aggregation of the TDP‐43 in cells and trigger apoptosis. Recently, by applying the amyloidogenic sequences deriving from TDP‐43 and Huntingtin protein (Htt), we had designed both the photocontrollable probes and therapeutic peptides. With these developing tools, we aim to delineate the roles of misfolding proteins, create diagnosis platform, and contribute in potential therapeutic strategies against different neurodegenerative diseases. POS096 Redox regulation of the antiviral adaptor mavs activation through formation of self‐perpetuating fibers Natalia Zamorano 1, Audray Fortin1, Stéfany Chartier1, Espérance Mukawera1, Nathalie Grandvaux1 1CRCHUM ‐ Université de Montréal MAVS is a central signaling hub activated in response to cytosolic sensing of viral nucleic acids to coordinate multiple signaling cascades essential to trigger an antiviral defense. Dysregulation of MAVS‐dependent pathways are also associated with autoimmune/chronic inflammatory diseases. MAVS is a transmembrane mitochondrial and peroxisomal protein that forms prion‐like self‐perpetuating fiber‐like polymers. In addition to being functional, MAVS aggregates are atypical and differ from amyloids fiber in that instead of ß‐sheets, they exhibit a six‐helix bundle structure composed of three a‐helices on the inside surrounded by three other external. Compelling evidence support a role of reactive oxygen species (ROS) in the regulation of MAVS‐dependent pathways. First, MAVS localizes is organelles strongly involved in the metabolism of ROS. Additionally, recent work from our laboratory and others unveiled that ROS derived from NADPH oxidase or mitochondria activities are essential for MAVS‐dependent response, including aggregation. The molecular mechanisms of action of ROS remain poorly understood. Redox post‐translational modifications of proteins (ox‐PTMs), notably on cysteine (Cys) residues, have recently emerged as key processes to regulate protein structure and function. Using maleimide‐derivative switch methods to label Cys ox‐PTMs, we found that MAVS possesses Cys that can undergo oxidation. Furthermore, analysis of MAVS behavior under oxidative stress conditions strongly support a role of MAVS Cys ox‐PTMs in the regulation of polymers formation. Structure‐function analyses are currently performed to further characterize the redox regulation of MAVS activation through formation of prion‐like self‐perpetuating fiber‐like polymers. POS107 Unraveling the Promising Action of Promethazine against Amyloid Fibrillation of Human Lysozyme: Implication towards Systemic Amyloidosis Saima Nusrat 1, Rizwan Hasan Khan 1 1Aligarh Muslim University The amyloid protein depositionhas a dominant role in different neurotoxic and aggregation related disorders like Alzheimer's disease, Parkinson's disease, systemic amyloidosis etc.The presentstudy emphases on the amyloid inhibitory as well disaggregating action of promethazine (PRM), a neuroleptic and antihistamine drug, against human lysozyme (HL) fibrillation and associated cytotoxicity.Exploiting biophysical, biochemical, microscopic and cytotoxicity assays we revealed that PRMis effectivein inhibiting and disaggregating HL amyloid fibrils, and displays a cytoprotective action against amyloid associatedcytotoxicity. Biophysical techniques like rayleigh scattering (RLS), ThT and ANSfluorescence measurement,circular dichroism (CD) and dynamic light scattering (DLS) measurementsrevealed the inhibitory action of PRM. The inhibition constant (Ki) and IC50 value of PRM was estimated to be (2.40 ± 0.18) x 103 and 158.48 ± 1.30 µM, respectively. Additionally, microscopic techniques (TEM and FM) displayed the nonexistence of fibrillar species when HL was co incubated with PRM. The cytotoxicity assay accomplished on SHSY5Y neuronal cell lines confirmed the decrease in cytotoxicity in the presence of PRM. PRM was established to be more potent against HL pre formed fibrils and the value of depolymerizationconcentration (DC50) was evaluated as 22.34 ± 0.89 µM and likewise lessened the cytotoxicity of disaggregated species as well. Consequently, PRMmightwork as a favorabletherapeutic inhibitors designed against amyloid related diseases. POS119 The Levinthal Problem in Amyloid Aggregation: Sampling of a Flat Reaction Space Jianhan Chen 1, Jeremy Schmit2, Zhiguang Jia1 1University of Massachusetts, Amherst, 2Kansas State University The formation of amyloid fibrils has been associated with many neurodegenerative disorders, yet the mechanism of aggregation remains elusive, partly because aggregation timescales are too long to probe with atomistic simulations. A microscopic theory of fibril elongation (Schmit, J. Chem. Phys. 2013) argues that the conformational search over H‐bonding states is the slowest step in aggregation and suggests that this search can be efficiently modeled as a random walk on a rugged one‐dimensional energy landscape. This insight motivated a multi‐scale computational algorithm for simulating fibril growth, where a large number of short atomistic simulations are performed to compute the system diffusion tensor in the reaction coordinate space predicted by the analytic theory. Ensemble aggregation pathways and growth kinetics are then computed from Markov State Model (MSM) trajectories. The algorithm is deployed here to understand the fibril growth mechanism and kinetics of Aß16–22 and three of its mutants. The order of growth rates of the wild‐type and two single mutation peptides (CHA19 and CHA20) predicted by the MSM trajectories is consistent with experimental results. The simulation also correctly predicts that the double mutation (CHA19/CHA20) would reduce the fibril growth rate, even though the degree of rate reduction with respect to either single mutation is over estimated. This artifact may be attributed to the simplistic implicit solvent model. These trends in the growth rate are not apparent from inspection of the rate constants of individual bonds or the lifetimes of the mis‐registered states that are the primary kinetic traps, but only emerge in the ensemble of trajectories generated by the MSM. POS131 Characterization of Lipid Binding by the Functional Amyloid Protein Orb2A Maria Soria 1, Silvia Cervantes1, Thalia Bajakian2, Ansgar Siemer1 1University of Southern California, 2Drexel University Orb2 is an mRNA binding protein and translational regulator found in D. melanogaster, which is able to form functional amyloid‐like aggregates in the fruit fly brain. Both isoforms A and B are found in these aggregates, but it is isoform A that is thought to initiate the aggregation process. This mechanism must be regulated to avoid toxic amyloid‐related species, such as those that occur in amyloid diseases. Previous data suggested that Orb2A might be able to bind lipid membranes. As membrane binding affects the aggregation of many other amyloid‐forming proteins, we investigate the potential for membrane binding of Orb2A, as well as how lipid membrane binding affects amyloid aggregation. We use circular dichroism and electron paramagnetic resonance (EPR) to identify how and where Orb2A binds lipid vesicles. We also use transmission electron microscopy and EPR to track amyloid formation over time with and without lipid vesicles present. We show that Orb2A binds to anionic small unilamellar vesicles (SUVs) using an N‐terminal amphipathic helix, and that charge is important for vesicle binding as well as a high degree of membrane curvature. We also show that the presence of anionic SUVs inhibits amyloid formation, which opens the door for a regulatory role for membranes in the formation of functional Orb2A amyloid fibrils. POS141 The Neurotransmitter Noradrenaline binds α‐Synuclein and modulates its Structure and Aggregation Properties Priyanka Singh 1, Rajiv Bhat1 1Jawaharlal Nehru University Parkinson's disease (PD) is characterized by deterioration of dopamine (DA) neurons of the substantia nigra pars compacta along with a substantial loss of noradrenaline (NA) neurons of the locus coeruleus which is the major source of NA in the brain. We have, thus, investigated the interaction of NA with a‐Synuclein (a‐Syn), the major protein constituent of lewy bodies that are the pathological hallmark of PD. The tissue expression of noredrenaline has been reported to be high in hippocampus, neocortex, striatum, thalamus and cerebellum regions of the brain which are the sites where a‐Syn is predominantly expressed, suggesting that NA might interact with a‐Syn. It is possible that NA could bind to a‐Syn and modulate its aggregation propensity and kinetics that could affect the onset of Parkinson's disease. We have evaluated the thermodynamic parameters of binding of NA with a‐Syn using isothermal titration calorimetry and steady state fluorescence and have investigated the conformational and aggregation aspects using circular dichroism, DLS and fluorescence spectroscopy. Binding isotherms of NA with a‐Syn have been observed to be exothermic in nature with apparent binding constant to be in millimolar range suggesting weak interaction. NA significantly suppresses a‐Syn aggregation in a concentration dependent manner by promoting alpha helical structure formation and the species formed are of smaller size and different morphology as shown by TEM. These species have also been observed to be toxic to human neuroblastoma cells as studied by MTT cytotoxicity assay. The studies signify the role of noradrenaline in PD and could help in the development of alternative strategies to cure Parkinson's disease. POS221 Sulfated glycosaminoglycans induce an amyloid aggregation of a non‐pathogenic neuropeptide Mathew Sebastiao 1, Isabelle Marcotte1, Steve Bourgault1 1Université du Québec à Montréal Numerous studies have reported that glycosaminoglycans (GAGs) accelerate amyloid assembly of peptides and proteins whose aggregation is associated with amyloid‐related diseases. GAGs are unbranched polysaccharides that are abundant at the cell surface and in the extracellular matrix. These sulfated polysaccharides have been associated with virtually all amyloid extracts analyzed from patients afflicted with protein misfolding diseases. Strikingly, GAGs not only mediate amyloid assembly of aggregation‐prone polypeptides but also the fibrilisation of numerous non‐amyloidogenic protein. The mechanism by which GAGs favor peptide self‐assembly, however, is still a matter of active debate. We used a non‐amyloidogenic, highly soluble peptide, PACAP27, as a model to investigate this mechanism. PACAP27 is a 27‐residue neurohormone that is non‐pathogenic, stable in solution, and non‐aggregating. In presence of low molecular weight heparin (LMWH), it readily forms amyloid‐like fibrils. Interestingly, PACAP27 rapidly adopts an a‐helical structure upon binding to sulfated GAGs and this conformation can be observed during the lag phase of the amyloid reaction. After a prolonged incubation, secondary‐structural transitions into a ß‐sheet‐rich conformation are observed. The amyloid nature of these assemblies were confirmed by atomic force microscopy, transmission electron microscopy, circular dichroism spectroscopy and thioflavin‐T fluorescence. To study whether a‐helical structures are essential to GAG‐mediated amyloidogenic pathways, two synthetic variants with reduced helical folding propensity were designed. Conformationally restricted peptides were able to form amyloid fibrils despite increased resistance to a‐helix formation. Lag phases from kinetics were observably reduced in both variants, suggesting that the formation of helical structures in the presence of GAGs is not an obligatory step in the mechanism of amyloid fibril formation. POS225 Tyrosine nitration and histidine carbonylation modulate λ6 immunoglobulin light chain structural stability and amyloidogenecity Ximena Zottig 1, Steve Bourgault1 1Université du Québec à Montréal, UQAM Light chain amyloidosis (AL) is the most common form of systemic amyloidosis, which originates from plasma cell over proliferation. This lethal disease is primarily characterized by an overproduction of immunoglobulin light chains (LC) and followed by pathological deposition of amyloid fibrils in the extracellular space of vital organs causing organ dysfunction. Non‐enzymatic post‐translational modifications (PTMs) can profoundly affect protein properties and have been shown to contribute to the pathogenesis of several protein misfolding diseases. However, few is known about PTMs effects on LC amyloidogenicity. Here, we investigated the impact of oxidative PTMs, particularly carbonylation by hydroxynonenal (HNE), oxidation and nitration, on the structure, thermodynamic stability and aggregation of Wil, a LC variable domain of the λ6 germline. We initially identified the residues that are prone to oxidative chemical modifications by LC‐MS/MS analysis performed after pepsin digestion. Subsequently, we noted that HNE‐carbonylation at specific His residues and nitration of precise Tyr side chains modulate Wil propensity to self‐assemble and to form ThT‐positive fibrillar aggregates. Nitration appears to accelerate the formation of aggregates with low cross–sheets quaternary structure. This effect has been associated with a decrease in thermodynamic stability. In contrast, HNE‐conjugation on specific His imidazole group did not affect the structural stability although it altered the conformational conversion driving the aggregation process. No effect on LC Wil aggregation and structural stability has been noted for oxidation Wil PTMs. Thus, both the thermodynamic stability and the physicochemical and structural properties have to be considered concomitantly when evaluating the amyloidogenic propensity of a LC variable domain in the context of AL. POS283 Why and how FVFLM peptides can be used as model systems to inhibit beta‐amyloid aggregation Maksim Kouza 1, Andrzej Kolinski2, Irina Buhimschi3, Andrzej Kloczkowski1 1Battelle Center for Mathematical Medicine, The Research Nationwide Children's Hospital, Ohio, USA, 2Faculty of Chemistry, University of Warsaw, Pasteura 1, 02–093 Warsaw, Poland, 3Center for Perinatal Research, Research Institute at Nationwide Children's Hospital, Columbus OH 43215, USA, Preeclampsia, a pregnancy‐specific disorder, shares typical pathophysiological features with protein misfolding disorders including Alzheimer's disease [1]. Despite much progress in understanding the protein aggregation process, the factors governing aggregation rates and stability of oligomers have not been fully understood. In our work, we performed all‐atom explicit solvent simulations with the GROMOS43a1 force field to reveal the relationships between the aggregation rate, mechanostability and binding energy of amyloidogenic peptides [2,3]. We studied two model peptide oligomers composed of FVFLM peptides which are overrepresented in urine of women with preeclampsia and KLVFF peptides from beta amyloid. The simulations demonstrated that mechanostability of peptide oligomers strongly correlates with binding energy. The larger mechanostability the stronger binding energy. The extent of the superior mechanostability of FVFLM as compared to KLVFF was quantified by subjecting both to steered molecular dynamics. The binding free energy of FVFLM and KLVFF systems, calculated from the potential of the mean force curve obtained through extensive umbrella sampling simulations, clearly established FVFLM as a better binder than KLVFF. Our study indicates that oligomer formation times are strongly correlated with stability: the faster the aggregation rate of a peptide the stronger has been noted to be its mechanical stability and binding free energy. Our study shows that peptide binding affinity might be accurately predicted using either mechanical stability or oligomer formation rates. References: 1. Buhimschi, I.A. et al., Science Translational Medicine 6, 245ra292 (2014) 2. Kouza M., et al., Physical Chemistry Chemical Physics, 19, 2990–2999 (2017) 3. Kouza M., Banerji A, Kmiecik M., Kolinski A., Kloczkowski A. and Buhimschi I, Molecules (2017) POS354 Inclusion Body Formation of Cu,Zn‐Superoxide Dismutase 1 in Escherichia coli Dalia Naser 1, Hilary Simon1 1University of Waterloo, 1992 Mutations in human Cu, Zn‐superoxide dismutase (SOD1) have been linked to the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS). Increasing evidence suggests ALS onset and/or progression may be due to the toxic misfolding and aggregation of mutant SOD1 protein. Overexpression of SOD1 in E. coli results in the formation of insoluble intracellular protein aggregates, referred to as inclusion bodies (IBs). SOD1 undergoes multiple post‐translational modifications in vivo, including binding one Cu and one Zn ion per monomer, the formation of an intramonomer disulfide bond, and dimerization to form the mature (holo) protein. We find that generally the less stable, immature and nonmetallated form of SOD1 has an increased propensity to aggregate in vitro. There is a wide range in the extent of IB formation for ALS‐linked SOD1 mutants under different conditions of temperature and added metal. IB formation is decreased in growth conditions of low temperature with the addition of Zn, indicating the protein may be partially rescued by binding of Zn. We consider the implications of these findings for the role of mutant SOD1 aggregation in disease. POS367 Polyamines enhances aggregation of folded proteins: a case study on bovine carbonic anhydrase Rimpy Kaur Chowhan 1, Laishram Rajendrakumar Singh1 1Dr. B. R. Ambedkar Center For Biomedical Research, University of Delhi, India Polyamines (PAs) are ubiquitously expressed cellular hydrocarbons containing more than one a‐amino groups. They participate in multiple cellular mechanisms including cell division,protein synthesis,etc. PAs physiological levels are very tightly regulated, as their low levels could inhibit cell progression while at higher concentrations they aid in carcinogenesis. However, in conformational diseases their levels are known to escalate and aggravate disease conditions by increasing the rate of protein aggregation. Since, PAs in normal cells(low concentrations) or cancer cells(high concentrations) have never been reported to induce protein aggregation, we speculated that only disordered protein's aggregation is enhanced by PAs. To verify, we investigated effect of human PAs on the aggregation of bovine carbonic anhydrase (BCA), a fully folded protein, using various probes including UV/Vis, fluorescence and CD spectroscopy, electron microscopy,etc. Surprisingly, we found that PAs enhances BCA aggregation,indicating protein's structural integrity to play no role in PA mediated aggregation modulation. This intrigued our curiosity to understand the mechanisms employed by PAs to enhance protein aggregation. Firstly, we investigated importance of PA's charged state in aiding its aggregation enhancing property by evaluating BCA aggregation profile in presence of PAs at pH conditions greater than pK of PAs a‐amino groups. Secondly, we analysed difference in extent of aggregation promoting propensity (measured in terms of final %aggregates,rate of aggregation, & lag time) of PAs with different hydrocarbon chain lengths(putrescine<spermidine<spermine) (See table 1). Result analysis revealed not charged state but hydrophobic residues of PAs to enhance protein‐protein interactions, and aggravate protein aggregation. The finding is believed to open up novel therapeutic targets for modulating protein aggregation, and thus conformational diseases. POS373 Exploring Amyloid‐like Aggregation Using a Mutant Domain of a Spidroin as a Solubility Tag Médoune Sarr 1, Nina Kronqvist1, Mara Reifenrath1, Gefei Chen1, Anna Rising1, Jan Johansson1 1Division for Neurogeriatrics, Center for Alzheimer Research, Department of NVS, Karolinska Institutet, Sweden Background Amyloidogenesis is associated with more than 30 diseases but the mechanisms involved remain largely unknown. ß17 is a de novo designed protein that favors ß‐sheet conformation and can form amyloid‐like fibrils (West et al. Proc. Natl. Acad. Sci. USA, 96, 1999), but its tendency to do so differs depending on subcellular localization in eukaryotic cells (Olzscha et al., Cell, 144, 2012, Woerner et al., Science, 351, 2016). Moreover, ß17 is difficult to express and purify from E. coli due to a pronounced tendency to aggregate, which makes it difficult to study experimentally. Aim: We investigated whether an N‐terminal mutant domain (NT*) of spider dragline silk protein (spidroin) (Kronqvist et al Nat Commun, in press) can be used as a solubility tag to prevent ß17 aggregation during expression and purification, and enable controlled aggregation of ß17. Methods: The fusion protein NT*‐ß17 was recombinantly produced in E. coli and the levels of soluble protein were analyzed by SDS‐PAGE. The fibril formation of ß17 and its secondary structure were determined by kinetic assays – using Thioflavin T (ThT) as a reporter – and by circular dichroism spectroscopy, respectively. Results: SDS‐PAGE revealed that high amounts of monomeric and soluble fusion protein were obtained. Proteolytic release of the NT* tag allows controlled ß17 aggregation as supported by ThT kinetic assay. Furthermore, structural studies showed that ß17 adopts a pH dependent ß‐sheet conformation. Conclusion: These findings support that NT* prevents aggregation of ß17, allowing structural studies to be performed. Our method may facilitate investigation of amyloid formation of other proteins, which could aid identification of potential anti‐amyloid strategies. POS382 Resveratrol Interacts with the Conformations Populated at the Early Stages of Human Lysozyme Fibrillation and Modulates the Pathway towards less‐toxic, Off‐pathway Aggregates Fatima Kamal Zaidi1 , Rajiv Bhat1 1Jawaharlal Nehru University, New Delhi, India The prevalence of devastating pathologies associated with amyloidogenic disorders have prompted the search for compounds that could inhibit the fibrillogenesis of the disease associated proteins and mitigate the toxicity associated with the on‐pathway intermediates of fibrillation. Polyphenols such as resveratrol have exhibited potent amyloid‐modulating properties towards a number of amyloidogenic proteins and in the present study its effect was investigated on the amyloid fibril formation of human lysozyme (HuL), associated with non‐neuropathic systemic amyloidosis. Using a range of biophysical techniques, it was shown that resveratrol interferes with an early event in the fibrillation pathway of HuL and potently inhibits its amyloid fibril formation by binding with weak to moderate affinity (Ka=104–107 M‐1) to the conformations populated at the early stages of the pathway, alongwith stabilization of these conformations. Molecular docking approaches revealed binding of resveratrol to the aggregation‐prone regions of HuL through a combination of hydrogen bonding and hydrophobic interactions, without perturbing its activity. The marginal decrease in HuL lifetime observed in the presence of resveratrol through time‐resolved fluorescence measurements indicated the involvement of complex formation between HuL and resveratrol and structure and activity analyses showed retention of the near‐native conformation of HuL in the presence of resveratrol. ANS binding analysis, seeding experiments and MTT cytotoxicity analysis revealed that in the presence of resveratrol the fibrillation pathway was modulated towards less‐hydrophobic and less‐toxic, off‐pathway aggregates. These results demonstrate the potential of resveratrol as a remarkable modulator of HuL aggregation and toxicity, and suggest its potential as a possible therapeutic agent against lysozyme amyloidosis. POS383 Qualitative and Quantitative Study of Amyloid‐like Structures in Urine of Pregnant Women with Preeclampsia Using Diazo Dyes V.A. Sergeeva1,3,4 , A.E. Bugrova2,4, N.L. Starodubtseva1,2, A.S. Kononikhin1,2, M.I. Indeykina3,4, Z.S. Khodzhaeva2, K. Muminova2, I.A. Popov1,2,3,5, V.E. Frankevich2, E.N. Nikolaev1,3,4,5, G.T. Sukhikh2 1Moscow Institute of Physics and Technology, Moscow, Russia, 2V. I. Kulakov Research Center for Obstetrics, Gynecology and Perinatology, Ministry of Healthcare of the Russian Federation, Moscow, Russia, 3V.L. Talrose Institute for Energy Problems of Chemical Physics, Russian Academy of Sciences, Moscow, Russia, 4Emanuel Institute for Biochemical Physics, Russian Academy of Sciences, Moscow, Russia, 5Orekhovich Institute of Biomedical Chemistry, Russian Academy of Sciences, Moscow, Russia. Purpose: Congo Red Dot (CRD) test is a new promising method of diagnosing preeclampsia (PE). It does not require specific lab equipment so may be implemented even at home. If it would demonstrate high specificity and sensitivity, it would effectively solve the problem of diagnosing and preventing severe forms of PE. Experimental description: Urine samples from pregnant women with preeclampsia and other hypertensive disorders and a control group of healthy pregnant women were collected at the V. I. Kulakov Research Center of Obstetrics, Gynecology and Perinatology. All patients included in the study provided a written informed consent. All procedures and study methods were approved by the Commission of biomedical ethics at the V.I. Kulakov Research Center for Obstetrics, Gynecology and Perinatology. The samples were processed according to the standard protocol suggested by I. Buhimschi et al.[1] using two diazo dyes: Congo Red and Evans Blue. Briefly, the urine sample was stained, incubated and dotted onto a nitrocellulose membrane. After drying the membrane was washed with alcohol. The results were processed first qualitatively as a yes/no answer, then quantitatively by the ratio of intensities prior and after washing. Results: The qualitative study of CRD showed a 60% sensitivity and 95% specificity. The quantitative study of Evans Blue Dot ‐ test and CRD‐test with cut‐off equal to 15% showed sensitivity of 70% and 80% and specificity of 85% and 80% respectively. Conclusion: The CRD‐test is statistically significant, but it requires improvement in order to be applicable to Caucausian women. Key Words: Preeclampsia (PE), Congo Red Dot (CRD) test. Acknowledgements: Amyloid‐like structures extraction and investigation was performed in the frame of Russian Science Foundation (grant No. 16‐14‐00181). Mass spectrometric measurements were performed using the equipment of “Human Proteome” Core Facility of the IBMC. Correspondence: address for correspondence – [e-mail], [e-mail]. Reference: [1] I.A. Buhimshi et al. Protein misfolding, congophilia, oligomerization and defection amyloid processing in preeclampsia. Sci. Transl. Med. 6, 245ra92 (2014) POS465 Endocytic proteins display prion‐like behavior that supports endocytic site assembly Ruben Claudio Aguilar 1, McKeith Pearson1, Arpita Sen2 1Purdue University, 2UC Berkeley Endocytosis is an essential process required for nutrient uptake and regulation of plasma membrane composition and signaling. Despite the knowledge accumulated along the years about this process, the mechanisms that mediate spatial‐temporal recruitment of proteins to endocytic sites are still not fully understood. However, this project aims to change this situation and to introduce the concept of prion‐like behavior as a force driving the assembly of endocytic complexes. Evidence collected in our lab indicates that similar to prions, the yeast homolog of the endocytic protein epsin Ent1 oligomerizes in vitro and displays cooperative binding to biological membranes in vitro and in vivo (Fig.1 A, B). Interestingly, Ent1 contain poly‐glutamine stretches and a Prion Forming Domain (PFD) [1, 2] and has been shown to display prion‐like aggregation in the presence of the well‐known prion conformation‐inducer [RNQ+]1. Further supporting the idea of a link between prion‐like behavior and endocytosis, the Hsp70 chaperone Ssa1 that is responsible for breaking prion‐complexes [3] is also a well‐known factor involved in the disassembly of the endocytic machinery. In summary, this project introduces the concept of prion‐driven interactions to the process of endocytic site assembly. In addition, our experimental results led to develop a quantitative mathematical model based on Fig.1C. Finally, we found that the mammalian epsin‐family member Epsin‐4, as well as AP1802 and Dab2 (adaptor proteins) also show PFDs, suggesting that prion‐like behavior plays an evolutionary role in vesicle trafficking. References: 1. Sen et al., (2017). Proceedings of the IEEE 102 (2): 385–393. 2. Alberti, et al. (2009). Cell. 137(1):146–158. 3. Schwimmer and Masison. (2002). Mol Cell Biol.;22(11):3590–3598. POS475 NMR Studies of the Inhibition of Insulin Fibril Formation by Rosmarinic Acid Qiuchen Zheng 1, Noel Lazo1 1Clark University Recent studies have shown that some naturally occurring polyphenols inhibit the formation of amyloid fibrils by several proteins including those associated with disease. The mechanism for the inhibition is not well understood. In this work, we first show that rosmarinic acid inhibits the formation of amyloid fibrils by insulin. To investigate the mechanism for the inhibition, we then used solution‐state 1H NMR. Analysis of NOESY spectra indicates that rosmarinic acid binds to insulin dimer and that it interacts with surface residues that have been identified to be important in the initial events of fibril formation. Together, our results show that rosmarinic acid inhibits fibril formation by stabilizing insulin dimers. POS479 The Effect of Small Peptides on the Aggregation of Amyloidogenic Sequences Sarah Petty 1, Andrew Mullin1, Sam Michelhaugh1, Benjamin Fitzgerald1, Christopher Fernandez1, Phallika Mon1, Mary Stuckey1, Michelle Schefter1 College of the Holy Cross, Massachusetts, USA, The misfolding and aggregation of the neuronal protein a‐synuclein is linked to the onset and progression of Parkinson's disease. Deletions from the amino acid sequence of a‐synuclein to form, for example, the Δ67–71 mutant have been seen to prevent aggregation of the protein. Certain short peptides based on this deleted sequence, (GGAVV), have the ability to aggregate in isolation, whereas others remain monomeric and unstructured as in the full‐length, intrinsically disordered a‐synuclein. In this work we examine the ability of the disordered stretches of GGAVV to reverse the aggregation of stable beta‐sheets. Peptides were synthesized using solid phase Fmoc chemistry and were capped at both termini to eliminate charge effects. Fourier transform infrared spectroscopy was used to determine the secondary structure by examining the frequency of the Amide I mode. Combinations of disordered and structured peptides were mixed at different concentrations and in different ratios and the IR spectra of the mixtures were compared to the mathematical sum of the individual spectra. We will report primarily on the ability of the disordered peptide, GGA to reverse the aggregation of the beta‐sheet forming peptide, AVV under various conditions. We will discuss the scope of this structural disruption by GGA having studied it with several other beta‐sheet peptides derived from a‐synuclein and other amyloidogenic proteins. Finally, we will show that this is not a unique property of GGA, and that other peptides derived from a‐synuclein can also reverse beta‐sheet formation in aggregated peptides. POS494 Exploration of the Folding Space of Amyloid‐ß: Identifying Specific Structural Features from Multiple Molecular Dynamics Simulations Simiao Lu 1, Christian Blouin1 1Dalhousie University Increases in amyloid‐ß 42 (Aß42) proteins was widely believed to be the trigger for a cascade of events in the brain that leads to Alzheimer's disease (AD). The accumulation results in the self‐assembly of monomers into neurotoxic oligomers, fibrils and plaques. This aggregation is preceded by a “misfolding” step of the monomer for which it is difficult to gather experimental evidence to elucidate the mechanism. We hypothesize that there exists consistent structural features in Aß despite the peptide's assumed intrinsic disorder. The structure and dynamics of Aß42 were studies based on 3 starting structures, over multiple replicates using standard and annealing molecular dynamics simulations (MDSs) in aqueous solution. Combining clustering, principal components analysis of cartesian coordinates, secondary structure analysis, and frequency contact matrices, we identified common characteristics of the Aß42 monomer. Although disordered, the Aß peptide has a consistent secondary structure propensity, a close interaction of its termini and an amphipathic nature. By including all samples from all simulations, we also constructed a relative free‐energy landscape that captures the conformational space of the Aß42 monomer. The study of intrinsically disordered structures using MDS is a challenging problem. We demonstrate in this work that it is possible to extract insight from a wide sampling even though the solution is made of a set of highly diverse tertiary structures. POS496 HspB1 and Hsc70 engage distinct tau species and have different inhibitory effects on amyloid formation Hannah Baughman 1, Amanda Clouser2, Amanda Clouser2, Rachel Klevit2, Abhinav Nath1 1University of Washington Department of Medicinal Chemistry, USA, 2University of Washington Department of Biochemistry, USA Tau is an intrinsically disordered microtubule‐associated protein that self‐associates to form amyloid fibrils in several neurodegenerative disorders, including Alzheimer's Disease. The cell's defense system against such diseases consists of a network of molecular chaperone proteins that act to prevent aberrant protein aggregation. To gain a better understanding of the strategies that have evolved to prevent tau aggregation and the roles of individual chaperone proteins, we characterized the interactions between tau and two molecular chaperones, HspB1 and Hsc70. HspB1 is an ATP‐independent small heat shock protein that functions as a holdase, whereas Hsc70 is a member of the Hsp70 family of refoldases. Using techniques including fluorescence spectroscopy, negative‐stain electron microscopy, and nuclear magnetic resonance spectroscopy, we have characterized the mechanisms by which each chaperone binds and alters the aggregation kinetics of tau. Although both chaperones bind the same aggregation‐prone region of tau, their effects on aggregation kinetics differ. HspB1 interacts transiently with tau species that occur early in the aggregation process, prior to the appearance of fibrillar material, and delays tau aggregation but does not prevent it. By contrast, Hsc70 forms a tight complex with aggregation‐prone tau and engages multiple tau species, including oligomers and fibrils. Hsc70 can fully prevent tau aggregation and is a highly potent substoichiometric inhibitor. These results demonstrate that chaperones from different families target distinct species along the tau aggregation pathway and may play complementary roles in the prevention of pathological aggregation. POS502 Understanding how ß‐Synuclein alters a‐Synuclein Fibril Formation Xue Yang 1, Jean Baum1 1Rutgers, The State University of New Jersey, USA Alpha‐Synuclein (aS) aggregation is related to many neurodegenerative diseases like Parkinson's disease that affects over five million people worldwide. Beta‐Synuclein (ßS) co‐localizes with aS and is a natural modulator of fibril formation. Using ThioT aggregation assays, Atomic Force Microscope (AFM), Circular Dichroism (CD) and Electrospray Ionization Mass Spectrometry (ESI‐MS) we characterize aS fibrils alone and aS and ßS co‐incubated fibrils. ThioT aggregation profiles show that fibril formation by co‐incubation has longer lag times and less ThioT binding than aS incubated alone. By ESI‐MS, we show that the main component of the co‐incubated fibril belongs to aS. CD and AFM show that co‐incubated fibrils have less ß‐sheet structure and are approximately 0.330 ± 0.058 µm shorter. These results show that co‐incubation of aS and ßS exerts an influence on aS by altering the fibril morphology. The relationship between these homologous proteins could be used to develop new approaches to therapeutic drugs against Parkinson's disease. POS509 Investigating the Mechanisms and Determinants of the Interactions of Amyloid‐Forming Peptides with Membranes Eleanor Vane 1, Abhinav Nath1 1Department of Medicinal Chemistry, University of Washington, USA Amyloid‐forming peptides display a variety of membrane‐disrupting and ‐remodeling activities, including membrane leakage, vesicle fusion, and vesicle aggregation. Membrane leakage, in particular, has been suggested as a general mechanism for the toxicity of pre‐amyloid states. Intriguing recent reports have suggested that, rather than being purely dysfunctional, certain amyloid‐forming peptides may carry out important antimicrobial functions, both by direct bacterial cytolysis and by inducing bacterial agglutination. Therefore, studying the interactions of amyloid peptides with membranes may be key to understanding their roles in health and disease. To understand the general principles that govern the interactions of amyloid‐forming peptides with membranes, we have focused on an amyloidogenic fragment of prostatic acid phosphatase (PAP248–286). In its amyloid state, this peptide is termed “semen‐derived enhancer of viral infection” (SEVI), and greatly increases HIV infectivity. Using a variety of biophysical techniques, we have discovered that PAP248–286 not only causes liposome disruption, but can also form large, fibrillar lipid‐peptide co‐aggregates. Both activities are dependent on lipid composition and environmental conditions. Low concentrations of PAP248–286 are able to cause leakage of partially anionic liposomes in a dose‐dependent manner. The lipid‐PAP248–286 co‐aggregates form only with anionic liposomes at specific peptide to lipid ratios. The importance of electrostatic interactions is expected given the cationic nature of PAP248–286, and is consistent with similar activities of other pre‐amyloid toxins. Both membrane leakage and vesicle co‐aggregation studies could illuminate potential mechanisms and determinants of PAP248–286's antimicrobial activity. This work could also guide further investigation into the membrane activity and potentially beneficial functions of other disease‐associated amyloid‐forming peptides. POS514 Modulation of TDP‐43 aggregation by ionic cosolutes Kevin Hadley 1, Yulong Sun2, Alison Medina Cruz2, Ying Dai2, Avi Chakrabartty2 1University Health Network, Canada, 2University of Toronto, Canada The 43 kDa TAR‐DNA binding protein (TDP‐43) is a soluble, homodimeric, intracellular protein involved in the regulation of mRNA processing and comprised of two oligonucleotide binding domains (RNA recognition motifs, RRM) and a large C‐terminal unstructured region. In the context of neurodegenerative disease, neuronal inclusions containing aggregated TDP‐43 are observed in about half of patients with frontotemporal dementia (FTD) and the majority of patients with amyotrophic lateral sclerosis (ALS). Consequently, identifying the factors that drive misfolding and aggregation of TDP‐43 is an important step to understanding the development of TDP‐43 pathology. We have refined methods for purifying a N‐terminally YFP‐tagged TDP‐43 (yTDP‐43) under denaturing conditions, followed by refolding in low‐ionic‐strength buffer. Under these conditions, yTDP‐43 forms a native‐like dimer that remains stable and soluble even at high concentrations. Misfolding and aggregation are rapidly induced by the presence of moderate concentrations (>100 mM) of NaCl and other sodium salts. Strikingly, divalent cations (Mg2+, Ca2+) induce extremely rapid misfolding and aggregation even at modest concentrations (<10 mM). Aggregation is inhibited by the presence of oligonucleotide binding partners, suggesting either direct or allosteric involvement of the RRM domains in maintaining stability and solubility. POS532 Coarse‐Grained Model of Fragments of Amyloid‐Beta peptide Apichart Linhananta 1, Robert Girardin1 1Lakehead University, Ontario, Canada Amyloid‐beta (Aß) peptides are 36 to 43 amino acid residues, implicated by the amyloid cascade hypothesis as one of the cause of Alzheimer's disease (AD). In the brain, Aßforms small peptide aggregates, called oligomers, leading to ß‐sheet fibrils that, with time, forms the 3D amyloid plaque that is the hallmark of AD. The structure of fibrils consists of parallel and/or anti‐parallel sheets wound in a wide array of complex three‐dimensional structures. Though parallel ß‐sheets are more common in AD, anti‐parallel sheets are believed by some researchers to be associated with early onset AD. This submission presents our effort in building a coarse‐grained model of the formation of micro‐crystal fibril of fragments of Aß. For computational efficiency, an amino acid is represented as a spherical bead, located at the center of its Ca carbon. This allows the study of systems of up to 200 peptides. In the spirit of Go models, the intra‐peptide dihedral and van der Waals interactions, as well as inter‐peptide interactions are biased to the experimental crystal structures. Two Ca beads on different peptides can also interact by a Mercedes‐Benz‐type hydrogen bond, which stabilizes ß‐sheet. MD simulations observed that Aß fragment models that are biased to anti‐parallel ß‐sheets condensed to fibrils at lower temperature than models biased to parallel ß‐sheets. Finally the kinetics of fibril formations are presented, and the implication to AD are discussed. POS538 A polar ring endows improved specificity to an antibody fragment. Zachary P. Schaefer 1, Lucas J. Bailey2, Anthony A. Kossiakoff3 1University of Illinois Urbana‐Champaign, USA, 2Invenra, USA, 3Biochemistry & Molecular Biology, The University of Chicago, USA Engineering monovalent Fab fragments into bivalent formats like IgGs or F(ab')2 can lead to aggregation presumably because of nonspecific off‐target interactions that induce aggregation. In an effort to further understand the molecular determinants of nonspecific interactions for engineered antibodies and natively folded proteins in general, we focused on a synthetic Fab with low nanomolar affinity to histone chaperone Anti‐silencing factor 1 (Asf1) that demonstrates off‐target binding through low solubility (∼5 mg/mL) in the multivalent F(ab') 2 state. Here, we generated phage display‐based shotgun scanning libraries to introduce aspartate as a negative design element into the antibody paratope. The antibody‐combining site was amenable to aspartate substitution at numerous positions within the antigen binding loops and one variant, Tyr(L93) Asp/His(L94) Asp/Thr(H100b) Asp, possessed high solubility (>100 mg/ml). Furthermore, the mutations decreased nonspecific interactions measured by column interaction chromatography and ELISA in the multivalent antibody format while maintaining high affinity to the antigen. Structural determination of the antibody‐antigen complex revealed that the aspartate‐permissive residues formed a polar ring around the structural and functional paratope, recapitulating the canonical feature of naturally occurring protein‐protein interactions. This observation may inform future strategies for the design and engineering of molecular recognition. 02. BIOINFORMATICS POS209 Bioinformatics discovery of chemical diversity in enzyme superfamilies Kai Hu 1, Benjamin Allen1 1The Pennsylvania State University, USA Enzymes exhibit great diversity in terms of reaction and chemical mechanism: while the same reaction can be catalyzed with different cofactors, the same set of cofactors can also be used toward different reactions. With recent developments in computational biology, we were able to investigate such diversities via conservation analysis regarding functionally important regions in the enzyme. The first enzyme we studied is class I ribonucleotide reductase (RNR). Class I RNRs can be divided into several subclasses according to their chemical mechanisms. To figure out potential alternative mechanisms, we performed sequence conservation analysis for sites around the active center in all queries that are annotated as class I RNR in protein database. By comparing sequence patterns with well‐characterized subclasses, we successfully identified sets of variants with distinct patterns. Further structural modeling analysis on one set of the variants (termed as NrdF*) suggests a novel chemical mechanism that is different from any canonical ones. Complementation test of NrdF*s in E.coli has confirmed their in vivo enzymatic activity and more detailed mechanistic studies are being performed. In addition to RNR, we applied the same pipeline and examined Fe/aKG‐dependent enzyme superfamily as well. Based on conservation patterns, we proposed critical sites that might be responsible for alternative chemistries performed by different subgroups. Our strategy leveraging structural biology and bioinformatics exhibits power in identifying alternative mechanisms in class I RNRs by prioritizing a list of novel RNR candidates. We can also apply our methods to other enzyme systems to identify critical sites/patterns that are associated with certain specific reactions. POS252 Sequence Based Analysis of LEA Proteins Expressed by Artemia franciscana. Brett Janis 1, Michael Menze1 University of Louisville1 Computed conformations of late embryogenesis abundant (LEA) proteins belonging to three LEA classifications (groups 1, 3, and 6) were analyzed using several bioinformatics programs. Each protein was found in the anhydrobiotic (life without water) cysts of the crustacean Artemia franciscana, the only animal known to express LEA proteins from more than one classification group. LEA proteins are hypothesized to have limited function in the hydrated state, but to undergo conformational transitions during desiccation that activate them in order to confer desiccation tolerance in anhydrobiotic organisms. Results suggest that AfLEA1.1 (group 1), is intrinsically disordered in the hydrated state, but may undergoes conformational shifts at its termini in response to desiccation with increasing MoRF potential towards the c‐terminus. The group 3 LEA proteins AfrLEAI, AfrLEA2, and ArfLEA3m, showed more structural variety than expected within a single LEA group. AfrLEAI may form an amphipathic a‐helix bundle that can interact with phospholipid bilayers and/or monolayers. In contrast, AfrLEA2 has an unusually high mean net charge when compared to all other LEA proteins, and contains no repeating amino acid sequences. AfrLEA6, a group 6 LEA protein, contains several repeats of seed maturation protein (SMP) domains and a 40‐amino acid long region enriched in proline which suggests limited conformational flexibility. This study suggests a high degree of structural variability within LEA groups indicating that LEA classification groups may not correspond with potential functions of the proteins and bioinformatics tools may provide insights into their individual roles in anhydrobiosis. This work was funded by NSF IOS‐1456809/1457061. POS300 A Deep Learning approach for the prediction of residues constituted in the Allosteric Communication Paths Andrzej Kloczkowski1, Girik Malik1, Anirban Banerji1, Maksim Kouza1, Irina Buhimschi1 1Nationwide Children's Hospital, Ohio, USA In the last twenty years the static view of nature of proteins, based solely on their tertiary (and quaternary) structure has completely changed. It has been shown that protein structure determines its fluctuational dynamics, and the dynamics is a key element for proteins to perform their functions. One of the most interesting manifestations of protein dynamics is allostery, where binding an effector molecule at one site often results in long‐range conformational changes of protein structure. Allosteric communication mechanism has been widely used by nature for regulatory purposes. Recently we have attempted to decipher the characteristics of residues which constitute the Allosteric Communication Paths by studying the annotated proteins from the AlloSteric Database (ASD) belonging to four different classes (kinases, nuclear receptors, peptidases, transcription factors), trying to decipher the consistent patterns inherent in the allosteric communication subsystem (ACSS). The graph‐theoretical approach to ACSSs unveiled interesting patterns in terms of individual and collective effects of these residues, along with other physical effects. In the present study, we took a step further, trying to learn the pattern representations exhibited by ACSS resides using deep convolutional neural networks. Using these learned representations, we aimed to classify, if a given residue belongs to ACSS or not. Preliminary data obtained by us are encouraging, showing that deep learning improves prediction of residues constituting Allosteric Communication Paths. Our method will further improve in the future with the growth of the size of ASD. POS361 Identification of structural determinants of the transglycosylation function in the alpha‐amylase enzyme family through residue contact analysis Rodrigo Arreola‐Barroso 1, Gloria Saab‐Rincón1 1Institute of Biotechnology, UNAM, Mexico Many drugs contain sugar moeities, however, attaching sugars to other molecules is a cumbersome process through traditional Chemistry as it requires many steps to ensure regio‐, chemo‐, and stereospecificity of the reaction. Enzymes can perform this task in a single step, although they usually need nucleotide activated substrates. The alpha‐amylase family employs readily available starch as substrate, and while some of its members only break starch into smaller glucose chains through hydrolyisis reactions, others are able to transfer sugar units to other sugar chains (transglycosylation), and even to different hydroxylated acceptors. The capability to perform both reactions within the family opens an opportunity to identify structural determinants of the transferase reaction. In this work we analyze the residue contacts in the alpha‐amylase family to identify possible mutation sites far from the catalytic center to modify the preference to transfer (measured as the transfer of glucose units to butanol) over hydrolysis of alpha‐amylase enzymes. POS366 Domain Family Analysis Reveals Insights into the Structure and Function of Yeast SH3 domains Elliott Stollar 1, Tom Brown1, Raj Shevagani1, Nick Brown1 Eastern New Mexico University, USA There is a need to develop bioinformatics to predict protein function, especially for all members of a domain family who share a common fold yet may have unique functions. To illustrate our approach, we investigate if each member of the yeast SH3 domain family (Figure 1) encodes specific information to bind unique peptide targets. Our approach identifies important specific residues that show little conservation within an alignment of a yeast domain family members (paralogs) but are yet conserved in an alignment of a given domain's non‐redundant ancestors (orthologs). This is unlike most other sequence alignment approaches that make no distinction between domain orthologs and domain paralogs and instead group domain family sequences together, losing critical insights. With this approach, we find most of the yeast SH3 domain family members have evolved unique amino acid conservation patterns that suggests they bind peptide targets with high intrinsic specificity. For a minority, it predicts a less diverse binding surface that likely requires additional factors to bind targets specifically. Our predictions are consistent with previously determined protein structures and recent evolutionary SPOT binding data. Furthermore, important secondary binding sites, allosteric pathways and structural extensions have been identified within the family. This approach can be used to probe intrinsic binding specificity in any other interaction domain family that is maintained over evolution. POS406 Exploring Sequence Space Using Coevolution And Structural Tertiary Motifs Vincent Frappier 1, Amy Keating1, Vincent Frappier1, Amy Keating1 1Massachusetts Institute of Technology, USA We recently conceived and implemented a new approach called SCRATCHED that explores proteins structure sequence space. SCRATCHED combines two methods that have attracted recent attention: (1) the use of small, modular protein tertiary structural motifs to describe or build protein structures, and (2) rich information provided by deep multiple‐sequence alignments (MSAs). The method decomposes protein structures (or PPIs interfaces) into small modular overlapping structural tertiary motifs (TERMs) of 10 to 25 residues that are queried against a database of single‐chain protein structures. MSAs corresponding to each structurally matched region are obtained from the PFAM database and used to learn the sequence preference for each TERM. Using a Multivariate Normal Distribution (MND) statistical framework, fragments are “stitched” together. We have used statistical scores from SCRATCHED to benchmark the method on 2 independent protein binding affinity test sets containing over 8000 data points. Preliminary results show that its prediction power is comparable to other known structure‐based methods, but it has several benefits over other approaches. First, it does not require all‐atom modeling, and thus can rapidly evaluate huge sequence spaces after a model for a target structure is derived. Second, the method provides an intuitive way to visualize the entire sequence landscape associated with a given fold or complex. Finally, our formalism makes it easy to look at how the landscape changes as constraints are imposed on the identities of certain residues. POS490 Structural Flexibility of an enzyme upon the binding of a ligand at an active site: using estimates of entropy over ensembles of contact matrices Zaahirah Qazi 1, Christian Blouin, Stephen L. Bearne 1Dalhousie University, Canada Contact maps have been widely used to represent proteins structures. We extend the concept to the aggregation of contact matrices across homologous samples by defining frequency contact matrices (FCM). A FCM encodes the frequency of a contact between the side chains of homologous sites. Since the contact between sites is not constant, it is possible to derive sitewise contact entropy from these data. Using this tool, we analyzed the general sitewise contact entropy of a set of protein structures with and without a ligand bound. This work specifically investigates the effect of ligand binding on side chain contacts of residues close to active site. The StructureFunction Linkage Database (SFLD) was used to define a dataset of enzyme of enolase superfamily that was further divided into subgroups. We calculated the Shannon entropy of residue contacts across the FCM, as a measure of the side chain freedom of residues. The results show that the contact entropy of residues buried inside the protein is higher as compared to the entropy of the residues located at the periphery. The results also show that for ligandbound protein subgroups, the degree of freedom for residues that are close to binding site is similar to that for the proteins that have no ligandbound at the active site. POS530 Mapping Beta Turn Geometry and Its Side‐Chain Determinants Nicholas Newell 1 1Independent Researcher Beta turns, which constitute about a quarter of all protein structure, are commonly classified by their central Ramachandran angles into a small set of types that provides only a low‐resolution picture of the variety of turn geometries and the favored conformations of important amino acid‐specific features. This work addresses this limitation by applying to beta turns the methods which the author recently used to map helix‐terminal structures (BMC Bioinformatics, 2015). A least‐squares, Cartesian‐space backbone clustering algorithm partitions the turns to any desired resolution while finding best‐fit representative exemplar structures for each cluster. The distribution of all turns across conformations is then depicted as a heat map by displaying this set of exemplars with colors and widths set in proportion to the turn abundances in the corresponding clusters. Statistical feature detection is then applied to the sequences of the turns in each cluster to detect all significant amino acid‐specific features involving one, two, or three residues, and the distribution of the abundance and over‐representation of each feature across conformations is depicted in individual heat maps for each feature as the widths and colors of the cluster exemplars. The global turn map provides a unified, high‐resolution picture of turn structure in Cartesian space. The determination of the detailed conformational preferences for previously known and unknown amino‐acid specific features, including those involving Asp, Asn, Glu, Pro, and Gly, should aid in understanding the structural consequences of disease‐induced mutations (as the results of the helix termini study have already done), and should be useful in protein design. 03. CHAPERONES POS020 Chaperone Activity of the N‐terminal Sequence of a Human Small Heat Shock Protein Kathryn McMenimen 1, Kathryn McMenimen1, Elizabeth DeLeon1, Mahima Poreddy1, Emily Gliniewicz, Chenwei Wang1 1Mount Holyoke College, Massachusetts, USA Small heat shock proteins (sHsps) are a family of molecular chaperones broadly employed across many organisms to prevent aggregation of partially unfolded protein substrates. A defining feature of sHsps is their ability to form dynamic, polydisperse oligomers that exhibit subunit exchange under certain conditions. The physiological relevance of oligomerization and mode(s) of chaperone function remain undetermined. sHsps contain the canonical “α‐crystallin domain” (ACD). This domain is flanked by an N‐ and C‐terminal region (NTR and CTR, respectively) of varying length and sequence, believed to participate in substrate and quaternary interactions. Previous studies have identified “mini‐chaperones”, which are fragments of the ACD that demonstrate chaperone activity by interacting with substrate proteins, preventing aggregation. In order to identify other functional regions of sHsps, we purified the relatively unstructured N‐terminal sequence from human HspB1. Using two model substrates, malate dehydrogenase (MDH) and citrate synthase (CS) we have identified an 88‐residue sequence that exhibits chaperone activity in solution. To determine the importance of oligomeric organization (multivalent interactions) in chaperone activity we constructed gold nanoparticles (AuNPs) appended with the HspB1 NTR. These sHsp‐AuNPs exhibit chaperone activity, with the efficacy varying by substrate. In exciting results, we observe that sHsp‐AuNPs are more active chaperones for citrate synthase than unconjugated sHsp NTRs. Finally, our results indicate the importance of NTRs in chaperone activity and demonstrate the therapeutic potential of sHsp‐AuNPs. POS135 Identifying and Developing GroEL/ES Chaperonin System Inhibitors as Antibacterial Candidates Steven Johnson 1, Sanofar Abdeed1, Trent Kunkle1, Nilshad Salim1, Andrew Ambrose2, Eli Chapman2 1IU School of Medicine, Indiana, USA, 2UA College of Pharmacy, Arizona, USA Prolonged use and misuse of antibiotics has led to the emergence of drug resistant bacteria for which effective therapies are severely lacking. This is illustrated by the rise of multidrug‐resistant Staphylococcus aureus (MRSA) strains, which kill ∼10,000 people in the US each year. Countering the rise of bacterial superbugs requires developing new antibiotics that target previously unexploited biological pathways. Towards this goal, the objective of our research is to identify antibacterial candidates that target the bacterial GroEL/ES chaperonin systems. GroEL/ES chaperonin systems are a distinct class of molecular chaperones that are essential for cell viability. As these molecular machines assist the proper folding and functioning of numerous cellular proteins, blocking their chaperoning function leads to multi‐pathway collapse and the death of bacteria. We previously identified several hundred inhibitors of the Escherichia coli GroEL/ES chaperonin system from a high‐throughput screening campaign. Through counter‐screening a subset of these GroEL/ES inhibitors in a series of chaperonin‐mediated biochemical and cell‐viability assays, we recently identified hit inhibitors that are cytocidal to Gram‐positive bacteria, including MRSA. Subsequent medicinal chemistry derivatization of initial hit scaffolds has identified analogs that exhibit sub‐micromolar antibacterial effects with therapeutic windows >100‐fold compared to human liver and kidney cell toxicity. These GroEL/ES inhibitors function through unique mechanisms that appear to ultimately prevent GroEL from engaging with the GroES co‐chaperone, thereby blocking the refolding cycle. These promising in vitro results encourage the continued optimization of novel GroEL/ES‐targeting antibacterial candidates for efficacy testing in in vivo bacterial infection models. POS200 Hsp40 and Hsp70 Act as Holdases to Prevent the N/C Interaction in the Androgen Receptor Prior to Activation by Androgens Marta Marin‐Argany 1, Xavier Salvatella1, Bahareh Eftekharzadeh1, Jennifer Rauch2, Daniele Mungianu1, Giulio Chiesa1, Jesús García1, Ángel Nebreda1, Jason Gestwicki2 1Institute for Research in Biomedicine (IRB Barcelona), Spain 2University of California at San Francisco, USA The androgen receptor (AR) is a transcription factor activated by androgens that regulates the expression of the male phenotype and is regulated by androgens. Prostate cells depends on AR activation for their proliferation, thus AR is an important therapeutic target for prostate cancer. Prior to activation, the androgen receptor is bound to the molecular chaperones hsp40 and hsp70 at the cytosol. Androgen binding causes the dissociation of the complex and leads to the formation of an active state of the AR capable to translocate into the nucleus. We have studied the molecular interaction of hsp40/70 with the transactivation domain (NTD) of the AR at high resolution by using solution NMR. We found that hsp40/70 stabilize the inactive form of the AR by recognizing a specific N‐terminal motif, which also interacts with the C‐terminal ligand binding domain (LBD) upon androgen activation. Our results reveal that hsp40 and hsp70 act as holdases of the inactive state of the AR and prevent the interaction between the N and the C‐terminal domains, that is characteristic of the active state of this important drug target. POS285 Dynamics of Membrane Protein‐Chaperone Interaction Camille McAvoy 1, Fu‐Cheng Liang1, Emily Miaou1, Shu‐ou Shan1 1California Institute of Technology, California, USA Membrane proteins are prone to aggregation and misfolding in aqueous environments and demand effective protection from molecular chaperones during their biogenesis. Chloroplast signal recognition particle 43 (cpSRP43) is a small, ATP‐independent chaperone that is necessary and sufficient for preventing and reversing the aggregation of the light‐harvesting, chlorophyll‐binding proteins (LHCP), a class of membrane proteins involved in photosynthesis. Our objective was to explore the nature of substrate‐chaperone interaction and further elucidate the role of conformational change in this complex formation. Using a combination of mutagenesis, light scattering, and fluorescence anisotropy we established interaction sites in the chaperone‐substrate complex. Moreover, mutations in the bridging helix (BH) domain of cpSRP43 helped to establish the cofactor cpSRP54's role in modulating cpSRP43 conformation. Electron paramagnetic resonance (EPR) analysis further illuminated the nature of conformational change in the Ankyrin repeat and BH regions of cpSRP43's substrate binding domain (SBD). POS321 The AAA+ chaperone‐protease ClpXP recognizes and degrades FtsZ polymers through a multivalent recognition strategy in Escherichia coli Marissa Viola 1, Jodi Camberg1 1University of Rhode Island, Rhode Island, USA The ATP‐dependent chaperone‐protease ClpXP degrades the tubulin homolog FtsZ during cell division, promoting subunit exchange at the division septum. ClpXP comprises a hexameric ClpX ring that recognizes, unfolds, and translocates substrates to ClpP, a serine protease, for degradation. FtsZ polymers assemble into a ring‐like structure at midcell during division. FtsZ contains a globular polymerization domain (residues 1–316), a disordered linker (residues 317–369), and a structured C‐terminus (residues 370–383). ClpX recognizes FtsZ in the disordered linker (residues 352–358) and the extreme C‐terminus (residues 375–383). Purified FtsZ contains a mixture of monomers and dimers. To determine if both recognition sites are utilized for degradation of monomers, we engineered a chimeric protein containing Gfp fused to the 67 C‐terminal residues of FtsZ (Gfp‐ZC67), containing the linker and C‐terminus, and monitored degradation by loss of fluorescence. Gfp‐ZC67 degradation is inhibited by the “XB” peptide, which contains residues important for the direct interaction between the SspB adaptor and the ClpX N‐domain and known to impair FtsZ degradation. Incubation of the XB peptide with Gfp‐ZC67(3527A), which contains mutations in the linker degron, also prevents degradation by ClpXP. A chimera lacking the C‐terminal degron (Gfp‐ZC67(ΔC9)) is not degraded by ClpXP. These results suggest that Gfp‐ZC67 is an N‐domain dependent ClpX substrate and that the linker degron is dispensable for degradation of FtsZ monomers. In contrast, degradation of fluorescent FtsZ(3527A) polymers with an impaired linker degron is significantly reduced, suggesting that the linker degron is important for polymer recognition. These data suggest that ClpXP uses two sites for enhanced recognition of FtsZ polymers, whereas FtsZ monomers utilize one. POS351 Hsp90 of Escherichia coli modulates assembly of FtsZ, the tubulin homolog in E. coli Anuradha Balasubramanian 1, Monica Markovski1, Sue Wickner1 1Laboratory of Molecular Biology, National cancer institute/NIH, USA Heat shock protein 90 (Hsp90) is a highly conserved ATP dependent molecular chaperone involved in remodeling, activating and stabilizing client proteins. More than 300 client proteins of Hsp90 have been identified. Only several clients of the E. coli Hsp90 homolog, Hsp90Ec, have been identified and the function of Hsp90Ec is unknown. However, when Hsp90Ec is overexpressed, cells become filamentous. The filaments exhibited distinct nucleoids, indicating that Hsp90Ec overexpression did not affect chromosomal segregation. To assess if overexpression of Hsp90Ec interfered with the cell division machinery, we tested if FtsZ, a tubulin homolog essential for cell division, assembled into ring‐like structures at future sites of cell division as it does in cells not overexpressing Hsp90Ec. We were unable to detect FtsZ rings in Hsp90Ec overexpressing cells, but observed that FtsZ was present at normal levels in cells overexpressing Hsp90Ec. We tested the hypothesis that Hsp90Ec prevents FtsZ polymerization. Using purified proteins and fluorescently labeled FtsZ, we observed by fluorescence microscopy that FtsZ formed polymers in the absence of Hsp90Ec, but not in the presence. Additionally, we showed that light scattering by FtsZ polymers was inhibited when Hsp90Ec was added prior to polymerization and that Hsp90Ec client‐binding defective mutants exhibited reduced ability to inhibit FtsZ polymerization. In summary, our data show that Hsp90Ec, when overexpressed, inhibits divisome assembly in vivo and prevents FtsZ polymerization in vitro. The results suggest that Hsp90Ec modulates of cell division by interacting and holding FtsZ, possibly slowing cell division during heat stress and other stresses. POS381 The RavA‐ViaA chaperone‐like system modulates the activity of respiratory chain complexes Vaibhav Bhandari 1, Keith Wong1 1University of Toronto, Canada The AAA+ proteins of the MoxR family have been proposed to have chaperone‐like activity in modulating protein assembly or in the insertion of cofactors into substrate proteins. Genetic analysis by our group has revealed that genes encoding for the MoxR AAA+ ATPases are often found in close proximity to those of proteins containing a von Willebrand Factor A (VWA) domain. In order to gain further insight into the relationships among these groups of proteins, functional analysis was performed on the Escherichia coli MoxR representative protein RavA and its VWA domain partner ViaA. Experimentally, we found that both the RavA and ViaA are functionally associated with anaerobic respiration in E. coli. Expression analysis of ravA and viaA genes showed that they are co‐expressed with several anaerobic respiratory genes, many of which are regulated by the anaerobic transcriptional regulator Fnr. Indeed, the expression of both ravA and viaA was found to be dependent on Fnr in anaerobically grown cells. Furthermore, ViaA was found to physically interact with FrdA, the flavin‐containing subunit of the anaerobic electron transport complex fumarate reductase (Frd). Both RavA and the Fe‐S‐containing subunit of the Frd complex, FrdB, regulate this interaction. Importantly, Frd activity was observed to increase in the absence of RavA and ViaA. This indicates that RavA and ViaA modulate the activity of the Frd complex, signifying a potential regulatory function of RavA‐ViaA during bacterial anaerobic respiration with fumarate as the terminal electron acceptor. POS385 Bri2 BRICHOS molecular chaperone activity is decoupled from its ability to inhibit amyloid fibril formation Gefei Chen 1, Axel Abelein1, Axel Leppert1, Simone Tambaro1, Henrik Biverstål1, Jenny Presto1, Jan Johansson1 Karolinska Institutet, Sweden Objectives The BRICHOS domain contains about 100 amino acids and associates with neurodegenerative and amyloid diseases, e.g. Alzheimer's disease (AD). In our in vitro and in vivo studies Bri2 BRICHOS was much more efficient than proSP‐C BRICHOS in inhibition of amyloid‐ß peptide (Aß) fibrillation. Recent data show that Bri2, but not proSP‐C, BRICHOS is able to efficiently suppress destabilized model protein aggregation. We hypothesized that the different quaternary structures of Bri2 and proSP‐C BRICHOS underlie their different abilities to work as molecular chaperones. Methods Human Bri2 BRICHOS proteins were recombinantly produced in E. coli, and analyzed by chromatography, mass spectrometry, western blotting, Thioflavin T assay, NMR spectroscopy, CD spectroscopy and chaperone activity assays. Results Here we found that the Bri2 BRICHOS dimer assembles into larger polydisperse oligomers, which are partly linked by inter‐chain disulfide bridges. Interestingly, Bri2 BRICHOS oligomers efficiently inhibit aggregation of thermodenatured citrate synthase (CS), whereas the monomers and dimers are inefficient. Conversely, the small size species are much more efficient than the large oligomers in suppressing Aß42 fibril formation, by blocking the secondary nucleation and elongation process. Furthermore, incubation of isolated monomeric Bri2 BRICHOS at 37°C and in the presence of mouse serum resulted in formation of larger oligomers with even number of subunits. Such oligomers potently inhibit CS aggregation, but their ability to prevent Aß42 fibril formation is reduced. Conclusions The decoupling of molecular chaperone activity and anti‐amyloid ability of the Bri2 BRICHOS domain in vitro suggests that its activity in vivo is regulated by environmental conditions. POS423 Chaperone–client‐interactions: From basic principles to roles in health and disease Sebastian Hiller 1, Björn Burmann1, Irena Burmann1, Roland Riek2, Silvia Campioni2, Juan Gerez2, Pratibha Kumari2, Stefan Rüdiger3 1Biozentrum, University of Basel, Switzerland, 2ETH Zurich, Switzerland, 3Utrecht University, Netherlands Molecular chaperones are essential in cellular protein homeostasis. Central aspects of chaperone function are still not well understood at the atomic level, including how chaperones recognize their clients, and in which conformational states clients are bound. We employ high‐resolution NMR spectroscopy as the main method to address such questions. Initial work on the periplasmic holdase Skp provided the first atomic‐level description of a natural full‐length chaperone–client complex [1]. An extension to the Spy–Im7 model system revealed how the chaperone Spy selectively recognizes the flexible, locally frustrated regions of partially folded client Im7 in a highly dynamic fashion. The interaction sites are identical for further chaperones, highlighting the general principles that govern client recognition [2]. We then utilize our mechanistic insights to investigate the role of chaperones in Parkinson's disease. Parkinson's is a common neurodegenerative disorder, pathologically manifested by intracellular accumulation of aggregates of the protein a‐Synuclein. Systematic investigations on an array of chaperones identified a general interaction motif at the a‐Synuclein amino‐terminus. This interaction is preserved inside living mammalian cells. In‐cell NMR experiments show that targeted knockdown of Hsp70 and Hsp90 chaperones in HEK‐293 cells triggers membrane‐interaction of a‐Synuclein and concomitant aggregate formation. Post‐translation modification of a‐Synuclein by methionine‐oxidation or specific tyrosine‐phosphorylation – known hallmarks of Parkinson's disease – destroy the chaperone interaction. Our data establish molecular chaperones as the dominant interactors of a‐Synuclein in healthy cells, mechanistically demonstrate their regulative function and provide novel therapeutic avenues for treatment of Parkinson's disease. References: [1] Burmann et al, Nat. Struct. Mol. Biol. 20, 1265 (2013). [2] He et al, Sci. Adv. 2, e1601625 (2016). POS434 Characterization of the role of Hsp70 system and HspB1 on disaggregase activity in humans Conrado de Campos Gonçalves 1,2, Jason C. Young1, Carlos H. I. Ramos2, Jason C. Young1 1McGill University, Canada, 2University of Campinas, Brazil Chaperones of Hsp70 family are engaged in many biological processes important for proteostasis, such as folding of polypopetides to the native state, prevention of aggregation and refolding of misfolded proteins. In metazoan cells under stress conditions, protein aggregates are frequently formed and they must be effectively eliminated in order to avoid potential pathological conditions. Further understanding of the disaggregation process in humans is very important since this activity is the cellular defense against several neuro‐degenerative disorders that are caused by deposition of aggregates. Information about how metazoans cope with accumulation of aggregates was limited, since these organisms do not express the AAA disaggregases found in yeast and bacteria. However, recent studies have shown that Hsp70, class A and B DNAJ proteins, and Hsp110 form a machinery with disaggregation activity. Moreover, Hsp26, a sHSP from yeast, seems to play a role in the disaggregation process. Therefore, our main objective is to investigate the Hsp70 system and the role of the human sHSP HspB1 on disaggregase activity. First, we analysed the ability of HspB1 and phosphorylation‐mimic mutants (S15D, S78D/S82D and the triple) to prevent luciferase aggregation by measuring light scattering after 45°C incubation. We observed that the triple mutant is the most efficient to bind luciferase and maintain solubility. Also, we performed in vitro disaggregation assays using aggregated luciferase and we show that Hsp70, Hsp110, DNAJA2, DNAJB1 and triple mutant HspB1 are important for disaggregation of luciferase. POS443 Small molecule inhibition of Hsp70 involving its interaction with substrate Yogita Patel 1, Jason Young1, Michael Wong1, Gabriela Chiosis2 1McGill University, Canada 2Memorial Sloan Kettering Cancer Center, USA Hsp70 is a ubiquitously expressed molecular chaperone that assists in protein folding and disaggregation. These ATP dependent processes require a J‐domain protein to stimulate ATP hydrolysis and a nucleotide exchange to displace ADP. Hsp70 is also upregulated in many cancers and is important for cancer survival. Therefore, small molecule inhibitors of Hsp70 show strong promise for eventual clinical use. We are currently characterizing novel, allosteric Hsp70 inhibitors. In HEK293 cells, the compounds decrease Hsp70‐dependent refolding and solubility of heat‐denatured luciferase. In vitro, using purified proteins, the compounds slow down ATP binding to Hsc70. Furthermore pulldown assays show the inhibitors increase luciferase binding to Hsc70, suggesting the compounds are impairing release of substrate. Insights into the mechanism of action of these Hsp70 inhibitors will provide tools to understanding the biological role of Hsp70 in cancer and other proteostasis disease contexts. POS445 The Role of DNAJB1 in Chaperone‐Mediated Disaggregation in the Mammalian System Kipunsam Lee 1, Jason Young1, Yogita Patel1, Michael Wong1 1McGill University, Canada Protein aggregates are toxic to cells. A number of neurodegenerative diseases result from the accumulation of mutant, misfolded protein aggregates. The molecular chaperones that are key in managing the misfolded proteins in cells are Hsp70 and its co‐chaperones. Hsp70 mediates protein refolding and directs proteins for degradation, either by the ubiquitin‐proteasome system (UPS) or autophagy. However, disaggregation of proteins in mammalian cells is not well understood. Recently, disaggregation by a complex of human chaperones, assisted by a yeast small heat shock protein, was observed in pure protein assays (1, 2). In our studies, we aim to demonstrate disaggregation activity and elucidate the mechanism of the core disaggregation machinery in mammalian cells. Thus far, we have results that show Hsp70, Hsp110 and DNAJB1 are involved with disaggregation in cells and we therefore hypothesize that DNAJB1 has an important role in mammalian disaggregation system. To examine the mechanism of the involvement of DNAJB1, a DNAJB1 knockout HEK293 cell line and DNAJB1 mutants with abolished interaction with other chaperones have been generated. In addition, we will also address how disaggregation is related to degradation via UPS and autophagy. The study of the disaggregation system in cells will allow us to have a better understanding of the protective mechanisms mammalian cells use against toxic aggregates. POS450 Structural Studies of the R2TP Complex Thiago Seraphim 1, Walid Houry1 1Department of Biochemistry, University of Toronto, Canada R2TP is a central complex modulating cellular processes that drive cell cycle, protein and genome homeostasis. It is also involved in chromatin remodeling, phosphatidylinositol 3‐kinase‐related kinases signaling, apoptosis and tumorigenesis. In 2005, our group discovered the R2TP complex interacting with the Hsp90 molecular chaperone and assisting in the assembly of box C/D small nucleolar ribonucleoproteins (snoRNPs) through Nop58, a core snoRNP factor. In yeast, R2TP is formed by four proteins: Rvb1 and Rvb2, which are members of the AAA+ superfamily of proteins; Pih1, a 40 kDa phosphopeptide‐binding protein; and Tah1, a 13 kDa tetratricopeptide motif‐containing protein that connects R2TP to Hsp90. Despite the studies regarding this important cellular complex, there is a lack of information about the structure of the R2TP complex and its interaction with snoRNPs complexes. In order to fill this gap, we used structural techniques to determine how R2TP assembles and interacts with snoRNPs. Using a combination of electron microscopy, small angle X‐ray scattering, nuclear magnetic resonance and pull‐down experiments, we determined the structure and interfaces driving the assemble of R2TP. We found that Rvb1 and Rvb2 assemble into a hexameric ring with alternating subunits and Pih1‐Tah1 is an elongated heterodimer in solution. We identified that these two heterocomplexes interact with each other via Rvb insertion domain and Pih1 N‐terminal domain. We also found that Pih1 N‐terminal domain also binds to the C‐terminus of Nop58. We propose a model for the R2TP assembly and mechanism, where the complex binds to Nop58 to protect it from aggregation and subsequently loads it onto snoRNP complexes. POS526 Characterization of Disease‐Associated Small Heat Shock Protein Mutants Christopher Woods 1, Rachel Klevit1 1University of Washington, USA A hallmark of life is the ability to tolerate and withstand stressors of a variety of sources, including ischemia, oxidation, and acidosis. Adverse cellular conditions can promote local unfolding of proteins, potentially leading to aggregate formation. Small heat shock proteins (sHSPs) are among the first responders to cellular stress and function as ATP‐independent chaperones that delay formation of aggregates by maintaining client proteins in a soluble, refolding competent state. All sHSPs share a domain architecture characterized by a structured central conserved a‐crystallin domain (ACD) with N‐ and C‐terminal sequence extensions of variable length, conservation, and amino acid identity. Most evidence indicates that the extensions are heterogeneous and largely disordered. While sHSP protomers are small (∼20 kDa), human sHSPs exist as large polydisperse oligomers (400–600 kDa) that defy traditional methods of structure determination. Missense mutations in the gene encoding HSPB5, the prototypical human sHSP (also known as aB‐crystallin), have been associated with autosomal dominant inheritance of diseases including cataract and myopathy. To compare and contrast the effects of such mutations on the structure and function of HSPB5, we have characterized three disease‐associated mutations located within the conserved a‐crystallin domain of HSPB5 (D109H, R120G, and D140N) by nuclear magnetic resonance (NMR) 1H‐15N heteronuclear single quantum coherence spectroscopy (HSQC), negative‐stain electron microscopy, and in vitro functional assessment. Although each mutant HSPB5 forms expanded oligomers, the effects observed on ACD structure and chaperone function vary substantially. Our current working model for the relationship between HSPB5 structure and chaperone function will be presented. 04. CHEMICAL BIOLOGY POS053 Selective covalent derivatization of hexahistidine tag in recombinant proteins Artem Melman1 1Clarkson University, USA We report on a new family of reagents for selective covalent derivatization of hexahistidine‐tagged recombinant proteins. The reaction involves reversible formation of a ternary metal complex between the reagent and imidazole function of hexahistidine tag through a bivalent metal cation followed by addition‐elimination reaction between Baylis‐Hillman ester functionality in the reagent and a non‐coordinated imidazole ring in the hexahistidine tag. These reagents can be used for introduction of fluorescent labels, “click” cycloaddition functionalities, or biotinylation of recombinant proteins using the equimolar amount of the reagent and a protein in diluted neutral aqueous solutions starting from 100 nM concentrations. This methodology was successfully extended for derivatization of surfaces and nanoparticles with the proteins. This approach appears to be a general protein bioconjugation technique suitable for derivatization of any hexahistidine‐tagged recombinant protein using a simple uniform procedure. POS056 The Synthesis of Keratan Sulfate Glycosaminoglycans by A Glycosynthase Approach Xiaohua Zhang 1, David Kwan1 1Concordia University, Canada Glycosaminoglycans serve an important role in cell communication. Keratan sulfate is an example of a glycosaminoglycan that exhibits varied biological functions, such as neuronal development, and maintenance of corneal matrix structure. It is also known to restrict neural regeneration after injury through the formation of glial scars, and to be overexpressed in some cancers. Elucidating the mechanism by which keratan sulfate mediates signalling by determining the interactions of ligands and binding proteins with specific keratan sulfate sub‐structures will lead to therapeutics that may be valuable in treating chronic diseases like neural damage and some forms of cancer. To do this, homogeneous structures of keratin sulfate with discrete length and defined patterns of sulfation are synthesized using glycosynthases, which are engineered from Keratanase II by mutation of the active‐site glutamate or aspartate residue that are involved in catalyzing the formation of the oxazoline intermediate. Sequence alignment analysis of several candidates among glutamate and aspartate residues that are conserved among GH111 enzymes reveal six residues that could be replaced by alanine. Preliminary results showed three of these six mutants are inactive to react with the natural substrate keratane sulfate. These active‐site inactive mutants could work as glycosynthases that can synthesize homogeneous keratan sulfate oligo‐ and polysaccharides of defined length and sulfation pattern, thus they can be used to probe the signals and interactions of specific keratan sulfate structures to determine their biological roles, focusing on neurological function and pathologies including cancers. POS109 Structural Symmetry of Proteins ‐ Why Do Proteins Stop Shy of Perfect Symmetry Maayan Bonjack 1, David Avnir1 1The Hebrew University of Jerusalem, Israel The majority of proteins form oligomers which have rotational symmetry. Despite the many functional advantages of symmetric packing, the vast majority of protein oligomers are only nearly symmetric. A key question in the field of proteins structure is therefore, if symmetry is so advantageous, why do proteins stop shy of perfect symmetry? The answer to that question is apparently multi‐parametric, and involves minimization of the free energy, the dynamics of the protein, the effects of surroundings parameters, and the mechanism of oligomerization. As a first step to address this question, we conversed the currently used vague qualitative descriptive language of the near‐symmetry into an accurate quantitative measure. We developed quantitative methods, which are capable of analyzing the whole protein, its backbone or its selected portions, and which are capable of visualizing the various levels of symmetry deviations in the form of symmetry maps. As a second step, we are analyzing the various parameters that affect symmetry distortions. We have explored the enthalpic effect and proved that in order to minimize the enthalpy of the amino‐acid interactions at the contact zones of the oligomeric subunits, giving away symmetry is required. We have also looked into the oligomerization mechanism parameter and found that it has an important role in affecting the final oligomer symmetry. Protein oligomers memorize their restricting evolutionary pathway of creation, which is translated into symmetry distortions. POS128 Probing Cation‐Pi Interactions of Lysine Reader Proteins Using In Vivo Unnatural Amino Acid Mutagenesis Stefanie Baril 1, Stefanie Baril1, Amber Koenig1, Mackenzie Krone1, Eric Brustad1, Marcey Waters1 1University of North Carolina, Chapel Hill, USA Regulation of gene expression is critical to cell development and function. Lysine methylation of histone proteins is a well‐recognized means of epigenetic control and dysregulation of lysine methylation has been implicated in multiple cancers. Proteins that interpret methylation states of lysine are referred to as reader proteins. In humans, these readers utilize a cage of aromatic residues that interact with the N‐methyl groups of methylated lysine (MenK) residues. Not surprisingly, cation‐pi interactions have been implicated as major contributors to MenK recognition. Dougherty and coworkers were the first to study cation‐pi interactions using unnatural amino acids (UAAs). Here we expand on Dougherty's approach by using in vivo UAA mutagenesis of a trimethyllysine (Me3K) reader protein. Two tyrosine residues in the aromatic cage of Heterochromatin Protein 1 (HP1) of Drosophila melanogaster were mutated to UAAs with various electron‐withdrawing and ‐donating groups. Me¬3¬K recognition was monitored by isothermal titration calorimetry and a correlation between binding and electrostatic potential of the UAAs’ R‐group was observed at both residues. Protein crystallography confirmed that changes in binding were not due to changes in structure. One position (Y24) appeared to exhibit more of a pronounced effect on binding than the other (Y48). This is likely due to the orientation of the Me3K peptide within the pocket. This provides, to our knowledge, the first example of differential, residue‐specific cation‐pi effects observed in a reader protein. Currently we are investigating how cation‐pi interactions contribute to HP1's recognition of the dimethyllysine ligand and applying this methodology to other reader proteins which have been implicated in human diseases. POS139 Characterizing the Functions of Structural Genomics Proteins through Computed Chemical Properties and Biochemical Validation Caitlyn Mills 1, Ramya Parasuram1, Penny Beuning1, Mary Jo Ondrechen1 1Northeastern University, Massachusetts, USA The Protein Structure Initiative resulted in over 14,000 Structural Genomics (SG) protein structures deposited in the PDB. Many of these structures have incorrect putative functional assignments or are of unknown biochemical function. The addition of better functional predictions for SG proteins will add substantial value to the already accumulated structural information. Our approach incorporates structure‐based computed chemical properties through local site prediction followed by local structural matching. First, Partial Order Optimum Likelihood (POOL) is used to computationally predict the catalytically important residues in a protein structure. Next, Structurally Aligned Local Sites of Activity (SALSA) analyzes a superfamily and develops spatially‐localized consensus signatures for the proteins of known function in each functional family based on POOL‐predicted residues and functionally characterized residues of importance. Finally, the POOL‐predicted residues for each SG protein are compared to each consensus signature and the alignments are scored to determine degree of similarity at the local active site. This research focuses on two enzyme superfamilies: Crotonase Superfamily (CS) and 6‐Hairpin Glycosidase Superfamily (6‐HGS). In the CS, we have computationally determined the function of many SG proteins that are currently misannotated and have provided better putative functional assignments for some. In addition, we have assayed a number of SG proteins and verified that our function predictions are correct. In the 6‐HGS, we have computationally demonstrated that our method can distinguish substrate specificity within a functional family. The main goal of this research project is to provide a validated approach to functional annotation for wider application by the community, including drug discovery and biofuel production. Support from NSF‐CHE‐1305655. POS158 Development of a Chemical Toolbox for Dissecting Activator‐Coactivator Interactions In Vivo Meghan Breen 1, Anna Mapp1 1University of Michigan, USA Dysregulation of transcription is observed in nearly all human diseases, and thus a better understanding of how transcription is regulated could open new avenues for drug development and therapeutic intervention. Several coactivators, such as the CREB binding protein (CBP) and the Mediator complex, utilize KIX domains as an interaction surface for transcriptional activators. KIX domains contain two activator interaction surfaces that have been shown in vitro to communicate allosterically. Many questions still remain, however, including if this allosteric network modulates transcriptional activation in vivo. To address this question, we are using the KIX domain of the Med15 subunit of the fungal Mediator complex as a model system and are developing a toolbox of probes that modulate activator‐Med15 KIX interactions. A library of Med15 mutants containing non‐native cysteine residues has been screened using Tethering, a site‐directed screening strategy, to identify ligands for both Med15 KIX activator binding surfaces. Identified ligands are being characterized according to their relative ability to act as chemical co‐chaperones that stabilize unique conformations of Med15 KIX and modulate activator binding orthosterically or allosterically. The suite of chemical co‐chaperones will then be used in Saccharomyces cerevisiae to evaluate the functional consequences of allosteric modulation of activator binding through transcription activation assays. We anticipate that results from these studies will clarify the role of allostery in activator‐KIX interactions and will expand our understanding of how activator‐coactivator interactions are regulated to control eukaryotic gene transcription. POS179 Kinetic Study of APH(3’)‐IIIa Inhabition Mechanism by Isothermal titration calorimetry (ITC) Yun Wang 1, Justin Di Trani1, Anthony Mittermaier1 1Department of Chemistry, McGill University, Canada The enzyme aminoglycoside phosphotransferase (APH) inactivates aminoglycoside antibiotics by phosphorylation, thereby conferring bacterial resistance. APH inhibitors could potentially re‐sensitize resistant bacteria, and are therefore of clinical interest. Enzyme kinetic studies of APH suggest that the substrate ATP is required to bind first, followed by aminoglycoside, and that the product phospho‐aminoglycoside dissociates rapidly while ADP dissociates slowly. The previous study suggested that the accumulation of ADP might compete with ATP for the enzyme active site. However, the inhibition process cannot be studied by the commonly used coupled assay method; in which the product ADP isconverted to ATP by the pyruvate kinase.We therefore developed a new method using Isothermal titration calorimetry (ITC) to study the kinetic and mechanism of APH product inhibition by ADP. We demonstrate for the first time that ADP is a potent competitive inhibiter of APH and determined KiADP via a multiple injection ITC assay, giving the unexpected result that APH(3’)‐IIIa binds more tightly to ADP than ATP. POS183 Bio‐valorization of waste engine oils Manel Ghribi 1, Marc Beauregard1, Fatma Meddeb‐Mouelhi1 1University of Québec at Trois‐Rivères, Canada Used engine oils are toxic to the fauna and the flora as they can alter natural processes. According to the US Environmental Protection Agency, 1 gallon of used engine oil can contaminate 1 million gallons of freshwater so the toxicity of these oils remains a constant threat. To manage this problem, Phoenix Environment (Québec, CA) recovers used motor oil, some components of which could be recovered or decontaminated biologically. In this project, we identified the classes of molecules present in representative stocks of waste oils and their proportions by GC‐MS, FTIR, RMN and TGA analyses. We also isolated and screened microorganisms found at Phoenix. The screening of bacteria was based on their ability to grow on culture media containing up to 20% used motor oils. These microorganisms were genetically identified. Subsequently, we have exposed those bacteria to a media containing used engine oil. When exposed to the used oil, fluorescent compounds were metabolized by certain bacteria. This suggests the existence of metabolic pathways potentially involved in oil‐derived fluorescent molecules processing .Those bacteria also have the ability to produce green bio‐products (bioplastics/biopolymers) using used engine oils as a nutrient source. FTIR analysis showed that one biopolymer produced extracellularly belongs to petrochemical bio‐polymers which can have several industrial applications. Moreover, those bacteria showed the ability to produce polyhdroxyalkanoate (PHA) intracellularly. Based on these preliminary results, we hope to identify biological avenues that could help to better manage used engine oil wastes. We are working to establish a partnership with Phoenix that could lead to bioplastic production from used engine oils. POS186 Interaction Between the Intrinsically Disordered Androgen Receptor N‐terminal Domain and RAP74‐CTD and How We Can Disrupt It Marta Frigolé‐Vivas 1, Xavier Salvatella1, Elzbieta Szulc1, Eva De Mol1, Claudio Di Sanza1, Paula Martínez1, Maciej Majewski2, Carla García1, Jesús García1 1Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Spain, 2Departament of Physical Chemistry, Faculty of Pharmacy, Universitat de Barcelona, Spain Androgen Receptor (AR) is a hormone‐activated transcription factor. AR N‐terminal domain (NTD) is intrinsically disordered. Its function is to recruit the basal transcription machinery in order to express genes related to the development of the male phenotype (Gelmann, E.P. 2002). AR‐NTD recruits the basal transcription machinery by directly interacting with RAP74‐CTD subunit of RNA‐polymerase II (McEwan, I.J. 1997). We have found that a short motif in AR‐NTD folds into an alpha‐helix conformation when bound to RAP74‐CTD. The affinity between RAP74‐CTD and chemically modified peptides spanning the AR interacting motif has been measured by solution Nuclear Magnetic Resonance and in cell Proximity‐Ligation Assay. In order to have low micro molar affinity between these two proteins, AR‐NTD should have high helical content and be phosphorylated at specific serine residues (DeMol, E. Manuscript in preparation). As AR over activation is the main driver for prostate cancer (PC) and castration‐resistant prostate cancer (CRPC), inhibiting AR‐NTD – RAP74CTD protein‐protein interaction (PPI) (Gelmann, E.P. 2002) could represent a novel therapeutic approach for the treatment of PC and CRPC. In my poster I will show i) the biophysical and cellular characterisation of AR‐NTD – RAP74‐CTD PPI and ii) the first results of the drug discovery process aimed at finding small molecule hits able to disrupt the aforementioned interaction. POS190 DNA Damage Specificity and Activity of Y‐family DNA Polymerases DinB and Pol Kappa Nicole Antczak 1, Morgan Packer1, Penny Beuning1 1Northeastern University, Massachusetts, USA DNA damage is a constant threat from both endogenous and exogenous sources. Most damage is repaired but unrepaired damage can be bypassed by Y‐family DNA polymerases in a process called translesion synthesis. Y‐family DNA polymerases E. coli DinB and human pol kappa specifically bypass minor groove adducts on the N2 position of deoxyguanine and are blocked by major groove adducts. Previously we have shown that a single point mutation in the active site loop of DinB eliminates discrimination against major groove adduct N6‐furfuryl‐dA. In this work, we created chimeric polymerases by swapping the loop region adjacent to the active site using site‐directed mutagenesis to assess the importance of the loop in damage specificity and activity. Using primer extension assays with DNA substrates containing the minor groove adduct N2‐furfuryl‐dG, major groove adducts etheno‐dA and N6‐furfuryl‐dA, as well as undamaged templates, we found the loop regions of DinB and pol kappa near the catalytic site are important for damage bypass specificity and accuracy of nucleotide incorporation. Specifically, human pol kappa is more tolerant of substitutions in its active site loops than DinB is. By creating loop swaps, an increase in the understanding of the preferential bypass of major groove adducts by DinB and human pol kappa can be accomplished. POS217 Optimizing lignocellulosic biomass processing: A novel and high throughput approach for xylan polysaccharides tracking at the surface of fibers Vinay Khatri 1, Fatma Meddeb‐Mouelhi1 1University of Quebec at Trois‐Rivieries, Canada Background: Xylan has been identified as a physical barrier which limits cellulose accessibility by covering the outer surface of fibers and interfibrillar space. Therefore, tracking xylan is a prerequisite for understanding and optimizing lignocellulosic biomass‐based processes (biofuel production). Results: In this study, we developed a novel xylan tracking approach using a two‐domain probe called OC15 which consists of a fusion of Cellvibrio japonicus carbohydrate‐binding domain 15 with the fluorescent protein mOrange2. The new probe specifically binds to xylan with an affinity similar to that of CBM15. The sensitivity of the OC15‐xylan detection approach was compared to that of standard methods such as X‐ray photoelectron spectroscopy (XPS) and chemical composition analysis (NREL/TP‐510–42618). All three approaches were used to analyze the variations of xylan content of lignocellulosic biomass fibers. XPS, which allows for surface analysis of fibers, did not clearly indicate changes in xylan content. Chemical composition analysis responded to the changes in xylan content, but did not give any specific information related to the fibers surface. Interestingly, only the OC15 probe enabled the highly sensitive detection of xylan variations at the surface of fibers. At variance with the other methods, the OC15 probe can be used in a high throughput format. Conclusions: We developed a rapid and high throughput approach for the detection of changes in xylan exposure at the surface of lignocellulosic fibers. The introduction of this method into the lignocellulosic biomass‐based industries should revolutionize the understanding and optimization of sustainable alternative fuel technologies. POS250 Design of Allosterically Regulated Protein Kinase and Phosphatase Switches Matthew Bienick 1, Indraneel Ghosh1, Sean Campbell1, David Lasansky1 1University of Arizona‐ Chemistry and Biochemistry, USA Selectively modulating the activity of a desired enzyme in vivo is a major goal in protein design and can aid in the development of methods for understanding and rewiring cell‐signaling pathways. Protein kinases and phosphatases are complementary enzymes that catalyze the addition and removal of phosphate groups upon substrate proteins, respectively. Kinases and phosphatases are implicated in almost every signaling pathway and their deregulation is implicated in diseases such as cancer and neurodegeneration. The high structural homology of kinases and phosphatases presents a challenge in designing selective inhibitors for understanding their cellular roles. Though powerful genetic knockdown or knockout tools exist, they are susceptible to compensatory cellular mechanisms. We have addressed this problem by designing a potentially general allosteric approach for gating kinase and phosphatase activity. We have utilized the well‐studied protein‐protein interactions between Bcl‐2 and BH3 domain proteins and their small molecule inhibitors. We have designed a system where specific BH3 peptides, 20 to 25‐residues, are inserted into an enzyme, at predetermined non‐homologous positions. BH3 domains, such as Bad, are unstructured but adopt a rigid, a‐helical conformation upon the addition of a protein binding partner, such as Bcl‐xL. Thus, in our system, Bcl‐xL acts as a poison and allosterically inhibits the function of the Bad‐inserted‐enzyme. Subsequently, the addition of a small molecule inhibitor, ABT‐737, binds to and displaces Bcl‐xL, acting as an antidote, thus restoring enzymatic activity. We have shown that this method allows for controlling the activity of both kinases and phosphatases with a small molecule in a dose dependent fashion both in vitro and in cellulo. POS284 Investigating the Functionality of Procaspase‐6 and Caspase‐6 by Various Nucleotides Ishankumar Soni 1, Kevin Dagbay1, Jeanne Hardy1 1University of Massachusetts at Amherst, USA Caspase‐6, a dimeric cysteine aspartate protease, is involved in multiple biological pathways including apoptosis and neurodegeneration. As a result, various researches have been conducted in understanding caspase‐6's regulation by biological as well as chemically synthesized molecules. Our research focuses on the direct interactions between nucleotides and (1) procaspase‐6: full‐length zymogen form as well as (2) caspase‐6: active cleaved form. Our preliminary results – ATP and GTP acyl probes bind to procaspase‐6 (but not caspase‐6) – motivate us to investigate in (1) where does ATP bind to procaspase‐6, (2) how does ATP modulate the functionality of procaspase‐6 and caspase‐6, and (3) how do other nucleotides (or their derivatives) modulate the functionality of procaspase‐6 and caspase‐6. Using site directed mutagenesis, we demonstrate that Tyr‐198 residue present at the dimer interface of procaspase‐6 is a putative binding site for ATP acyl probe as well as ATP. From our gel‐based in vitro activity assays, we establish that exogenous ATP can attenuate the self‐proteolytic activity of procasase‐6. At last, using gel‐based in vitro activity assays, we have derived the functionality effects of procaspase‐6 by dATP, ADP, AMP, GTP and diadenosine tetraphoshate (Ap4A). POS314 Identification and optimization of inhibitors of dihydrofolate reductases B, trimethoprim‐resistant enzymes Jacynthe Toulouse 1, Joelle Pelletier1, Dominic Bastien1, Delphine Forge2, Brahm Yachnin3, Daniel Deon4, Marc Gagnon4, Edward Ruediger4, Kévin Saint‐Jacques5 1University of Montreal, Canada, 2University of Mons, Belgium, 3McGill University, Canada, 4IRIC, University of Montreal, Canada, 5Université de Sherbrooke, Canada The widespread use of the antibiotic trimethoprim (TMP) in clinical applications against infections provides positive pressure for the development of antibiotic resistance. TMP inhibits the bacterial chromosomal dihydrofolate reductase (Dfr). The selective pressure produced by TMP has resulted in the emergence of an alternative family, the TMP‐resistant plasmid‐borne DfrB. There is little knowledge of the prevalence of dfrB genes. A library of hundreds of TMP‐resistant samples from clinical E. coli infections was screened in silico for the seven known dfrB genes. The relatively new dfrB4 gene was found as a mobile genetic element in a plasmid with multiple resistance genes. Previously, we reported the first generation of selective inhibitors towards DfrB1(Bastien et al. (2012)). These complex symmetrical bis‐benzimidazole inhibitors inhibit in the low micromolar range(Ki = 2–4 µM). We have built upon our previous work by developing a second generation of inhibitors in which we demonstrated, the tolerance to a variety of linker compositions, an increased potency, while optimal length of the inhibitors remained constant. The 1.75‐Å‐resolution crystal of DfrB1 with one inhibitor has helped uncover the mode of binding. In order to circumvent risks associated with taking two drugs, we screened a class of folate‐like compounds called bisubstrates. Bisubstrates are known inhibitors of a bacterial enzyme found in the folate pathway. We found bisubstrate inhibitors of DfrB1 in low micromolar range(Ki=10–20 µM). Amino acid sequences of DfrB1 and of DfrB4 are highly similar (82% identity). Based on this similarity, successful DfrB1 inhibitors will be tested as potential DfrB4 inhibitors. POS316 Developing a screening platform by Surface Plasmon Resonance (SPR) for the characterization and discovery of enzyme inhibitors Sarah Melissa Jane Abraham 1, Jacynthe L. Toulouse1, Nathalia Bukar1, Dominic Bastien1, Natalia Kadnikova1, Jean‐François Masson1, Joelle N. Pelletier1 1University of Montreal, Canada The objective of the research is to develop a more sensitive screening method based on a portable Surface Plasmon Resonance (SPR) device, an emerging technology. Our target enzyme is the R67 dihydrofolate reductase (R67 DHFR), which confers bacterial resistance to the antibiotic trimethoprim. Here, the target enzyme is linked to a very thin gold surface with specific plasmonic (optical) properties that are proportional to the mass of bound molecules. This can allow monitoring of binding events to the surface‐linked R67 DHFR, and thus permit identification of inhibitors. However, the mass of a typical inhibitor (i.e. 500–1000 g/mol) is too low to result in a significant SPR signal. Therefore, a competitive assay will be developed: a gold nanoparticle carrying a substrate analog will bind the surface‐immobilized R67 DHFR, giving a strong SPR signal due to its high mass. Then, upon screening for potential inhibitors, the bound nanoparticles will be displaced from the target enzyme if a molecule provides sufficient affinity. Thus, it is possible to indirectly monitor the binding of an inhibitor to the target. This project aims firstly at testing and validating of the SPR screening approach applied to R67 DHFR, and then applying this methodology to the screening of new inhibitors. POS340 Protein Dynamics and DNA‐Binding Specificity of the Eukaryotic Transcription Factor Pax5 Cecilia Perez‐Borrajero 1, Florian Heinkel1, Mark Okon1, Lawrence P. McIntosh1 1University of British Columbia, Canada Pax5 drives B‐cell development, and thus its DNA‐binding Paired domain is a hotspot for disease mutations linked to carcinogenesis. Using nuclear magnetic resonance (NMR) spectroscopy combined with molecular dynamics (MD) simulations, we have characterized the DNA binding mechanism of Pax5. In solution, the Paired domain is composed of two helical bundle subdomains joined by a flexible linker. Upon binding DNA, an N‐terminal ß‐hairpin folds, and the entire domain, including the linker, becomes more rigid and protected from amide hydrogen exchange (HX). Both subdomains contribute to specific DNA binding, resulting in an equilibrium dissociation constant more than three orders of magnitude lower than exhibited by the separate subdomains (nM versus µM). The isolated N‐terminal subdomain, which has very high DNA‐binding specificity, is dynamic as evidenced by HX and MD simulations. In addition, using isothermal titration calorimetry (ITC), we found that its interactions with DNA are driven by favorable enthalpy changes that counteract large losses in entropy. In contrast, the less flexible C‐terminal subdomain associates with a diverse range of DNA sequences by relying on non‐specific electrostatic contacts. The distinct physicochemical and DNA‐binding properties of the subdomains point to a mechanism in which the C‐terminal subdomain provides initial low‐affinity non‐specific contacts that allow Pax5 to associate with DNA rapidly. On the other hand, the dynamic N‐terminal subdomain is responsible for setting the specificity of cognate Pax5 sites. Overall, our studies indicate that conformational plasticity is needed for Pax5 to form high affinity interactions with its cognate DNA sites, and allow the protein to efficiently scan genomic DNA. POS359 Mapping of the binding sites of naphthalene‐based inhibitors on Trypanosoma brucei RNA editing ligase 1 Vaibhav Mehta 1, Reza Salavati1 1McGill University, McGill University The X‐ray crystal structure of Trypanosoma brucei RNA editing ligase 1 (TbREL1) in complex with ATP, paved the way for the discovery of C35 (V2) and V4 naphthalene based inhibitors of the adenylylation step by competing with ATP for the active site (Durrant et al, PlosNTD 2010; Moshiri et al, JBC 2010). While these compounds inhibit the in vitro adenylylation step of recombinant protein TbREL1 (rTbREL1) in a 1–10 uM range, they interfere with editosome‐RNA interactions in the context of native TbREL1 in purified editosomes (Moshiri et al, JBC 2010 for C35; unpublished for V4). The differences that we observed between the effects of compounds in the recombinant and native TbREL1, underscores their low efficiency. In fact, when we measured the inhibition constants (Ki) against the rTbREL1, the equilibrium constants were 500–1000 times larger than the dissociation equilibrium constant (Kd) for ATP (12 nM), suggesting that the lower affinities for rTbREL1 are one of the reasons for off‐targeting. In this work, by varying the predicted binding‐site residues of rTbREl1, we determined which residues are important for inhibitor activity. While F209, R288 and R309 appear to be crucial for C35 interaction with the ligase, K87, V88, F209 and R309 are important for V4 binding. Our data suggest partially overlapping binding sites for the two inhibitors with distinct contributions from the residues surrounding the active site of TbREL1. These results provide an important guideline for the design of more potent and specific TbREL1 inhibitors. POS369 Genomic targeting of epigenetic probes using a chemically tailored Cas9 system Glen Liszczak1, Tom Muir1 1Princeton University, USA Recent advances in the field of programmable DNA‐binding proteins have led to the development of facile methods for genomic localization of genetically encodable entities. Despite the extensive utility of these tools, locus‐specific delivery of synthetic molecules remains limited by a lack of adequate technologies. Here we combine the flexibility of chemical synthesis with the specificity of a programmable DNA‐binding protein by using protein trans‐splicing to ligate synthetic elements to a nuclease‐deficient Cas9 (dCas9) in vitro and subsequently deliver the dCas9 cargo to live cells. The versatility of this technology is demonstrated by delivering dCas9 fusions that include either the small‐molecule bromodomain and extra‐terminal family bromodomain inhibitor JQ1 or a peptide‐based PRC1 chromodomain ligand, which are capable of recruiting endogenous copies of their cognate binding partners to targeted genomic binding sites. We expect that this technology will allow for the genomic localization of a wide array of small molecules and modified proteinaceous materials. POS400 Intein Zymogens: Conditional Assembly and Splicing of Split Inteins via Targeted Proteolysis Josef Gramespacher1, Tom Muir1 1Princeton University, USA Naturally split inteins have found widespread use in chemical biology due to their ability to ligate separately expressed polypeptides through a process called protein trans‐splicing (PTS). However, due to the autocatalytic nature of PTS, significant effort has gone towards engineering inteins to require an external input or trigger to initiate the splicing reaction. In this study, we harness PTS by rendering the association of the split intein fragments conditional upon the presence of a user‐defined protease. We show that these intein ‘zymogens’ can be used to create protein sensors and actuators that respond to the presence of various stimuli, including bacterial pathogens, viral infections and light. Furthermore, we show that this design strategy is compatible with several orthogonal split intein pairs, thereby opening the way to the creation of multiplexed sensor systems. POS422 Spectroscopy of CISD Proteins Mary Konkle 1, Audrey Rex2, Michael Menze3, Nilay Chakraborty4 1Ball State University, Illinois, USA, 2Eastern Illinois University, Illinois, USA, 3University of Louisville, Kentucky, USA, 4University of Michigan ‐ Dearborn MitoNEET was the first member of the CDGSH iron‐sulfur domain (CISD) protein family discovered in 2004. Two additional family members, CISD2 and CISD3, were later discovered. While the structures of mitoNEET and CISD2 are strikingly similar, 74% sequence conserved, the functional impact of knock‐out models is quite different. A plasmid with the mitoNEET or CISD2 gene and an antibiotic resistance gene was obtained for the transformation into the Escherichia coli strain of C43(DE3) competent cells. Interestingly, consistent differences in the isolation and purification of CISD2 were noted relative to mitoNEET. Because the pH environment has a significant impact on iron‐sulfur protein structure/function, the pKox of CISD2 was determined by spectroscopic methods within the pH range between 6 and 11. This technique could not be used for mitoNEET due to protein instability. Fluorescence spectroscopy was employed to further characterize the binding of mitoNEET and CISD2 to the redox cofactors NADH and NADPH. The results presented here contribute to the larger trend of how small chemical changes can have large functional impacts on biomolecules. POS435 Selective Inhibition of E. coli DNA and RNA Topoisomerase Dev Arya 1, Nihar Ranjan1 1Clemson University, South Carolina, USA DNA topoisomerases are important class of enzymes that help in regulating DNA topology. They are involved in several cellular functions such as removing supercoils, strand breakage during recombination, chromosome condensation as well as disentangling of intertwined DNA. Eukaryotic DNA topoisomerases I and II have gained significant attention as drug targets particularly in cancer treatment. On the other hand, bacterial DNA gyrase and topoisomerase IV have been targets of some established antibiotics. Therefore, controlling DNA topoisomerase functions has been envisioned for developing new anticancer and antibacterial agents. The emergence of resistance to anti‐bacterials has necessitated the search of novel molecules that could help tackle these issues. We have recently reported that a Hoechst 33258 derived bisbenzimidazole with a linker containing a long alkyl group showed excellent DNA topoisomerase I inhibition. In this presentation, I will discuss the bacterial DNA and RNA topoisomerase poisoning inhibition of bisbenzimidazoles by systematically a] varying the alkyl chain length b] comparing the antibacterial and topoisomerase activity of bisbenzimdiazoles with mono‐benzimidazoles. We will then explore the relationships between the topoisomerase I poisoninginhibition, antibacterial activity and DNA binding of these compounds. References: 1. Berg, J. M., Tymoczko, J. L., and Stryer, L. (2002) Biochemistry. Fifth ed., W.H. Freeman, New York. 2. Champoux, J. J. (2001) DNA TOPOISOMERASES: Structure, function, and mechanism. Annu. Rev. Biochem. 70, 369–413. 3. Ranjan, N., Fulcrand, G., King, A., Brown, J., Jiang, X., Leng, F., and Arya, D. P. (2014) Selective inhibition of bacterial topoisomerase I by alkynyl‐bisbenzimidazoles. Medchemcomm. 5, 816–825. POS451 Fluorogenic Sensors for Carbonylation in Live Cells Kamalika Mukherjee 1, Tak Chio2, Anthony Sorrentino2, Dan Sackett3, Susan Bane2 1Massachusetts General Hospital and Harvard Medical School, USA, 2State University of New York at Binghamton, USA, 3Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, USA Oxidative stress induced carbonylation of biomolecules such as proteins and lipids is a hallmark for diseases such as cancer and diabetes and serves as a disease biomarker. Existing methodologies for detecting biomolecule carbonyls are endpoint assays, which require lengthy downstream processing of cell lysates or fixing for immunocytochemistry. Here we present a one‐step carbonyl detection, visualization and quantification methodology that can be performed in live cells. Three synthetic hydrazine‐functionalized coumarins have been synthesized and are shown to be biocompatible sensors that enable real time analyses of proteins and lipids in the cellular milieu. These fluorophores readily internalize into live cells, promptly react with carbonyls in the cellular milieu and are non‐toxic at the concentrations used for our studies. Each of these fluorescent derivatives has a distinct photochemical property but shares a unique ‘turn‐on’ ability. Upon conjugation with carbonyls, these sensors exhibit an increase in fluorescence that is conveniently detected by commonly available instrumentation. Here we demonstrate the sensors’ utility in different oxidative stress models, including serum starvation, metal catalyzed oxidation and peroxide induced stress. The intracellular distribution of carbonylated biomolecules in each live cell model is visualized using fluorescence microscopy. Quantitative analysis of cellular carbonylation using a plate reader on live cells or lysates with these fluorophores is also presented. The fluorescent tag remains intact through SDS‐PAGE of cellular lysate, indicating that these probes may also find application in proteomics. POS455 Investigating Novel Allosteric Modulators of Glutamate Dehydrogenase 1 Emma Scaro 1, Mary Konkle1, Sarah Gisondi1, Michael Menze2, Nilay Chakraborty3 1Eastern Illinois University, Illinois, USA, 2University of Louisville, 3University of Michigan – Dearborn, USA Glutamate dehydrogenase 1 (GDH1) is an enzyme that is key to metabolic control in mammalian cells. Numerous molecules that exert allosteric control over GDH1 activity have been identified and include ADP, GTP, leucine and palmitoyl‐CoA. MitoNEET is a recently discovered mitochondrial [2Fe‐2S] protein that is a binding partner of the anti‐diabetic drug pioglitazone. MitoNEET contains a unique three cysteines and one histidine ligation of the metal cluster. However, the cellular function of mitoNEET is currently unknown. Putative protein‐binding partners of mitoNEET were analyzed for cellular localization, cofactors, and function in order to elucidate the function of mitoNEET. One result of the pull‐down assays, GDH1, was evaluated as a protein‐binding partner for mitoNEET. Enzyme kinetic assays were used to study how different binding partners, both proteins and small molecules, affect GDH1 function. Allosteric regulators were added to the enzyme to compare the GDH1 function in the presence of the binding partners such as mitoNEET and TZDs. POS457 DNA‐protein conjugates for electrochemical biosensing applications Xiaomeng Wang 1, Alexis Vallée‐Bélisle1 1Université de Montréal, Canada Protein‐oligonucleotide conjugates (POCs) possess unique properties with broad applications ranging from biomedical diagnostic assays to fundamental research on molecular recognition. The future of this class of molecules is bright, but the tools for making them are far from generic. Different approaches of conjugation, including non‐covalent and covalent attachment, typically require modification of the protein. Here, we explore a cheap and universal covalent labelling approach to synthesize a wide range of POCs from non‐modified proteins. By using a heterobifunctional cross‐linker, we have successfully attached a thiol‐modified, redox‐labeled single‐stranded DNA (ssDNA) to lysine residues of a bacterial fimbriae protein. Using careful choice of reaction conditions (e.g. stoichiometry, time, buffer), we show that we can synthesize a range of conjugated proteins containing different numbers of DNAs. Future efforts will investigate site‐specific labelling for different research topics in our lab. Possible approaches to achieve this goal include kinetic control, or by using a NTA‐labeled ssDNA1 that can non‐covalently attach to a poly‐ hisdine tag on a protein. This ssDNA1 will hybridize with an ester‐functionalized ssDNA2 which will then react with the spatially proximate lysine on the protein, followed by displacement of ssDNA1 to leave the covalently‐attached ssDNA2. POS505 Novel Metal‐Specific Inhibition of Histone Deacetylase 8 Hannah Foley 1, Kelsey Diffley1, Christian Perez2, Seth Cohen2, Carol Fierke1 1University of Michigan, USA, 2University of California, San Diego, USA Post‐translational modifications (PTMs) are important regulators of gene expression. Acetylation is a reversible PTM that is modulated by the opposing activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs). The HDAC family of enzymes includes metal‐dependent HDACs that require an active site divalent metal ion for catalysis, either Zn(II) or Fe(II). Of the HDAC metalloenzymes, HDAC8 is the best biochemically characterized isozyme, yet the identity of its in vivo divalent metal species is still unclear. Development of metal‐specific inhibitors is one promising approach that may help elucidate the in vivo metal species bound to HDAC8. Here we used a biochemical activity assay to screen fragment libraries of HDAC8 inhibitors. These inhibitors were structurally based on parent compounds discovered in a previous screen to preferentially inhibit HDAC8 with Fe(II) bound in its active site rather than Zn(II). We demonstrated that all screened compounds lower the IC50 for inhibition of Fe(II)‐HDAC8 when compared to parent compounds while still maintaining specificity for Fe(II)‐ compared to Zn(II)‐HDAC8. This work provides a basis for developing metal‐specific HDAC8 inhibitors with increased affinity and specificity that may be useful for identifying the metal species bound to HDAC8 in vivo. POS516 Novel Non‐Hydroxamate Inhibition of Histone Deacetylase 8 Kelsey Diffley 1, Carol Fierke1, George Murphy III1 1University of Michigan – Ann Arbor, USA Histone deacetylases (HDACs) have become an attractive target in cancer therapies. There are currently four FDA approved HDAC inhibitors. Presently, inhibitor design is based on targeting the divalent metal present in the active site of HDACs through functionalization around a hydroxamic acid warhead. However, the hydroxamate moiety has poor pharmacokinetics and these inhibitors have high toxicity. Using a fragment‐based metalloenzyme inhibitor library focused on non‐hydroxamate compounds1, we have identified compounds with novel functional groups which inhibit HDAC8. A subset of these compounds additionally showed inhibition that was dependent on the HDAC8 bound metal (Fe(II) vs Zn(II)). While the fragments show promise for further development into HDAC inhibitors, the mechanism of inhibition needs to be determined. There are three likely mechanisms by which the compounds are inhibiting HDAC8: (1) chelating metal in a manner similar to EDTA; (2) stripping metal directly from the active site; or (3) acting as competitive inhibitors by binding in the active site. We developed a method to determine the extent of metal chelation utilizing inductively coupled plasma‐mass spectrometry (ICP‐MS) to measure the presence of Fe(II) after incubation with the inhibitors. Several compounds were identified as competitive inhibitors and illicit further study. References: 1. Agrawal et al. Chelator Fragment Libraries for Targeting Metalloproteinases. (2010), ChemMedChem, 5: 195–199 POS517 SmgGDS‐607 regulation of small GTPase prenylation is nucleotide‐dependent Desirée García‐Torres1, Carol Fierke1, Benjamin Jennings1 1University of Michigan, Ann Harbor, USA Several small GTPases, including members of the Ras, Rho, and Rap families, are modified on their C‐terminus by protein prenylation. Protein farnesyltransferase (FTase) and protein geranylgeranyltransferase‐I (GGTase‐I) catalyze the attachment of a 15‐carbon farnesyl or a 20‐carbon geranylgeranyl moiety, respectively, to a cysteine residue near the C‐terminus of substrate proteins ending with a CAAX sequence. Proper prenylation and subsequent processing of some small GTPases are known to be important for correct membrane localization and cellular functions. Recent work in cells has indicated that SmgGDS (small G‐protein GDP dissociation stimulator) proteins can bind small GTPases and regulate their entry into the cellular prenylation pathway. To further characterize the potential role of SmgGDS proteins in regulating small GTPase prenylation, we tested whether SmgGDS splice variants could inhibit the in vitro activity of GGTase‐I or FTase for small GTPases. SmgGDS‐607 inhibited the activity of both enzymes and this was determined to occur through GTPase substrate sequestration rather than direct enzyme inhibition. Nucleotide‐status (GTP‐ vs. GDP‐bound) is known to alter the structure of small GTPases; therefore, its effect on the interaction with SmgGDS proteins and protein prenylation was tested. SmgGDS‐607 reduced the in vitro geranylgeranylation and farnesylation of RhoA‐GDP significantly more than RhoA‐GTP. However, SmgGDS‐607 could still bind RhoA‐GTP albeit with weaker affinity. Currently, we are looking into other small GTPases, such as KRas, to further understand how SmgGDS proteins regulate protein prenylation. Increased understanding on SmgGDS proteins regulation and the trafficking of prenylated proteins may offer new insights into targeting prenylation of small GTPases for treating human diseases. 05. COMPUTATIONAL MODELING/SIMULATION POS022 Odorant receptors activation dynamics involves specific sequence motifs Claire de March 1, Claire de March1, Elise Bruguera1, Jérémie Topin2, Jérôme Golebiowski2, Hiroaki Matsunami1 1Duke University, North Carolina, USA, 2Université de Nice Sophia Antipolis, France The complexity of the odor chemical space and the number of odorant receptors (ORs) make understanding odor coding an enormous challenge. More specifically, being able to predict the behavior of an OR in front of an agonist, an antagonist or a non‐agonist remains to be done.[1] Using a joint approach combining molecular modeling and experimental data on several ORs[2], we have built a model that can capture the active or inactive state of these proteins when bound to ligands with different potencies.[3] By the aim of computational tools combined with site‐directed mutagenesis, we predict the activation of human OR7D4 by its strong agonists, androstenone and androstadienone, and its inactivated form by a non‐agonist the (Z)‐2‐decenal. For the first time, a robust computational model of a G Protein‐Coupled odorant Receptor captures a differential activation mechanism upon ligand binding, consistent with in vitro data. This suggests that an unprecedented metric, the activation mechanism, can be used for predicting OR activation. This model is used to identify residues responsible for the dynamics activation from the binding cavity to the G protein coupling site. These residues belong to highly conserved motifs, suggesting that the results are valid for all mammalian OR. Mutations at these sites confirm their crucial functional role. Such powerful approaches will help unravel odor‐coding in the nervous system and facilitate the understanding of general rules of neuronal activation induced by an odor. References: [1] C. A. de March et al., FFJ 2015, 30. [2] C. A. de March et al., Prot Science 2015, 24. [3] C. A. de March et al., JACS 2015, 137. POS054 Active Site Distortion in the Dishevelled PDZ Domain Charles Sader 1, Jie Zheng1 1UCLA, USA Elevated Wnt signaling has been implicated in a number of diseases, but we are specifically interested in its role in retinal neovascularization as well as elevated intraocular pressure leading to damage of the trabecular meshwork. Dishevelled (Dvl), a cytoplasmic protein involved in Wnt signaling, is a major regulator of development through control of cell proliferation and differentiation. Inhibition of abnormal Wnt signaling by targeting the PDZ domain of Dvl has therapeutic potential; however, improvement of binding affinity and specificity of small‐molecule inhibitors has been a challenge. The purpose of this study was to expand upon current structure‐activity relationships based on small‐molecule and peptide ligands of Dvl. Specifically, our goal was to determine the correlation between the amount of distortion in the active site with binding affinity across a range of ligands and use this information to guide future design efforts. Molecular dynamics simulations of Dvl PDZ domain in the free and ligand‐bound states were performed in a box of explicit water. These simulations reveal an increase in pairwise distances between C positions along the active site binding groove upon ligand binding. Observed changes in backbone dihedral angles support distortion of the alpha2 helix toward a concave active site. Taken together to represent distortion, the structural deviations in the active site of the PDZ domain upon binding, quantified as root‐mean‐square deviation, correlate moderately well with binding affinity. POS062 Exploring the conformational space of anti‐apoptotic proteins of the Bcl‐2 family Luis Caro‐Gomez1 1Instituto Politécnico Nacional, Mexico Apoptosis is a natural process required for the removal of redundant cells during development, potentially dangerous cells and those in senescence. Cell death dysregulation has been implicated in a variety of human diseases such as cancer, autoimmunity, and neurodegenerative disorders. This process is regulated by several proteins that belong to Bcl‐2 family. Members of this family are grouped according to their homology and participation in the apoptotic mitochondrial pathway (pro‐ and anti‐apoptotic proteins). The anti‐ apoptotic Bcl‐2 proteins (e.g., Bcl‐2, A1, Bcl‐XL and Bcl‐W) pro‐apoptotic proteins (e.g., Bax, Bak and Bok) or only the BH3 domain (e.g., Bid, Bin and Bik). Computational studies are of great interest to describe the folding/unfolding properties of proteins and to explain the interaction properties with other proteins or small ligands which can be used in rational drug design. The aim of this project is to describe structural properties of proteins Bcl‐2, Bcl‐2A1, Bcl‐2ΔTM and Bcl‐2A1ΔTM and possible conformational changes associated with interaction with other proteins through methodologies in silico (using molecular dynamics simulations (MD) at different temperature conditions. This information will be useful in better understanding the contribution of each domain to the function of the mentioned proteins. The most important results indicate that FLD and TM contribute in maintaining the overall conformation of the protein when proteins are thermally stressed. Furthermore, the stabilizing effect of both domains is additive. POS111 Molecular Modelling of the Orai Calcium Channels Tugba Nur Ozturk 1, Guillaume Lamoureux1 1Concordia University, Montreal, Canada Store‐operated calcium entry is one of the primary mechanisms of calcium influx into cells. In many cell types, it is mediated by the activation of calcium release‐activated calcium (CRAC) channels in the plasma membrane. CRAC channels are composed of Stromal Interaction Molecules (STIMs) and Orai proteins. Orai proteins form the pore subunit of the CRAC channels and are activated by STIMs, which act as an intracellular calcium sensor. Although it is believed that the functional form of Orai channels is tetrameric [Thompson and Shuttleworth, Sci. Rep. 3, 1961 (2013)], the recent crystal structure of Orai [Hou et al., Science 338, 1308 (2012)] exhibits a hexameric form with a threefold, quasi‐sixfold axial symmetry around the pore of the channel. In this study, we examine the stabilities of the hexamer and tetramer assemblies of Orai proteins by performing all‐atom molecular dynamics simulations. To explore the functional significance of both forms of the Orai channel, we analyze the contacts between the consequent subunits, the distribution of water molecules inside the ion pore, the stability of the pore radius and the conformational changes of the pore‐lining residues involved in the gating mechanism. To investigate ion selectivity, we generate the free energy profiles for permeation of calcium, potassium and sodium ions using umbrella sampling. POS159 A Single Mutation Enables LAO Binding Protein to Visit a Closed State Without Ligands Diego S. Granados 1, Alejandro Sosa‐Peinado1, Jesus Banda‐Vazquez1 1Laboratorio de Fisicoquímica de Proteínas. School of Medicine, UNAM. México., The modest success while changing functions of existing proteins with traditional strategies like grafting make evident we are not understanding to what degree subtle perturbations on the natural interactions of a protein will have drastic effects on functional features that rely on dynamics. One way to gain insight on this is understanding the conformational dynamics of our systems of study by computational means. Methods We performed Molecular Dynamics simulations (MD) to study dynamical effects of mutating the site 117 on LAO‐BP (PDB ID: 2LAO), a periplasmic binding protein of Salmonella typhi which naturally recognizes positive aminoacids on a nanomolar range and shows an open to close transition only in presence of ligands. We evaluated the effect of four substitutions on the 117 site: L117K, L117Q, L117R and L117E. We generated structures for each mutant using I‐tasser server. The simulations were done using amber suite and ff99SB‐ildn forcefield with TIP3P water and a total simulation time of 500 ns was obtained for each protein simulated. Analysis routines were performed using cpptraj and the R library on structural analysis called Bio3D. Results We have found that changing the naturally occurring residue in this position (leucine) for a lysine on L117K mutant alters dramatically the behaviour of this protein making possible the sampling of closed state without ligands. Additional mutants don't show this alteration suggesting that a interaction between K117 and D11 makes this possible on L117K. This spontaneous mechanism of closing could be an explanation of the new affinities gained by this mutant reported on related experimental works by our group. POS176 A Novel Algorithm to Compute and Visualize Protein Coarse‐grained Energy Landscapes for Folding Prediction David Becerra 1, Zheng Dai1, Jerome Waldispuhl1 1School of Computer Science and McGill Centre for Bioinformatics, Montreal, Canada The protein‐folding (PF) problem aims to predict the physical and dynamical process that transforms an unfolded protein into a functional 3D structure. To‐date, the approaches to obtain this information rely on time‐consuming molecular dynamic (MD) simulations. We propose a novel dynamic programming algorithm that combines statistical ensemble modeling techniques with evolutionary‐based sequence information to compute accurate coarse‐grained representations of the conformational landscape of proteins. This representation models conformations as ensembles of fully folded structures containing a set of interacting secondary structures, where the interactions are computed using a Boltzmann‐based energy function. This landscape is then used to predict dominant folding pathways. To present the predictions to the wider biology and computer science communities, we also developed a graphical tool to convey the content of the energy landscape through an interactive exploration of networked data. The proposed algorithm was applied to a benchmark of 145 proteins and demonstrated excellent results in terms of residue‐contact and folding pathway prediction. Particularly, the algorithm on average has greater precision than state‐of‐the‐art contact residue algorithms for proteins without homology‐based templates and the predicted folding routes agree with pathways elucidated by experimental studies and MD simulations. The proposed PF method represents an alternative to high computational‐cost approaches and will allow for large‐scale studies of folding dynamics annotations in proteomes. POS227 Mechanisms of Activation of Nuclear Receptor Liver receptor homolog‐1 by Synthetic Agonists and Peroxisome proliferator‐activated gamma coactivator 1‐a Transcriptional Coactivator Denise Okafor 1, Suzanne Mays1, Richard Whitby2, Devrishi Goswami3, Jozef Stec1, Autumn Flynn1, Michael Dugan1, Nathan Jui1, Patrick Griffin2 1Emory University, Georgia, USA, Emory University, 2University of Southampton, UK, 3Scripps Research Institute, California, USA Liver receptor homolog 1 (NR5A2, LRH‐1) is an orphan nuclear hormone receptor that regulates diverse biological processes, including metabolism, proliferation, and the resolution of endoplasmic reticulum stress. LRH‐1 has great potential as a therapeutic target for metabolic diseases and cancer but development of LRH‐1 modulators has been difficult. Chemical scaffolds exist that are capable of activating LRH‐1, however the mechanisms of activation are unknown. X‐ray crystallography and other structural methods are used to explore receptor‐ligand interactions associated with LRH‐1 activation by a set of related agonists with similar efficacies but dramatically different binding modes. Molecular dynamics simulations elucidate the important roles of pi‐stacking and polar interactions in mediating differing binding modes, and subsequently different mechanisms of action for the two agonists. A network of conserved water molecules near the ligand‐binding site, important for activation by both agonists, is explored. Additionally, the mechanism of LRH‐1 coactivation by Peroxisome proliferator‐activated gamma coactivator 1‐a (PGC1a), a coactivator for LRH‐1, is explored in comparison with Nuclear Receptor Coactivator‐2 (Tif2). LRH‐1 binds PGC1a with higher affinity than Tif2. Molecular dynamics reveal that PGC1a induces correlated atomic motion throughout the activation function surface of LRH‐1, while Tif2 induces weaker signaling at the activation function surface, instead promoting allosteric signaling from the Helix 6/ß‐sheet region of LRH‐1. This work i) reveals complexities associated with LRH‐1 agonist development, ii) offers insight into rational design strategies, and iii) illuminates strategies for selective therapeutic targeting of PGC1a dependent LRH1 signaling pathways. POS287 Structural, Dynamic, and Electrostatic Influences on Catalysis in Wild Type Human Phosphoglucose Isomerase and the H100L Variant Shanadeen Begay 1, Penny Beuning1, Mary Ondrechen1 1Northeastern University, Massachusetts, USA Human phosphoglucose isomerase (hPGI) is important in glycolysis, catalyzing the reversible isomerization of glucose‐6‐ phosphate and fructose‐6‐ phosphate. It has been shown that distal amino acids, residues 11–13 Å away from the site of reaction, contribute to catalysis, such that single‐site, conservative mutations at these residues result in significant loss of catalytic activity. It is shown that H100, a third‐shell residue, indirectly interacts with other catalytic residues; the H100L variant has a larger (630‐fold) effect on catalytic efficiency than some second‐shell residues that interact directly with the first‐shell catalytic residues. The dynamics of the H100L variant from the perspective of overall structural changes show minor fluctuations. The molecular dynamics simulations of wild‐type PGI and of the H100L variant were carried out to compare the electrostatic and hydrogen bonding network around the active site to determine the extent of contribution of structural changes. Together with electrostatics, dynamics contributes to an observed decrease in catalytic efficiency. These results suggest that H100 is involved in imparting the necessary chemical and electrostatic properties to the catalytic base E358 and to R273. Funded by NSF‐MCB‐1517290, and an NSF Postdoctoral Research Fellowship in Biology (SCB). POS298 In Silico Studies of the Concentration‐dependent Protein Dissociation from DNA Min‐Yeh Tsai 1, Peter Wolynes1, Bin Zhang2, Weihua Zheng1, Peter Wolynes1 1Rice University, Texas, USA, 2Massachusetts Institute of Technology, USA Fis protein is a DNA‐binding protein that can regulate over two hundreds of genes in Escherichia coli (E. coli). The regulation profile of the genes during E. coli cell growth follows a growth phase‐dependent regulatory trend, where Fis expression level decreases dramatically from ∼50,000 molec/cell at early exponential phase to 100 molec/cell at stationary phase. Recent experimental studies have shown that the dissociation of Fis protein from DNA is accelerated by increasing the bulk concentration of the Fis protein (facilitated dissociation), suggesting the importance of the dissociation rate in understanding gene regulation. The objective of the study therefore is to understand the molecular mechanism of dissociation of Fis protein from DNA. We use a coarse‐grained protein/DNA model to explore the binding landscapes of protein dissociation from DNA. Our simulations uncover a partially dissociated Fis conformation on DNA where only single domain remains bound to the DNA. The simulations support a three‐state sequential kinetic model (N⇄I→D) for facilitated dissociation, where N, I, and D refer to the bound state, intermediate, and dissociated states, respectively. This proposed kinetic model explains the concentration‐dependent dissociation. POS358 Conformation and dynamics of the Zinc Finger of NEMO and diseased‐associated mutants Freddie Salsbury 1, Ryan Godwin1 1Wake Forest University, North Carolina, USA Zinc‐finger proteins are regulators of critical signaling pathways for various cellular functions, including apoptosis and oncogenesis. NEMO, also known as IKK‐γ, is one such zinc finger that is the regulatory portion of the IκB kinase, which is involved in the celluar response to inflamation. We will present our results, from microsecond scale all‐atom molecular dynamics, on how binding site protonation states and zinc coordination influence protein conformations, dynamics, and ultimately function as well as how disease‐associated mutants also affect the dynamics and conformations of the zinc finger NEMO. Our analysis focusing on understanding conformational change and dynamics via several techniques incluldung clustering analysis, correlation analysis, and network analysis of the hydrogen bond networks. POS362 An Accurate and Efficient Atomic Four‐body Knowledge‐Based Potential to Distinguish Native Protein Structures from Non‐Native Folds Majid Masso1 1George Mason University, Virginia, USA Methods: Atomic coordinates were obtained for single chains of 1417 diverse proteins for training. A four‐letter alphabet (C,N,O,S) designated atom types (hydrogens excluded). Delaunay tessellation was performed on each structure, whereby atoms were treated as vertices to generate a convex hull enclosing hundreds of space‐filling, non‐overlapping, irregular tetrahedra. Each tetrahedron identifies at its four vertices one of 35 possible types of interacting atomic quadruplets. Relative frequency of occurrence F_ijkl was calculated for each atomic quadruplet type (i,j,k,l) using the observed tetrahedra, and rate P_ijkl expected by chance was determined from a multinomial reference distribution. The energy of interaction scores, given by S_ijkl = – log(F_ijkl/P_ijkl), collectively define the atomic four‐body potential. Subsequently, total potential (tp) of any protein can be computed by adding up scores of all tetrahedral interacting atomic quadruplets derived from its structure tessellation. Results: We evaluated 129 benchmark sets in Decoys‐‘R’‐Us (see Figure) and compared performance with 12 physics‐ and knowledge‐based potentials based on native rank, Z‐score, correlation coefficient, and fractional enrichment. Scoring 3rd, we tied CHARMM19 and surpassed AMBER force field potentials. Using a similar four‐body potential based on a six‐letter alphabet (add M = metals, X = other non‐metals), we predicted binding energies for 25 known HIV‐1 protease‐inhibitor complexes as tp_complex – tp_target (r^2 = 0.72). Experimental and predicted correlation proved robust upon identifying 115 additional complexes (r^2 = 0.64). Conclusion: Results suggest an accurate and efficient atomic four‐body statistical potential for protein structure prediction and assessment. POS389 Characterization of the Interactions of a Lignostilbene‐a,ß‐dioxygenase with both Resveratrol and Lutein Substrates Anthony Zara 1, Fang Huang1, John Allingham1, Michele Loewen1 1Queen's University, National Research Council, Kingston, Canada Apocarotenoids form a family of small molecules derived from the oxidative cleavage of larger C40 carotenoid compounds. Classic examples include vitamin A, produced by the oxidative cleavage of ß‐carotene, and the plant hormone abscisic acid, produced by the cleavage of epoxycarotenoids. Such reactions are catalyzed by a family of non‐heme, iron‐coordinating enzymes known as carotenoid cleavage oxygenases (CCOs). With the aim of elucidating factors mediating the unique and diverse substrate and cleavage specificities mediated across this family of enzymes, a bacterial CCO homolog from the bio‐control agent Pseudomonas brassicacearum (PbLSD) was selected for structural and functional characterization. In vitro enzymatic assays highlighted strong activity against the lignostilbene resveratrol, with weak activity against the C40 carotenoid lutein. Subsequent in silico docking studies using a recently obtained structure (PDB ID#: 5V2D), predicted strong binding interactions for both substrates. However, while further inspection of the docked molecules in the crystal structure showed resveratrol in the active site with its single cleavable double bond coordinated directly above the reactive‐iron center, an unfavourable substrate orientation was observed for lutein. Further modeling of substrate binding pocket residues highlighted structural features that are likely contributing to this substrate selectivity. Further work by way of mutational studies will be performed to characterize these substrate interactions and their importance in catalytic activity. This work was supported by NSERC DG grants to MCL and JSA, and the NRC Bio‐based Specialty Chemicals Program to MCL. POS395 Modeling Cysteine and Methionine Binding to Aromatic Residues Esam Orabi 1, Ann English1 1PROTEO and Department of Chemistry and Biochemistry, Concordia University, 7141 Sherbrooke Street West, Montréal, Québec H4B 1R6, Canada Non‐covalent interactions of Cys and Met side chains with those of Phe, Trp, Tyr and His contribute to protein function and stability. Toward understanding the structural and energetic properties of these S‐aromatic interactions, we perform ab initio quantum mechanical calculations at the MP2(full)/6–311++G(d,p) level of theory on complexes of MeSH and Me2S with aromatics that model the side chains of the aromatic residues. Results reveal that in the most stable conformers, the S atom of MeSH and Me2S binds edge‐on to the five‐membered heteroaromatic rings but between edge‐on and en‐face to the phenyl rings. Complexation also modulates the ionization potential of the interacting fragments and the geometry of the complex controls the center of oxidation. For investigations in bulk water, the CHARMM36 all‐atom additive force field (FF) is calibrated for the S‐aromatic interactions and implemented in molecular dynamics simulations. The aqueous complexes are stable with binding free energies of −0.6 to −1.1 kcal/mol. N‐H···S or O‐H···S s‐type H‐bonding persists in ∼10% of the S‐imidazole and S‐phenol structures but to a lesser extent in MeSH‐indole (1.5%) and Me2S‐indole (4%). The most stable conformers adopt en‐face geometry except MeSH‐imidazole and Me2S‐phenol favor intermediate geometry. Our comprehensive investigation of S‐aromatic interactions in the gas phase and water provides a valuable data set for studying these interactions in proteins. POS398 Distinct Structural Dynamics of Monomeric, Dimeric and Tetrameric Glyceraldehyde‐3‐phosphate Dehydrogenase (GAPDH) Illuminate its Multiple Functions Vinod Parmar 1, Ann M. English1, Gilles H. Peslherbe1 PROTEO and Centre for Research in Molecular Modeling, Department of Chemistry and Biochemistry, Concordia University, Montreal, Canada GAPDH's oligomeric states exhibit distinct moonlighting functions and pathologies. For example, tetrameric GAPDH possesses dehydrogenase activity whereas aggregates of monomeric/dimeric GAPDH are associated with neurodegeneration. Since the subunits are structurally identical, we hypothesize that dynamics dictate GAPDH's functions. Consistent with the known behavior of the tetramer as a dimer of dimers, normal mode analysis reveals that one NAD+‐binding domain (NBD) in each dimer is more flexible. Both NBDs are more flexible in the dimer relative to the tetramer or monomer whereas the S‐loop, which covers the dehydrogenase active site, is most flexible in the monomer. Multivariate statistical analysis of molecular dynamics simulations further supports distinct dynamics with degree of oligomerization. Principal component analysis reveals that the NBDs exhibit differential flexibility in both tetrameric and dimeric GAPDH, which can be associated with negative cooperativity in NAD+ binding, and unmasks high fluctuations of the S‐loop in the monomer. Conversion of the S‐loop into an intrinsically disordered domain in the monomer may contribute to loss of glycolytic activity and may promote the binding of GAPDH to its multitude of protein partners, including the death factor, Siah1. Consistent with our hypothesis, the distinct essential dynamics of monomeric, dimeric and tetrameric GAPDH likely dictate its functions. POS405 Macromolecular Crowding Effects on Biomolecular Recognition: Progress Toward Building Accurate Yet Efficient Computational Models Mala Radhakrishnan 1, Rachel Kim1, Carla Perez1, Helena Qi, Donald Elmore1 1Wellesley College, Massachusetts, USA Macromolecular crowding within the cell has been shown to influence the thermodynamics of biomolecular processes, and it may be necessary to account for crowding effects when modeling such processes. When building a computational model, one should determine the optimal balance between accuracy and efficiency for the question at hand. We seek to explore this tradeoff as it applies to crowding effects on molecular recognition, with a particular focus on electrostatic effects driven by crowding. On one extreme, we use theoretical, interacting “toy” molecules and highly simplified models for crowding from which we can extract general principles about how physical properties of the interacting molecules and crowders affect binding energetics. On the other extreme, we use molecular dynamics simulations to consider atomistic representations of a DNA/peptide system with particular protein crowders, allowing us to directly evaluate models experimentally. Preliminary results from sampling models from within the accuracy/efficiency continuum show both general and system‐specific effects of crowding on molecular recognition. POS427 Effects of trimethylamineN‐oxide (TMAO) on the conformation of peptides and miniproteins. Cristiano Dias 1, Zhaoqian Su1 1New Jersey Institute of Technology, USA TrimethylamineN‐oxide (TMAO) is an organic compound that affect osmosis in cells and protect the native state of proteins. In this presentation, I will discuss computational studies to understand TMAO's protecting effect at the molecular level. In particular, I will show results from all‐atom molecular dynamics simulations of homo‐peptides (poly‐glycine, poly‐alanine, poly‐valine, and poly‐leucine) and the Trp‐Cage miniprotein in explicit solvent. Poly‐glycine is a model of the protein backbone whereas poly‐alanine, poly‐valine and poly‐leucine represent peptides with small and large non‐polar side chains, respectively. We find that TMAO favors compact conformations of poly‐glycine monomers consistent with its nature as a protecting osmolyte while it has little effect on poly‐alanine. However, TMAO favors expanded conformations of poly‐valine and poly‐leucine. Effects of TMAO on poly‐alanine, poly‐valine and poly‐leucine agrees with studies showing that TMAO has little effect on the interaction between small non‐polar residues whereas it weakens hydrophobic interactions between large non‐polar molecules. These results raise a question: If TMAO's collapsing effect on the backbone model (i.e., poly‐glycine) can be overcompensated by its effect on hydrophobic interactions causing peptides to swell, how does this molecule stabilize the compact native state of proteins? To answer this question, I will show results from other peptides and replica exchange molecular dynamics simulations of Trp‐Cage miniprotein. POS470 Computational Modeling of the Interface between a Multi‐junction DNA Motif and T7 Endonuclease I Benjamin Walcott 1, Tom Jing1, Ian Huntress1, Megan Kizer1, Joseph Bromley1, Keith Fraser1, Christopher Bystroff1, Xing Wang1 1Rensselaer Polytechnic Institute, Troy, New York, USA Paranemic crossover (PX) DNA, a four‐stranded multi‐crossover structure, has been implicated in recombination‐independent homologous pairing (RIHP). RIHP plays many roles in the in various biological processes; including repeat‐directed DNA modification and chromosome segregation. Errors in this segregation may cause aneuploidy, resulting in infertility and birth defects. To prove its role in RIHP, we propose the design of an anti‐PX ligand. As a template, we have selected T7 endonuclease I (T7 endoI), a junction‐resolving enzyme. The first step in this design is modeling the interface between T7 endoI and PX. For every possible scissile phosphate in PX, we used PyRosetta to simulate the interface between PX and the T7 endoI structure from PDB 2PFJ where it is complexed with a Holliday junction. The scissile phosphate in PX was superimposed on the scissile phosphate of the Holliday junction in 2PFJ. AtomPair constraints were used to maintain the interface between the scissile phosphate and the active site residues of T7 endoI. In general, the low energy configurations correspond to the phosphates T7 endoI was determined to bind experimentally. The structures of these simulated interfaces were then used to construct a model of the PX‐T7 endoI interface to be used for further modeling. POS474 Structural and functional roles of dynamically correlated conserved residues in Thymidylate kinase. Santosh Chaudhary 1, Kanagaraj Sekar1, Jeyaraman Jeyakanthan2 1Indian Institute of Science, India, 2Department of Bioinformatics, Alagappa University,India Thymidylate kinase is an essential enzyme in thymidine triphosphate (dTTP) biosynthesis pathway, hence, critical for DNA synthesis. Thymidylate kinase is widely studied due to its role in prodrug activation and is also exploited as a drug target. Homologous structures of thymidylate kinases exhibit similar topology. But the structural and functional roles of some conserved residues, present around the active site, remain unclear. In the present study, thymidylate kinase from Thermus thermophilus has been used as a model protein. Distinct conformations obtained in native and ligand bound crystal structures revealed that correlated motions of active‐site residues, P‐loop and LID region are required for the binding and positioning of the substrate TMP. In addition, the binding of both the substrates (ATP and TMP) at the active site was found to follow random bi‐bi mechanism. Functionally important movements of residues were studied through molecular dynamics simulations. Conserved arginine of DRX motif acts as a hub for the suboptimal paths present between ATP and TMP. The functional and structural importance of residues present in the suboptimal paths was assessed using enzymatic and thermal stability assays. Mutations of the conserved residues either resulted in loss of activity or affected the thermal stability of the protein. Molecular dynamics analyses of mutants provide insight into the mechanism of phosphoryl transfer reaction. POS482 Computed proton binding properties correlate with enzyme activities of D‐mannonate dehydratases Jaeju Ko 1, Mary Ondrechen2 1Indiana University of Pennsylvania, USA, 2Northeastern University, MA, USA Proton binding properties and their relationship to the enzymatic activities of six D‐mannonate dehydratases (ManDs) are reported. This study is an application of THEoretical Microscopic Anomalous TItration Curve Shapes (THEMATICS), a computational procedure for identifying active site residues of proteins. THEMATICS utilizes individual‐site titration curves of ionizable side chains. The individual‐site titration curves are computed from the three‐dimensional structure of each individual protein. The negative of the first derivative of the individual‐site titration curve is a probability density function and is used to compute the statistical moment values. We find that the fourth central moments of catalytically important residues correlate well with the kcat values of the ManDs. A high fourth central moment requires coupling of the proton binding events. Thus, a set of coupled proton binding events in the active site is likely an essential component of natural enzymes and is one of the properties that should be considered in enzyme design. POS534 In‐silico identification of SOD1 exposed dimer interface which binds a novel computationally designed HTB1 binding protein in ALS SOD1 mutants Efrat Ben‐Zeev 1, Niv Papo2, Victor Banerje3,4, Ofek Oren3,4, Ran Taube4, Stanislav Engel3,5 1The Nancy and Stephen Grand Israel National Center for Personalized Medicine, Weizmann Institute of Science, Israel, 2Ben‐Gurion University of the Negev, Israel, 3Department of Biotechnology Engineering and the National Institute of Biotechnology in the Negev, Ben‐Gurion University of the Negev, 4Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences, Ben‐Gurion University of the Negev, Israel 5Department of Clinical Biochemistry and Pharmacology, Ben‐Gurion University of the Negev, Israel Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease resulting in paralysis and death, usually within 3–5 years of diagnosis. Misfolding of Cu/Zn‐superoxide dismutase (SOD1) is emerging as a mechanism underlying motor neuron degeneration in individuals with amyotrophic lateral sclerosis (ALS) who carry a mutant SOD1 gene (ALS SOD). Molecular agents that specifically bind and neutralize misfolded and toxic SOD1 mutant proteins may help to attenuate the disease progression of familial ALS (fALS). The promiscuous natural human IgG‐binding domain, a hyperthermophilic variant of protein G (HTB1), was converted into a highly specific aggregation inhibitor (designated HTB1M) of two fALS‐linked SOD1 mutants, SOD1G93A and SOD1G85R. HTB1M displayed high binding specificity toward SOD1 mutants, inhibited their amyloid aggregation in vitro, prevented the accumulation of misfolded proteins in living cells, and reduced the cytotoxicity of SOD1G93A expressed in motor neuron–like cells. The computational modeling identified a specific HTB1M recognition site on the surface of SOD1 monomer. It binds to SOD1 via both its a‐helical and ß‐sheet domains involving some of the engineered mutations, e.g. R29, I33 and F44. HTB1M recognizes only those SOD1 conformations where the native dimer is disrupted or misfolded and thereby exposes the hydrophobic dimer interface. Hence, this mechanism where mutant SOD1 are engaged in interaction with HTB1M via their dimer interface stabilizing the monomeric form, may lead to enhanced in solubility of the monomer, thus preventing intermolecular interactions and/or redirecting aggregation to a non‐amyloid path. Our proposed mechanism and detailed amino‐acids binding interactions is a basis for future designed therapeutic candidates or as a research tool. 06. DESIGN/ENGINEERING POS016 Large‐scale prediction, characterization and modulation of protease enzyme specificity landscape using computation and experiment Sagar Khare 1, Sagar Khare1, Manasi Pethe1, Aliza Rubenstein1 1Rutgers University, New Jersey, USA Characterizing the substrate specificity of protease enzymes is critical for illuminating the molecular basis of their diverse and complex roles in a wide array of biological processes. Rapid and accurate prediction of their extended substrate specificity would also aid in the design of custom proteases capable of selectively and controllably cleaving biotechnologically or therapeutically relevant targets. A catalytic drug such as a programmed protease would have several advantages over binding‐based moieties such as antibodies. Proteases are multispecific enzymes that cleave multiple substrates of disparate sequence while not cleaving other sequences, thereby showing signs of both positive and negative selection. Thus, the specificity landscape of proteases determines their functions, and will be key to the use of designed therapeutic proteases to ensure proper targeting and minimizing side‐effects. We have developed a specificity modeling and design framework by combining in silico structure‐based modeling using the Rosetta macromolecular modeling approach and machine learning with experimental in vivo assays and Deep Sequencing to elucidate and predictively modulate specificity landscapes of proteases on a large scale. In our framework tens of thousands of substrates are experimentally evaluated and this information is used to guide computational design approaches to make predictions for the entire landscape (millions of sequences). I will describe the framework and its application for uncovering and designing the specificity landscape of the Hepatitis C virus protease, and its drug‐resistant mutants. POS017 A Place for Unpublished Gene‐to‐Protein Information: the Recombinant Proteomic Database Resource Peter Nollert 1, Mark Mixon1 1Bio Data Bridges, Seattle, Washington, USA The objective of this development project is to collect and expose currently inaccessible protein production information to reduce the effort for establishing new recombinant protein production systems. We have developed a web‐based Recombinant Proteomic Data Resource, allowing entry of a diverse set of recombinant protein production data, exploration of targets and configuration of new protein production systems. Biomedical researchers require highly engineered protein samples to investigate their biological structure and function. While detailed recombinant protein production information is routinely collected by researchers in a variety of “in lab” data formats, it is extremely difficult for scientists to share this information using conventional routes of publication and database submission. Our research shows that most proteomic information remains invisible to outside parties. We believe that protein research economics can be greatly improved if this hidden proteomic data is exposed in a manner that prevents wasteful unnecessary re‐treading of trial‐and‐error protein production efforts. In order tackle this issue we have built an online Recombinant Proteomic Data Resource that enables researchers to easily upload existing data, find and compare experimental details that have yielded productive or unproductive, but nevertheless instructive protein production results. We show components of search, exploration and configuration of new gene‐to‐protein experimental plans. We discuss how this tool helps to open the door to much improved community‐wide protein research and efficient design of experiments with higher likelihood of success in protein production. POS042 Combined Strategies to Achieve a Desired Ligand Binding Protein Specificity Jesús Banda 1, Alejandro Sosa‐Peinado1, Sooruban Shanmugaratnam1, Birte Höcker2, Rogelio Rodríguez‐Sotres1 1National Autonomous University of Mexico, Mexico, 2University of Bayreuth, Germany It is known that computational design of ligand binding is not a solved problem, since there is not a general approach. to succeed at every case of study. In the present work, using Salmonella typhimurium (S. typhimurium) LAO protein as scaffold, a binding protein only able to bind the positive amino acids l‐lysine, l‐arginine, l‐ornithine and l‐histidine, the combination of two design approaches led us to find a simple solution for achieving the novel L‐glutamine binding by LAO protein. Results: Both approaches, binding pocket grafting and a statistical coupling based strategy, each one with 10 mutations out of 238 possible at LAO sequence, allows the refinement for a second round of mutations (only a point mutation, actually) into LAO sequence after proper experimental protein expression and ligand binding measurements. There was no need for any high throughput mutational analysis, such as directed evolution or some other gene library approach. Conclusion: It was possible to, upon experimental and mutational comparison, of two design approaches taken, find a solution to achieve a desired ligand binding for the LAO protein. The new mutant, which only contains one point mutation according to the wild type scaffold sequence, presents a radically distinct binding profile, in which L‐glutamine is one of the ligands bound with the highest affinity according to the isothermal titration calorimetry (ITC) determined dissociation constant (Kd=1.5µM), which is in the same order of magnitude as the known wild type Escherichia coli (E. coli) L‐glutamine binding protein. POS067 Design of 2D and 3D arrays from engineered amyloid proteins Fernanda Bononi 1, Fernanda Bononi1, Michael Toney1 1Department of Chemistry ‐ UC Davis, USA Amyloid fibrils have been extensively studied due to their role in the development of diseases such as Alzheimer's and type II diabetes. More recently, their high strength has been exploited for development of nanomaterials. We recently presented a general approach for the synthesis of amyloid structures from naturally occurring ß‐solenoid proteins, most of which are antifreeze proteins. Three different types of amyloid fibrils were obtained from naturally non‐amyoloidogenic antifreeze proteins. They assemble under mild conditions, in contrast to most amyloid fibrils, and possess very high stability towards environmental stress. Following the design and synthesis of strong and resistant fibrils, work focused on engineering surfaces to create fibril‐based, higher‐order structures such as 2D and 3D scaffolds. These ordered protein‐based, rugged scaffolds will be useful for material applications such as nanoparticle ordering for photovoltaic and thermoelectric applications. Searches through various polymerization conditions led to partially ordered fibril assemblies in agreement with the design strategies. We are advancing these partial successes by engineering new interfaces with more sophisticated approaches. Hence, our current work focuses on utilizing new approaches such as protein fusions as well as well‐established approaches such as Rosetta design and engineered metal binding sites for creating new protein interfaces that allow the assemble of high order structures. POS110 Discovering light‐switchable protein interactions using phage display Jakeb Reis1 , Xiuling Xu1, Sherin McDonald McDonald2, Anna Jaikaran1, Andrew Woolley1, Maruti Uppalapati2 1Department of Chemistry, University of Toronto, Canada, 2Department of Pathology and Laboratory Medicine, University of Saskatchewan, Canada Nature provides a rich source of light‐responsive proteins that can serve as powerful optogenetic tools to study biological systems. However, many of these have no known binding partner so that optogenetic control of protein‐protein interactions is currently restricted to a few well‐defined natural systems or to an engineered system based on the AsLOV protein. In addition, the affinities and kinetics of native interactions are often sub‐optimal and are difficult to engineer in the absence of any structural information. Here, we present a general approach to discover de novo synthetic light switchable protein‐protein interactions using combinatorial protein engineering techniques. Using phage libraries based on small, disulfide‐free domains, and a novel codon optimization scheme to generate diversity in binding interfaces, we were able to generate well‐folded monomeric binding partners that specifically recognize either the light‐state or the dark‐state of the AsLOV protein as well as for photoactive yellow protein (PYP) a bacterial photo‐protein with no known binding partners. De novo binding partners formed 1:1 complexes with a range of affinities and on/off rates. The success of this approach implies these combinatorial libraries can be used to generate binding partners for other light‐switchable proteins and thereby create a palette of light‐switchable protein‐protein interactions, easily customizable for different optogenetic applications. POS118 Shifting the boundaries of experimental studies in engineering enzymatic functions: Combining the benefits of computational and experimental methods Maximilian Ebert 1, Simon Dürr1, Armande Ang Houle1, Guillaume Lamoureux2, Joelle Pelletier1 1Université de Montréal, Canada, 2Concordia University, Montreal, Canada While biocatalysis is thought to be the sustainable and benign complement to organic synthesis, past efforts to tailor enzymatic function towards desired reactions have faced a significant knowledge gap. Here we present two distinct, combined computational and experimental protocols to predict the conformation of catalytically‐relevant enzyme:ligand complexes, and to reveal substrate access channels even in the absence of a ligand‐bound structure. The applied Adaptive Biasing Force (ABF) method is broadly applicable for predicting mutational hotspots in a substrate‐specific manner and has the potential to drastically reduce the experimental screening effort to tailor an enzyme to new substrates of interest. Starting with a ligand‐free crystal structure, we successfully identified all residues known to be involved in palmitic acid binding to Cytochrome P450 CYP102A1 (BM3). The binding trajectory also revealed a new binding residue, Q73, which we confirmed experimentally. Mapping the substrate access channels of proteins represents a significant challenge. Like many other biocatalysts, P450s contain numerous channels thought to be populated by their substrates, products, solvents, and gases. We identified and predicted correctly multiple ligand migration channels for two bacterial P450s (BM3 and CYP102A5), using Implicit Ligand Sampling (ILS) and free molecular dynamics simulations. Furthermore, calculations of the free energy of gas migration through each channel revealed evidence of the evolution toward O2 binding in conjunction with protection against inhibitory CO and exclusion of N2. These results significantly enhance our understanding of gas migration in proteins and provide insights into the evolution of gas‐utilizing enzymes. POS121 Why protein oligomer complexes allow more precise regulation mechanisms over dimers and monomers? Dominic Lauzon 1, Alexis Vallée‐Bélisle1 1Laboratory of Biosensors & Nanomachines, Department of Chemistry, University of Montreal, Canada Proteins have mutated over millions of years and up to 75% of human enzymes listed have evolved into multimeric complexes. We already know that protein complexes can improve biological input by, for example, increasing the activity of enzyme or by helping regulation by combining specificity, allostery, activation and inhibition. On the other hand, less is known about the thermodynamic advantage or cost related to the use of protein complexes and how their assembly may regulate their function. In this study, we employ a synthetic biochemistry approach to compare the performance of monomeric, dimeric and trimeric complexes. We do so by designing a simple DNA structure (three‐way junction) that can be form using one, two or three DNA strands. DNA represents a material of choice because it enables to control every thermodynamic parameters of the structure through simple mutations (e.g. modify the trimer affinity without affecting the dimer affinity). This contrasts with protein systems where the impacts of mutations are often unpredictable. Using mathematical simulation and experimental studies, we show that trimeric complex can exhibit a much larger window of regulation mechanism compared to dimeric complexes or monomers. Our DNA trimers illustrate binding behaviors going from positive to negative cooperativity with Kobs that cover 4 fold of magnitude. We also identify the ratio of dimeric affinity over trimeric affinity as the key parameter for programming the thresholds and the cooperativity of trimer assembly. Results provided by this study shine a new light on possible regulation mechanism of trimeric system and may help understanding why some proteins have evolved into oligomers. POS130 PHOTOCONTROL OF A PROTEIN AFFINITY REAGENT USING AZOBENZENE SWITCHES Amir Babalhavaeji 1, Andrew Woolley1 1University of Toronto, Canada Photocontrol of protein activity is a powerful technique in biology. One method to obtain photoswitchable proteins is to couple the photoisomerization of azobenzene switches to conformational changes in the protein of interest. We have applied azobenzene photoswitches to a Fynomer, a small protein‐based affinity reagent based on the SH3 domain of human Fyn that has been engineered to bind and inhibit the activity of human chymase. Chymase is known to be involved in cardiovascular diseases as well as pathological inflammatory conditions. We introduced two cysteine residues by point mutations in the sequence of the chymase‐binding Fynomer. Subsequent crosslinking of the inhibitor with azobenzenes at those residues allows only the cis isomer of the azo moiety to be compatible with the well‐folded Fynomer. The dark‐adapted (trans azo) crosslinked Fynomer was partially unfolded and showed reduced inhibitory activity. Upon its irradiation with the switching light (370 nm), the inhibitor was largely folded and better inhibited the activity of chymase. Since the Fyn SH3 scaffold can be broadly used in phage‐display selection techniques to generate Fynomers to target virtually any protein, these results demonstrate the promise of azobenzenes for in vivo functional studies and photopharmacology. POS134 Tuning the Dynamics of Biomolecular Switches Using Induced Fit and Conformational Selection Mechanisms Carl Prévost‐Tremblay 1, Alexis Vallée‐Bélisle1 1Université de Montréal, Canada Regulation of biological processes requires precise tuning of activation and deactivation rates of biomolecular switches across a dozen orders of magnitude. Regulation of biomolecular switches typically relies on two distinct structure‐switching mechanisms. Activation by induced fit (IF) occurs when ligand binding to the inactive state induces a conformational change while activation by conformational selection (CS) requires spontaneous switching of the biomolecule to its active conformation prior to ligand binding. Despite 60 years of experimental and theoretical investigations, the distinct kinetic performances of these two mechanisms as well as the rationale behind their evolutive selection remains unexplored. Here we employ a synthetic fluorescent DNA switch system able to operate through either mechanism and allowing easy tuning and assessment of all its thermodynamic parameters as well as straightforward kinetic characterization. Our results reveal that IF yields activation rates several orders of magnitude faster than CS (>1000‐fold) due to an increase in the concentration of binding‐competent switch and an acceleration of the conformational change. We also find that IF is reversible and produces similarly faster deactivation rates. Finally, our results reveal that CS and IF enable programming of their activation and deactivation rates, respectively, through simple modulation of the conformational equilibrium. Together, these findings reveal how evolution may have taken advantages of IF and CS mechanisms to program activation and deactivation rates of biomolecular switches while providing a new structure‐kinetic relationship with key applications in bioengineering. POS146 Developing a drug against trichomoniasis Jose Vique 1, Claudia Benitez1 1Instituto Politecnico Nacional, Mexico Trichomonas vaginalis is a protozoan, the causal agent of trichomoniasis, the most common non‐viral sexually transmitted infection (STI) spread worldwide. Trichomoniasis is associated with infections in the genitourinary tract in both sexes and can provoques premature births. For over 40 years, the treatment against trichomoniasis is the provision of nitroimidazoles, commonly metronidazole and tinidazole. However, 5 to 20% of the patients show no improvement by this treatment. This highlights the need for new therapeutic regimens against trichomoniasis. Carbohydrates are the main nutrient source for T. vaginalis. Therefore, the enzymes in the glycolytic pathway on T. vaginalis like triosephosphate isomerase (TIM) are potential therapeutic targets. We performed molecular interaction simulations between a set of compounds obtained from libraries and triose phosphate isomerase from T. vaginalis. Subsequently, the compounds with higher probability of interaction were assayed in their ability to inhibit or destabilize the mentioned glycolytic enzyme. Some compounds selected by docking strategies were able to reduce the replication and viability of T. vaginalis cultures. These findings have important implications in the development of new therapeutic strategies against trichomoniasis. Finally, we propose a new potential drug against trichomoniasis. POS172 Engineering a Polymerase for the Forensic Analysis of Damaged DNA Samples Tim Coulther 1, Penny Beuning1, Mary Jo Ondrechen1 1Northeastern University, Massachusetts, USA Enzyme engineering seeks to create an enzyme, either de novo or by modification of a known protein, with a new desired function. Biocatalysis has advantages over conventional catalytic processes, but there are not always natural enzymes with the desired properties and activity. For example, many DNA polymerases can replicate DNA accurately at high speeds but are inhibited by damaged bases, such as the common oxidative lesion 8‐oxoguanine. Specialized polymerases may be well suited to bypass the damage, but are typically less accurate on undamaged DNA. A hybrid polymerase that retains the accuracy and speed of the replicative polymerases, while incorporating specific lesion bypass abilities, would be a useful biochemical tool. This research focuses on developing a high‐fidelity, damage‐tolerant DNA polymerase for the forensic analysis of damaged DNA. The 8‐oxoguanine lesion is mutagenic when it resides in the syn conformation, as this allows formation of a Hoogsteen pair with adenosine. Molecular dynamics and docking are being utilized to understand the lesion conformation and relative fidelity of model polymerases and engineered variants. Our computational method, THEMATICS, identifies functional residues and assesses whether variants retain the electrostatic properties of the natural polymerase for catalysis. The combination of these computational methods is being used to identify specific variants for biochemical characterization, including thermal stability, catalytic activity, lesion bypass capability, and fidelity. Supported by NSF‐MCB‐1517290 and the National Institute of Justice. POS175 Thermostabilization of VPR, a cold adapted subtilase, by proline substitutions into surface loops. Magnus Kristjansson 1, Kristinn Oskarsson1, Kristinn Oskarsson1, Arnor Aevarsson1, Magnus Kristjansson1 1Department of Biochemistry, Science Institute, University of Iceland, Iceland A structural factor that has been correlated with increased stability of thermophilic proteins is the increased number of proline residues located in surface loops, as compared to homologs adapted to lower temperatures. Increased number of proline residues located in loops may increase their rigidity, as proline residues would limit the number of possible configurations of these loop structures. Structural comparisons of VPR, a subtilisin‐like serine proteinase from a psychrophilic Vibrio sp., to related subtilases, including aqualysin I (AQUI), a close homologue from the thermophile Thermus aquaticus, show that AQUI contains five additional proline residues as compared to VPR, four of which are located in surface loops. We have substituted the four Pro residues into corresponding sites in the structure of VPR in an attempt to confer increased thermal stability of the cold adapted enzyme. We produced variants containing each of the mutations (N3P, I5P, N238P and T265P), as single, double, triple and quadruple variants, containing these substitutions. The proline mutations had significant effects on the thermal stability of the VPR variants. The single variants, containing either of the two substitutions near the N‐terminus (N3P and I5P) had the largest effect, and in combination they stabilized the double mutants by ∼ 5–7°C. When all four substitutions were present at the selected sites in loops of the VPR variant, the melting temperature was increased by ∼ 8–10°C, as compared to the wild type enzyme. The Pro substitutions however, did not significantly affect the catalytic properties of the enzyme. POS177 Rational Design of Proteins that Exchange on Functional Timescales Roberto Chica 1, James Davey1, Adam Damry1, Natalie Goto1 1University of Ottawa, Canada Proteins are the molecular machines of life, carrying out complex physical and chemical processes that often require concerted motions of local protein structural elements. Previous efforts to design new proteins for applications in research, industry, and medicine have focused on the creation of sequences that stably adopt a single target structure, ignoring the potential impact of protein dynamics in function. Although computational protein design has enjoyed considerable success in creating new proteins using this approach, most have failed to match the efficiencies that are found in nature because standard methods do not allow for the design of exchange between necessary conformational states on a functionally‐relevant timescale. Here, we develop a broadly‐applicable computational method to engineer protein dynamics that we term meta‐multistate design. We used this methodology to design spontaneous exchange between two novel conformations introduced into the global fold of Streptococcal protein G domain ß1. The designed proteins, named DANCERs, for Dynamic And Native Conformational ExchangeRs, are stably folded and exchange between predicted conformational states on the millisecond timescale, as evidenced by nuclear magnetic resonance structures and ZZ‐exchange experiments. The successful introduction of defined dynamics on functional timescales paves the way to new applications requiring a protein to spontaneously access multiple conformational states. POS188 Rational Control over Protein Oligomerization through Engineered Domain Swapping Neha Nandwani 1, Parag Surana1, Nahren Mascarenhas2, Jayant Udgaonkar1, Ranabir Das1, Shachi Gosavi1 1National Centre for Biological Sciences, India, 2Sacred Heart College, Tirupattur, India Domain swapping is a mode of protein self‐association where dimers or higher order oligomers are formed from monomeric proteins by exchange of secondary or tertiary structural elements. Even though domain swapping offers a facile way to induce oligomerization in proteins, lack of sequence similarity between proteins that are known to swap has impeded the identification of a sequence motif that can induce domain swapping in proteins rationally. We have identified and experimentally demonstrated (using X‐ray crystallographic and NMR spectroscopic techniques) that a five amino acid motif (QXVXG) conserved for function in the cystatin superfamily is sufficient to drive domain swapping in a protein which doesn't otherwise domain swap. Different parts of the same protein could be made to swap using this stretch, depending on the target loop in which it was engineered. We generated new and different folds from the same starting monomer using this motif. We could, for the first time, demonstrate that a double domain swapped dimer could be designed rather simply, a phenomenon that adds complexity to the linear polymerization achieved by domain swapping. We propose that QXVXG motif can be used as a simple trick to rationally design oligomers from simple monomeric proteins. The computational cost of engineering de novo interfaces between proteins is huge, and our strategy bypasses that by exploiting naturally evolved protein‐protein interaction interfaces. POS192 High specificity protein‐protein interaction networks by computational design Ravit Netzer 1, Sarel J. Fleishman1 1Weizmann Institute of Science, Israel Signaling and metabolic processes depend on highly orchestrated interactions among proteins, and networks of both specific and multi‐specific interactions play important roles. State of the art methods in computational specificity design are still not accurate enough to generate such networks. To overcome this challenge, I developed a design method that controls all of the molecular elements observed in natural high‐specificity networks, including the backbone conformation of the interacting proteins, their orientation, and sequence. I applied the method to the bacterial colicin toxin/anti‐toxin family, generating hundreds of unique designed pairs, from which I selected 58 for experimental testing. 20 of these formed the desired complexes in vivo and in vitro. The designs showed diverse specificity patterns: some were highly specific and bound their cognate partner 5–6 orders of magnitude tighter than other designs. Others showed multi‐specific binding to several designed partners. Overall, these cognate and non‐cognate interactions span all the biologically relevant interaction affinities, from sub‐nanomolar to millimolar dissociation constants. A crystal structure of a highly‐specific design showed atomic accuracy in orientation, backbone conformation, and the conformations of most of the designed sidechains. Computational analysis showed that the specific designs were rich with polar interactions and had rigid interfacial loops that were designed by the algorithm. By contrast, the multi‐specific designs had flexible loops at their interactions surfaces that promoted the promiscuous binding. These results suggest that control over backbone conformation enables the design of large networks of interacting proteins with high specificity or multi‐specificity, opening the way to engineering of insulated or cross‐reactive interaction networks, as desired. POS197 Development of red light switchable protein‐protein interactions using phage display. Jaewan Jang 1, G. Andrew Woolley1 1University of Toronto, Canada Optically controlled proteins enable precise spatial and temporal control of molecular function in complex living systems. Photoactive proteins, which change conformation in response to an irradiation with a certain wavelength of light, are the core of optogenetic tools. For use in whole animals, photoactive proteins that respond to near IR wavelength would be ideal. We have engineered a small GAF domain from cyanobacteria that is monomeric in solution. The protein binds biliverdin, which is present in mammalian cells and undergoes near‐IR driven conformational change that reverts in several minutes in the dark. This new photoswitchable domain can enable new applications of optogenetic in whole animals. POS208 Tuning enzymatic activity by combining Virtual Docking and Residue Interaction Networks Yossef Lopez de los Santos 1, Ying Lian Chew Fajardo1, Guillaume Brault1,2, Nicolas Doucet1,3,4 1INRS – Institut Armand‐Frappier, Université du Québec, Canada, 2Odyssey Biotech, Canada, 3PROTEO, the Québec Network for Research on Protein Function, Engineering, and Applications, Canada, 4GRASP, Groupe de Recherche Axé sur la Structure des Protéines, Canada Within the evolvability landscape of protein engineering approaches, the vast majority of mutations yield neutral, deleterious, or destabilizing effects. It has been demonstrated that stabilizing mutations are usually achieved by random mutagenesis, making the identification of improved protein variants an exhaustive and inefficient process. Herein we present a semi‐rational combinatorial approach supported by docking simulations and Residue Interaction Networks (RINs) to design smaller and smarter libraries of mutants. Lipase B from Pseudozyma antarctica (CalB) was selected as an industrially relevant model system. Since CalB displays very low activity towards bulky substrates, the main goal of this project was to develop CalB variants with enhanced synthetic activity towards aromatic substrates like cinnamic and salicylic acid. We used this combined approach to uncover the importance of residues in the CalB active‐site cavity and their contribution to the synthetic reaction (Docking), in addition to calculating the energetic contributions upon site‐directed mutagenesis (RINs). As a result, we improved the synthetic activity of CalB from 2% to more than 70% of the total substrate‐product conversion ratio. This strategy allowed us to obtain more than 5 CalB variants with enhanced activity toward two bulky substrates after only two rounds of directed evolution. The sequential incorporation of favorable mutations increased our chances of selecting improved CalB variants and reduced screening effort. The use of a ‘bottom‐up’ strategy such as the RINs allowed us to further understand the effects of mutations throughout the protein structure, a powerful tool for protein engineering purposes. POS218 Accelerating Characterization of Large Variant Libraries Using Multiplexing Techniques for Industrially Relevant Reactions Olivier Rousseau 1, Joelle Pelletier1, Maximilian Ebert1, Nicolas Moitessier2, Josh Pottel2, Sebastian Pechmann1 1University of Montreal, Canada, 2McGill University, Canada Conventional organic synthesis produces industrially relevant compounds, but it is still costly in time, money and has an environmental impact. Enzymes are an eco‐friendly alternative to achieve these reactions, however they can be labor intensive to engineer. Therefore, there is a constant need to develop faster high throughput methods to accelerate enzyme engineering. We tackle this problem with a multiplexing approach to create, screen and identify multiple variants at the same time. As a proof of concept, we use the transformation of indole to indigo by the enzyme P450 BM3 (BM3), an enzyme part of the large family of P450s able to catalyze the challenging regio‐, chemo‐ and stereoselective oxidation of non‐activated carbon atoms in a single step. It is a reaction of great industrial importance. We mutated BM3's active site at 49 positions by saturation based on semi‐rational design and screened the libraries using an easy colorimetric plate assay. The plate screening revealed 29 new positions able to produce indigo and a gradation of blue in the colonies indicated the efficiency of each variants. Therefore the variants were pooled in 3 categories: white, light blue and dark blue and were sent to next‐generation sequencing for rapid, cheap and massive identification. In collaboration with Sebastian Pechmann (UdeM), we wrote a script to parse the 12 million reads revealing 422 new identified variants for these 3 pools in a primary analysis. In a second round of screening, these methods will be used for novel conversions of industrial importance. POS245 Expression of marine adhesive protein repeats using yeast surface display Kristina Reinmets 1, Stephen Fuchs1 1Tufts University, USA Adhesive proteins from marine organisms are capable of withstanding a variety of harsh environmental conditions and provide a great model for studying and designing improved biological adhesives. Our approach has utilized a well‐established protein engineering method, yeast surface display, to express mussel adhesive protein Mefp1 repeats and evaluate their ability to mediate adherence to surfaces such as stainless steel. Surface display of adhesive peptides allows easy detection of cells presenting the peptides with desired properties, whereas previous work from our lab has shown that repetitive coding sequences offer a potential mechanism for imparting genetic diversity in yeast. Preliminary results demonstrate the expression of adhesive peptides on cell surface and indicate that expression of Mefp1 repeats on yeast surface mediates the adhesion to stainless steel in the absence of stabilizing post‐translational modifications such as tyrosine hydroxylation. Future work will be aimed at directed evolution of improved adhesive peptides using the inherent instability of repetitive sequences in conjunction with both random and site‐specific approaches to incorporate modified amino acids important for adhesion. POS247 Development of a Red Glutamate Sensor and Exploiting the Extracellular Matrix for its Targeted Localization Rochelin Dalangin 1, Robert Campbell1, Jiahui Wu1, Robert Campbell1 1Department of Chemistry, University of Alberta, Edmonton, Canada Abnormalities in neurotransmitter dynamics have been implicated in various neurological conditions, but there is a shortage of tools for visualizing neurotransmitter release. Genetically‐encoded fluorescent protein (FP) sensors have become powerful tools for studying neuronal circuits due to their specificity and high spatiotemporal resolution. There is only one practically useful FP‐based sensor for neurotransmitters: the membrane‐tethered green glutamate sensor, iGluSnFR. To enable multicolor imaging experiments and because red light allows for deeper tissue imaging, we developed a red glutamate sensor, GltR1, from iGluSnFR that shows a three‐fold decrease in fluorescence in the presence of glutamate. However, GltR1 does not traffic to the cellular membrane of neurons as efficiently as iGluSnFR. Hyaluronan is ubiquitously expressed in the extracellular matrix (ECM), which makes up ∼20% of the adult brain volume. Previous work has shown that a green FP fused to a hyaluronan‐binding domain can be used to image hyaluronan. Inspired by this, we are now investigating the approach of secreting GltR1, iGluSnFR, and a variety of red proteins into the ECM as fusions to a hyaluronan‐binding domain. This approach should provide an alternate approach for using FP‐based indicators to visualize neurotransmitter release, and may provide larger and more robust signals due to the larger volume of space occupied by the secreted indicators. POS249 Beyond point mutations – Directed evolution tools for efficient and systematic exploration of protein functional space. Pedro Tizei 1, Emma Harris2, Vitor Pinheiro1 1University College London, UK, 2Birkbeck, University of London, UK Loops play a central role in many protein catalysts and high‐affinity ligands. It has been repeatedly shown for high affinity ligands such as antibodies – both in nature and in laboratory settings – that changes in composition as well as loop length can have huge impact on function. Nevertheless, most directed evolution strategies cannot access that diversity. Methods for introducing diversity focus on fixed‐length libraries and in most cases, changes in length are discarded to make analysis of the output of selection possible. As such, insertions and deletions (indels) represent a vast region of sequence space that is not explored and that goes undetected. Here, using the the O‐loop in TEM‐1 ß‐lactamase effect on substrate specificity as a model system, we present a novel platform for DNA library assembly that can generate highly customizable libraries that vary both in length and composition. In addition, we present an alignment‐free sequence analysis strategy that maximizes the information that can be gained from selection from libraries that vary in both composition and length. Together, they enable sequence space to be efficiently navigated – as we demonstrate by isolating 5 O‐loop variants with different length from wild‐type and unrelated sequence that have activity against a non‐cognate substrate. Our assembly and analysis tools represent a powerful new tool for directed evolution and highly relevant to protein loop and linker optimization. POS253 Engineering of a Fluorescent Protein Based Calcium Sensor Abhi Aggarwal 1, Robert Campbell1, Landon Zarowny1 1Department of Chemistry, University of Alberta, Edmonton, Canada Genetically‐Encoded Fluorescent Calcium Indicators (GECIs), that modulate their fluorescence intensity in response to changes in calcium ion concentration, are powerful tools for the investigation of cell biology. These fluorescent indicators are vital when it comes to cellular imaging because they allow the non‐invasive study of cells, tissues, and sub‐cellular structures at a detail that was previously not possible. The focus of this project is engineering a new GECI that exhibits favourable characteristics, such as increased brightness and a higher fold change. To develop this new GECI, we started from mNeongreen, the brightest monomeric fluorescent protein currently available. An initial prototype construct was made using rational design, following the precedent of the GCaMP series of indicators. This construct was further improved using directed protein evolution with colony‐based screening of libraries of randomly generated variants. We observe the brightness of promising new variants and perform tests to see how new mutations have affected the brightness and the fold change of the variant. After 8 rounds of screening, our latest variant of mNeonGreen‐GECI exhibits a Ca2+‐dependent change of 13.8. When compared to the original construct, this indicator appears significantly brighter with higher contrast between Ca2+‐bound and Ca2+‐free states. Directed evolution is ongoing and we expect to produce a fluorescent indicator that will be used for in vivo imaging of intracellular Ca2+ dynamics. POS275 Frontier Microfocus Macromolecular Crystallography at the National Synchrotron Light Source II (NSLS‐II) Martin Fuchs 1, Wuxian Shi2, Babak Andi1, Yuan Gao1, Jean Jakoncic1, Edwin Lazo1, Nicolas Guichard1, Robert Sweet1, Alexei Soares1 1Brookhaven National Laboratory, USA, 2Case Western University, Ohio, USA Two new macromolecular crystallography (MX) beamlines at the National Synchrotron Light Source‐II, FMX and AMX, have seen first light in March 2016 and opened for general user operation in February 2017. The micro‐focusing Frontier MX beamline (FMX), when fully commissioned, will deliver a flux of ∼5e12 ph/s at 1 Å into a 1–20 µm spot, with its maximum flux density surpassing current MX beamlines by up to two orders of magnitude. It covers a wide wavelength range from 0.4–5 Å and features a next generation Eiger 16M pixel array detector with a 133 Hz framing rate. The high brightness and micro‐focusing capability of FMX is ideal for solving difficult crystallographic problems. The experimental station's highly flexible design will support a wide range of structure determination methods – serial crystallography on (sub‐)micron sized crystals, raster optimization of diffraction from crystals of complexes with large unit cells, rapid sample screening and room temperature data collection for difficult to freeze crystals. The associated wide variety of samples includes frozen crystals in loops and meshes, single crystals or up to 20 acoustically deposited crystals, crystals in crystallization plates, lipidic cupic phase‐plates and jets. Specialized sample holders for serial crystallography based on micro‐structured silicon chips are currently being fabricated at the BNL Center for Functional Nanomaterials. The new beamlines will push the frontier of synchrotron crystallography and enable users to determine structures from difficult to crystallize targets like membrane proteins, using previously untractable few‐micron‐sized crystals, and obtain higher quality structures. This research is supported by the US National Institutes of Health NIGMS grant P41GM111244. POS282 De novo design of antivirulence therapeutics based on genetically encodable, hyperstable constrained peptides Christopher Bahl 1, David Baker1,2 1University of Washington, USA, 2HHMI, USA Organisms from all domains of life produce ∼20–50 residue disulfide‐constrained peptides, with functions ranging from signaling to virulence and immunity. These peptides, which are stabilized in a functional conformation by disulfide bonds, possess many of the beneficial pharmacological properties of small molecule drugs (e.g. high stability, tissue penetrance), while retaining the high interaction specificity of larger biological drugs, such as antibodies. Thus, constrained peptides represent a largely untapped class of drug scaffolds, and they are genetically encodable. We have developed a generalized computational method for designing constrained peptides de novo, which provides access much more molecular diversity than is currently available from naturalistic observation. We used the method to design constrained peptides spanning nine different structural topologies with sequences unrelated to known genes. The designed peptides contain up to three disulfide bonds, can be expressed and purified from bacteria, and exhibit high thermal and chemical stability. Experimentally determined X‐ray and NMR models show that the design protocol has atomic‐level accuracy. The new method enables design of peptides with structures custom‐tailored to specific applications; current efforts to incorporate function are directed toward treating infectious disease by antagonizing the virulence‐promoting mechanisms of pathogenic microorganisms. Our two primary targets are: the biofilm‐regulating Lap system from Gram‐negative bacteria, and super‐antigenic enterotoxins secreted by Gram‐positive bacteria. In each case, existing structural and mechanistic information is being leveraged in conjunction with computational peptide design to engineer site‐specific protein‐protein interactions with an intended therapeutic effect. POS301 A Synthetic Design Approach to Understanding Serpin Specificity and Thermostability Emilia Marijanovic1, James Fodor1, Shani Keleher1, Benjamin Porebski2, Blake Riley1, Mary Pearce1, David Hoke1, Sheena McGowan1, Ashley Buckle1 1Monash University, Australia, 2University of Cambridge, UK Serine protease inhibitors (serpins) are one of the few protein families that fold into a metastable conformation. This metastability is crucial for their function as inhibitors, but renders them susceptible to misfolding and aggregation. Redesigning a serpin may provide insight into this balance between function and stability. Previously, we used protein engineering to develop a synthetic serpin, Conserpin, with enhanced biophysical properties. This serpin has a high thermal stability (Tm > 100°C), is more aggregation‐resistant than a1‐antitrypsin (a1AT), and is an active inhibitor. We explore the balance between function and stability in serpins by grafting regions from Conserpin to a1AT, and vice versa. (1) We engineer Conserpin to mimic a1AT by mutating residues in the reactive centre loop (RCL) to those of a1AT (creating ConserpinAATRCL). While these mutations did not affect the stability or X‐ray crystal structure of the serpin, ConserpinAATRCL possesses weak inhibitory activity compared to a1AT. The surface potential between ConserpinAATRCL and a1AT differs greatly, and molecular dynamics simulations show that the RCL is more dynamic in ConserpinAATRCL than in a1AT. These factors could play a role in the failure of ConserpinAATRCL to match the inhibitory activity of a1AT. (2) a1AT was engineered for stability and aggregation‐resistance by “grafting” regions of Conserpin onto a1AT. Circular dichroism thermal unfolding analysis shows that some of these grafts confer an increased thermostability, while retaining the activity of wild type a1AT. Taken together, our results suggest that a1AT can be engineered for increased stability and aggregation resistance, without compromising function. POS303 The Creation Of Stable Protein Coatings On Magnetic Nanoparticles For Biomedical Applications Anna Bychkova 1, Tatiana Danilova1, Mariia Lopukhova1, Alina Milyutina1, Elena Grishechkina2, Mark Rosenfeld1 1N. M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Russia, 2State Research Institute of Chemical Reagents and High Purity Chemical Substances, Russia Proteins are promising materials for creation of coatings on magnetic nanoparticles (MNPs) due to their biocompatibility, protection of magnetic cores from biological liquids influence and prevention of their agglomeration in dispersion. Magnetically targeted nanosystems with protein coatings are considered to be applicable in different areas of biology and medicine such as hyperthermia, magnetic resonance imaging, immunoassay, cell and molecular separation, a smart delivery of drugs to target cells. The study objective was to development of free radical approach to creation of functional bicomponent protein coatings on magnetic nanoparticles of various sizes Adsorption of a group of blood proteins including serum albumin and immunoglobulin G on MNPs was studied. A novel approach based on protein liability to free radical modification, leading to the formation of intermolecular covalent cross links has been used for obtaining coatings assembled from protein molecules on the surface of magnetite nanoparticles in dispersions. The properties of the coatings have been studied with the help of dynamic light scattering (DLS), UV/Vis spectrophotometry, antibody‐antigen test and the method of spectral‐fluorescent probes. The protein adsorption was shown to be dependent on the incubation time, protein/MNPs concentration, nanoparticles curvature, the temperature, ionic strenght and buffer characteristics, etc. The free radical linking of thrombin and immunoglobulin G on the surface of nanoparticles has been shown to almost completely keep native properties of the protein molecules as potential therapeutic products and biovectors. The reported study was funded by RFBR and Moscow city Government according to the research project No. 15–33‐70019 «mol_a_mos», by RFBR, according to the research project No. 16–34‐60244 mol_a_dk. POS317 Mispacking and the Fitness Landscape of the Green Fluorescent Protein Chromophore Milieu Thomas Jordan 1, Chris Bystroff1, Shounak Banerjee1, Julia Reimertz1, Emily Crone2, Donna Crone1, Christian Schenkelberg1, Chris Bystroff1 1Rensellaer Polytechnic Institute, Troy, USA, 2Colgate, USA The auto‐catalytic maturation of the chromophore in green fluorescent protein (GFP) was thought to require the precise positioning of the side chains surrounding it in the core of the protein, many of which are strongly conserved among homologous fluorescent proteins. In this study, we screened for green fluorescence in an exhaustive set of point mutations of seven residues that make up the chromophore microenvironment, excluding R96 and E222 because mutations of these positions have been previously characterized. Contrary to expectations, nearly all amino acids were tolerated at all seven positions. Only four point mutations knocked out fluorescence entirely. However, chromophore maturation was found to be slower and/or fluorescence reduced in several cases. Selected combinations of mutations showed non‐additive effects including cooperativity and rescue. The results provide guidelines for the computational engineering of GFPs. POS320 Directed Evolution of ß‐glucosidase from Paenibacillus polymyxa Janeala Morsby 1, Nicholas Panasik1 1Claflin University, South Carolina, USA The biofuel industry uses ß‐glucosidase to break down cellulose to a‐glucose and ß‐glucose for bioethanol production. However, sufficient heat is generated during this process to denature ß‐glucosidase. The biofuel industry spends ∼$145 billion on cooling techniques such as cloud chambers. The purpose of my research which is entitled ‘The Directed Evolution of ß‐ glucosidase from Paenibacillus polymyxa” is to design a thermostable ß‐glucosidase that can be used by the biofuel industry to breakdown cellulose into a‐glucose and ß‐glucose without the need for cooling. If I am successful, the biofuel industry can use this enzyme and spend little to no money on cooling techniques and as such, fuel will be more affordable. Error prone polymerase chain reaction was used to create a library of mutant ß‐ glucosidase DNA sequences. This library was transformed into and Escherichia coli strain (JM109) that lacks the ß‐galactosidase gene. The colonies were then screened with the chromogenic substrate X‐ Gal at 41 oC to identify clones that were active at temperatures where wild type is not active. Over 1000 colonies were screened and 3 thermostable mutants were identified. We present here phenotypic and biochemical characterization of this thermostable phenotype. POS330 Semi‐rational evolution of the Pseudomonas aeruginosa Rhamnosyltransferase 1 subunit A (RhlA) for the synthesis of industrially relevant rhamnolipids Carlos Eduardo Dulcey 1, Yossef Lopez de los Santos1, Éric Déziel1, Nicolas Doucet1 1INRS‐Institut Armand‐Frappier ‐ Université du Québec, Canada Rhamnolipids (RLs) are glycolipidic compounds produced by a few of bacterial species, especially Pseudomonas and Burkholderia spp. These compounds display excellent surfactant properties and environmental advantages. Nevertheless, their high production cost hampers their practical use in industry. In addition, bacterial strains that produce RLs generate a mixture of congeners with varying lipophilic chain lengths, therefore affecting their macromolecular behavior. Since the physicochemical characteristics of RLs are directly influenced by their molecular structure, modification or improvement of their surfactant properties can be achieved by controlling the length of their alkyl chains. RhlA acts as a key enzyme in the RL biosynthesis pathway. The enzyme catalyzes the esterification reaction between two units of hydroxylated fatty acids to form a dimer, ß‐3‐(3‐hydroxyalkanoyl) alkanoic acid (HAA), the dilipid precursor of RLs. HAA biosynthesis is the rate‐limiting step in RL biosynthesis. Here, we present a semi‐rational evolution approach to engineer RhlA from P. aeruginosa to increase in vivo HAA production and to modulate substrate selectivity. Using a structural homology model of the enzyme, we predicted a number of substrate‐interacting residues and performed intragenic suppression‐type mutagenesis to increase the catalytic efficiency of RhlA. We also employed a chimeric approach to identify protein domains involved in enzyme selectivity, in addition to performing site‐directed mutagenesis on residues located in the substrate binding pocket to modulate RL congener biosynthesis. Our results provide evidence that protein engineering approaches can be efficiently employed to improve RL production in P. aeruginosa. POS342 Identification of hotspot residues for M. tuberculosis alanine racemase Sudipta Majumdar 1, Cuong Diep1, Noriko Mikeasky1 1Indiana University of Pennsylvania, USA M. tuberculosis alanine racemase (MT_Alr), an anti‐tuberculosis drug target, is an obligate homodimer. Both monomers contribute to the overall composition of their active sites. Therefore, disruption of the dimer interface could be used to inhibit MT_Alr activity. Five potential hotspot residues (Asp135, Arg140, Lys261, Glu267, and Arg373) were identified using computational alanine scanning Robetta. The yeast two‐hybrid system is being used to assess their contribution to dimerization. MT_Alr was cloned into two plasmids and expression of one plasmid was confirmed in yeast by Western blot. The candidate residues will be mutated to alanine (individually or in combination) and their effect on dimerization will be determined in the two‐hybrid system. An unbiased random mutagenesis will also be carried out to find other potential hotspot residues. The experimental verification of key residues for MT_Alr dimerization will lead to the identification of potential ligand binding site at the dimer interface for inhibitor design. POS344 Using phage‐displayed peptide libraries to identify peptide ligands binding to bacteria as a means to characterize the gut microbiota. Shweta Shah 1, A. Gururaj Rao1, Gregory J. Phillips2 1Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, 2Department of Veterinary Microbiology and Preventive Medicine, Iowa State University Random peptide libraries can provide a diversity of peptide combinations and molecular structures that can recognize and bind to a variety of target molecules. Phage display is one of the most powerful and well established technologies for selectively screening for desired binding peptides from a combinatorial peptide library. In our work, we have used the phagemid vector pCANTAB 5E to construct a random 21 amino acid library fused to the pIII coat protein, achieving a diversity of ∼ 1x109. The library has been validated by screening against the anti‐FLAG monoclonal antibody, and isolating a peptide that matches the known flag epitope DYKDDDDK. We have previously successfully used the library in protein ‐protein interaction studies (Shah et al., 2013, PLoS One, 8(5): e64783; Meyer et al., 2013, Arch. Biochem. Biophys.535 (2):101–110). Our current effort is focused on exploring the use of phage‐peptide libraries in the context of the gastrointestinal (GI) microbiota i.e., identifying peptide ligands that are unique to specific bacteria that colonize the mammalian gut. To this end, in a pilot experiment we have screened our library against a commensal strain of Escherichia coli, which yielded binding peptides containing the consensus binding motif “SWLK/R”. Further, 21 mer phage library has been used to isolate binding peptides specific to Lactobacillus murinus, which is a member of the murine GI tract. One of our future goals is also to utilize this technique for isolating and identifying peptides that can distinguish between various bacterial species as new tools to study the structure and function of the GI microbiota. POS368 Designing Protein Polyhedra Using a Generalizable Symmetry‐Based Approach Ajitha Cristie‐David 1, Neil Marsh1, Ajitha Cristie‐David1, Neil Marsh1 1University of Michigan, USA Protein‐based nanostructures have many potential applications in medicine, and materials science. Here we describe a generalizable method that uses short coiled‐coil domains to assemble proteins into nanocages of various geometries. The approach requires minimal alteration of the protein and hence should preserve the structural and functional properties of the protein. Our strategy relies on the combination of 2 rotational symmetry elements, one provided by the natural, building block protein (BBP) and the other provided by the coiled coil, to specify a protein cage of the desired geometry. The oligomerization of the coiled‐coil brings together the copies of the BBP together, leading to the assembly of protein cages. By employing BBPs and coiled‐coils of different rotational symmetries it is possible to design cages with various sizes and geometries. Key to the success of this strategy is optimization of the linker between the BBP and the association strength of coiled‐coil strands. Using this strategy we have been able to successfully design an octahedral and a tetrahedral protein cages. Our current focus is on designing structurally more challenging icosahedral protein cages as well as elaborating the structure of the previously designed octahedral cage with additional protein domains. POS372 Design and Deployment of Enhanced Split Inteins Adam Stevens 1, Tom Muir1, Giridhar Sekar2, David Cowburn2 1Princeton University, USA, 2Albert Einstein College of Medicine, USA Protein splicing is a post‐translational autoprocessing event in which an intervening protein (intein) cleaves itself from a precursor protein while simultaneously ligating the adjacent residues (exteins) to form a native peptide bond. Although the biological function of inteins remains unclear, they have found widespread use as tools in chemical biology and protein engineering. Of special interest are naturally split inteins, in which two intein fragments are separately expressed and then efficiently undergo association and splicing in trans. However, applications of split inteins have been limited by a number of shortcomings, including issues of expression yield among protein‐intein fusions and decreased splicing rates under certain extein contexts (extein dependency). Through structural and mechanism‐guided approaches, we have engineered split inteins with increased thermostability and extein promiscuity. These enhanced split inteins were then applied to a number of protein engineering methods, such as the generation of head to tail cyclized proteins, the modification of a monoclonal antibody with a small molecule cargo, and the semisynthesis of cellular chromatin in isolated nuclei. Overall, we expect these engineered proteins to facilitate the further use and development of protein trans‐splicing based technologies and methods. POS376 Tracking Heme Loading of a Protein in Live Cells by Fluorescence‐Lifetime Imaging Microscopy (FLIM) Samaneh Dastpeyman1, Ann M English1 1Concordia University and PROTEO, Canada Heme biosynthesis is well characterized. The final steps occur in mitochondria but how heme is transferred to heme‐dependent proteins is not known, in part due to current technical limitations in monitoring the heme loading of specific proteins in cells. Cytochrome c peroxidase (Ccp1) is a heme‐based mitochondrial H2O2 sensor and labilization of its heme occurs when H2O2 levels spike in respiring yeast. This leads to the activation of catalase A (Cta1), which directly or indirectly receives Ccp1's heme. To fully understand this unprecedented H2O2‐triggered heme mobilization, we selected Ccp1 fused to green fluorescent protein (Ccp1‐GFP) as a probe of Ccp1 heme loading in live cells since heme efficiently quenches GFP fluorescence. We monitor fluorescence lifetimes as these are concentration independent unlike fluorescence intensities. In vitro, heme‐free recombinant apoCcp1‐GFP exhibits a lifetime of 2.86 ns whereas heme‐loaded holoCcp1‐GFP displays long (2.45 ns) and short (0.96 ns) lifetimes. The fractional amplitude of the short lifetime increases linearly at the expense of the long‐lifetime amplitude as apoCcp1‐GFP binds heme. These results allow us to estimate by FLIM the heme status of Ccp1‐GFP in 2‐ and 7‐day live yeast cells. Our study not only sheds light on the heme status of Ccp1 in vivo but also suggests a novel tool for unraveling intracellular heme trafficking. POS386 Engineering Antiviral Lectins by Computer‐Guided Design and Evolution. Giovanna Ghirlanda 1, Banu Ozkan1, Orkun Pinar1, Can Kazan1 1Arizona State University, USA Cyanovirin‐N (CV‐N) is an antiviral lectin with potent activity against enveloped viruses. In the case of HIV, antiviral activity of CV‐N is postulated to require multivalent interactions with the oligomannoses on the envelope protein gp120, achieved through a pseudo‐repeat of sequence that adopts two near‐identical glycan‐binding sites, and possibly involves a 3D‐domain‐swapped dimeric form of CV‐N. Work in our lab has shown that our flexible docking methods can dissect the contribution of single amino acids to binding target glycans, and predict mutations that increase binding affinity. We improved antiviral activity by designing a covalent dimer of CV‐N that increases the number of active glycan‐binding sites. Two native repeats were separated by the “nested” covalent insertion of two additional repeats of CV‐N, resulting in four possible glycan‐binding sites. The resulting Nested CV‐N folds into a wild‐type‐like structure as assessed by circular dichroism and NMR spectroscopy, and displays high thermal stability with a Tm of 59°C, identical to WT. All four glycan‐binding domains encompassed by the sequence are functional as demonstrated by isothermal titration calorimetry, which revealed two sets of binding events to dimannose with dissociation constants Kd of 25 µM and 900 µM, assigned to domains B and B’ and domains A and A’ respectively. Nested CV‐N displays a five‐fold increase in activity when compared to WT CV‐N in both a cellular assay and a fusion assay. This work demonstrates that rational design can be used to increase binding affinity and multivalency in antiviral lectins resulting in more potent activity. POS388 Computational Design of Novel Enzymes Guided By Evolutionary Data Gideon Lapidoth 1, Sarel Fleishman1 1Weizmann Institute of Science, Israel The ability to computationally design efficient, specific enzymes is a rigorous test of our understanding of the principles of catalysis and molecular recognition. Successful designs have to date shown several limitations: they only targeted simple reactions, involving two to three catalytic residues with often low efficiencies and selectivities, and impaired stability. We developed a new algorithm using Rosetta to combine compatible backbone fragments from natural enzymes of the same enzyme superfamily to generate novel conformations. The designs’ sequences are then optimized, guided by sequence conservation data to improve stability and expressibility. We used the algorithm to design novel TIM barrel fold enzymes belonging to the GH10 family capable of hydrolyzing xylan, an abundant plant polysaccharide, with Kcat/Km values similar to those of natural xylanases. The designed enzyme conformations differ from one another and from any other known natural xylanase conformations and have different substrate specificities. The algorithm is completely automated and can be applied to other enzymes of modular fold to efficiently and broadly explore the potential selectivities of the superfamily. POS409 Self‐assembling supramolecular nanostructure complexes constructed from protein nanobuilding blocks Ryoichi Arai 1, Naoya Kobayashi1, Naoya Kimura1 1Shinshu University, Japan Living organisms are maintained by various supramolecular complexes of self‐assembling biomolecules including proteins, nucleic acids, sugars, and lipids. The chemical reconstitution of living matter is one of the ultimate goals of chemistry and synthetic biology. Research on molecular design and dynamical ordering systems of self‐assembling artificial protein complexes is an important step toward achieving the goal and further applications. Recently, we designed and created a protein nanobuilding block (PN‐Block), WA20‐foldon, by fusing an intermolecularly folded dimeric de novo protein WA20 and a trimeric foldon domain from bacteriophage T4 fibritin (Kobayashi, N., et al., 2015, J. Am. Chem. Soc., 137, 11285). The WA20‐foldon, as a simple and versatile nanobuilding block, self‐assembled into several oligomeric nano‐architectures in multiples of 6‐mer. We also designed and created de novo extender protein nanobuilding blocks (ePN‐Blocks), by fusing tandemly two WA20 with various linkers, to construct self‐assembling cyclized and extended chain‐like nanostructure complexes. Moreover, to stabilize the de novo protein WA20 for extensive application of PN‐Blocks in nanotechnology, we designed and developed a WA20 variant, called SUWA (Super WA20), by several mutations for stabilization of helices and hydrophobic cores. Thermal denaturation experiment shows denaturation midpoint temperature (Tm) for SUWA is extremely high, 122 degrees C. We also constructed a PN‐Block, SUWA‐foldon, and its native PAGE and SAXS analyses suggest that the SUWA‐foldon self‐assembled into several homooligomeric complexes. Partial thermal denaturation and reconstruction experiments suggest dynamical ordering systems of the SUWA‐foldon supramolecular complexes. These results demonstrate that the PN‐Block strategy is useful for constructing self‐assembling protein complex nano‐architectures. POS446 Cas9 as a target for dynamics‐based rational design Kendra Hailey 1, Giulia Palermo1, Patricia Jennings1 1University of California, San Diego, USA Crystal, NMR, and cryo‐EM structures provide the critical starting points for understanding bimolecular function at the atomic level. However, to fully explain the mechanistic details within each system in vitro and ultimately in vivo, an expansive array of in silico (MD, aMD, QM/MM) and solution biophysical/biochemical techniques are employed to inform us on functionally important phenomena missed by inspecting structures alone. DXMS is a medium resolution solution method (NMR dynamics < DXMS < SAXS, FRET) that measures the deuterium incorporation into the peptide backbone over time, and the resulting data are then used to understand the native state dynamics of proteins on the second/minute time scales (protein folding intermediates, ligand‐induced conformational changes). The strengths of DXMS include a large sample size range (>2 MDa, e.g.), amenable to diverse sample compositions (PTMs, membrane environments, amyloid), and great ease of use (data collection and processing, semi‐HT). In combination with quench‐flow, DXMS can also be used to track non‐native state conformations within the system, including intermediate states within protein folding trajectories, as well as dynamic (potentially allosteric) pathways within and between molecules. Here, paired with specific FRET‐labeling schemes, we aim to elucidate the dynamics for each “native” state of Cas9 (apo‐, sgRNA‐bound, and sgRNA‐DNA‐bound), as well as follow each intermediate via stop‐flow (DXMS, FRET). We further aim to refine our data analyses in order to create a “higher‐throughput, rational design” workflow (below Figure), with Cas9 as our test system for discovering and selecting for new target functions. 07. DYNAMICS AND ALLOSTERY POS030 HtrA3 Serine Protease: Elucidating the Complex Regulatory Mechanisms of a Unique Cell Death Regulator Saujanya Acharya 1, Saujanya Acharya1, Kakoli Bose1 1ACTREC, India Introduction: High temperature requirement serine protease A3 (HtrA3) has been implicated in intrinsic and non‐classical apoptotic pathways through its proteolytic activity. It is proposed to be a tumor suppressor and hence a potential therapeutic target. Mature HtrA3 comprises an N‐terminal region, a serine protease domain (SPD) and a C‐terminal PDZ domain. Its complex mechanism of activation has been poorly characterized and understanding of its biological functions is limited by a single natural substrate. Objective: Our aim is to perform structural and functional analyses to decipher the role of different HtrA3 domains and critical residues in mediating protease activity and specificity in presence of its known and novel binding partners. This might aid in modulating its activity with desired characteristics. Methods: Cloning and purification of HtrA3 variants were done using molecular biology and biochemical tools. Conformational changes, stability, functional enzymology and interaction with its partners were studied using multidisciplinary tools. Results: Enzyme kinetics demonstrated that HtrA3 followed an allosteric model of activation. N‐SPD, SPD and SPD‐PDZ variants were inactive. A mutation in the N‐terminal region made the protein monomeric and inactive. Moreover, X‐linked inhibitor of apoptosis (XIAP) was identified as a novel binding partner. Conclusions: Through domain wise dissection of HtrA3, the importance of both N‐terminal region in trimerization and protease activation was demonstrated. It also highlighted the role of PDZ in modulating protein activity. HtrA3 might also be promoting apoptosis by binding and deactivating XIAP. POS049 Phospholipase A2: A Unique Paradigm of Allosteric Regulation by Membranes Varnavas D. Mouchlis 1, J. Andrew McCammon1, Edward A. Dennis1 1University of California, San Diego, USA Defining the details of the molecular recognition between water‐soluble enzymes and membranes is fundamental to understanding protein‐lipid interactions, molecular complementarity and membrane functioning (PNAS 2015, 112, E516‐E525). Phospholipases A2 (PLA2) including cytosolic (cPLA2), calcium‐independent (iPLA2) and lipoprotein‐associated (Lp‐PLA2), provide an ideal system for studying protein‐lipid binding and interactions. Hydrogen/deuterium (H/D) exchange mass spectrometry was used to identify peptide regions of these three enzymes that interact with phospholipid vesicles. Molecular dynamics simulations guided and validated by experimental H/D exchange data showed that the active sites of these enzymes are allosterically regulated by membranes. Membrane phospholipids bind to allosteric sites located on the interfacial surface of PLA2s shifting their conformation from the “closed” to the “open” state. This process facilitates extraction and binding of a phospholipid molecule in the active site where the hydrolysis occurs at the sn‐2 position of the phospholipid. This constitutes the first detailed study describing the binding and interaction mechanism of PLA2s with the membrane bilayer as well as how they bind a single phospholipid molecule in the catalytic site. These enzymes are implicated in chronic inflammatory diseases and understanding their association with membranes, mechanism of action and interactions with membranes and inhibitors at the molecular level will allow us to identify potent and selective inhibitors that can be further developed as novel anti‐inflammatory agents (J. Med. Chem. 2016, 59, 4403–4414 and Bioorg. Med. Chem. 2016, 24, 4801–4811). POS063 Allosteric Modulation of the Kinase/RNase IRE1a by Small Molecules and Scaffolding Kinases Hannah Feldman1, Shuhei Morita2, Feroz Papa2, Dustin J. Maly1 1University of Washington, USA, 2University of California San Francisco, USA Numerous cellular perturbations can overwhelm the homeostatic capacity of the endoplasmic reticulum (ER), causing the accumulation of unfolded proteins and activation of the unfolded protein response (UPR). The UPR restores ER homeostasis (adaptive), but under prolonged stress switches to a pro‐death (terminal) output. The protein kinase/RNase, IRE1a, containing dual kinase and RNase activities, has been shown to contribute to the transition from adaptive to terminal UPR‐outputs. During ER stress, unfolded proteins result in lumenal domain oligomerization, leading to kinase autophosphorylation and RNase activation. In cases of prolonged stress, IRE1a becomes hyperactivated leading to the endonucleolytic decay of hundreds of ER‐localized mRNA, contributing to cell death. I will present mechanistic studies probing the allosteric communication between the kinase and RNase domains of IRE1a. Inhibitors targeting the ATP‐binding site of IRE1a's kinase have divergent allosteric effects on IRE1a's RNase activity. Some inhibitors allosterically activate the RNase of IRE1a, while others, called KIRAs, allosterically inhibit RNase activity. We performed biochemical and structural studies providing insight into how ATP‐competitive inhibitors affect IRE1a's oligomeric state, which directly influences RNase activity. A comprehensive structural model of how different classes of inhibitors divergently modulate IRE1a's oligomeric state will be described. Furthermore, how different classes of inhibitors affect cell fate under ER stress will be presented. We recently discovered that the tyrosine‐kinase Abl, allosterically activates the enzymatic activities of IRE1a. Under ER stress, Abl co‐localizes with IRE1a at the cytosolic face of the ER membrane, promoting IRE1a autophosphorylation and stimulating RNase activation. Mechanistic studies into how Abl scaffolding activates IRE1a and molecular details of the IRE1a‐Abl complex will be presented. POS070 Study of allosteric communications in chimeric two‐domain proteins Kristyna Bousova1 1Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Czech Republic At present, studies of protein structures vary from the mapping of their basic functions to studies of the synergy between protein elementary units ‐ the protein domains. The information about mutual interactions of domains can be used to decrypt inter/intra domain communication and the allosteric modulation of a function. Our team study new synthetic protein chimeras composed of two different functional protein domains – PDZ3 domain (part of ZO‐1 [1]) acting as cytoskeleton and membrane protein, and TrpCage artificial protein molecule generated in silico [2]. These two domains are flexibly connected in two different orders: PDZ3‐TrpCage and reverse TrpCage‐PDZ3. We use various biochemical, biophysical and structural methods to describe properties of the De Novo created proteins ‐ analytical size exclusion chromatography, analytical ultracentrifugation, dynamic light scattering, thermal shift assay, circular dichroism, chevron plot kinetic studies and NMR. Our data suggest completely different behaviour of both studied chimeras. PDZ3‐TrpCage protein is characterized as a single domain protein in contrary to the TrpCage‐PDZ3 protein showing two domains character, lower stability and tendency to aggregate. We assume, the order of protein domains in cell specific multi‐domain proteins is the key factor of domain allosteric modulation and determines their specific function. References [1] Pan L, Chen J, Yu J, Yu H, Zhang M. The structure of the PDZ3‐SH3‐GuK tandem of ZO‐1 protein suggests a supramodular organization of the membrane‐associated guanylate kinase (MAGUK) family scaffold protein core. J Biol Chem. 2011; 286 (46): 40069‐74. [2] Kitazawa S, Fossat MJ, McCallum SA, Garcia AE, Royer CA. NMR and Computation Reveal a Pressure‐Sensitive Folded Conformation of Trp‐Cage. J Phys Chem B. 2017; 121 (6): 1258–1267 POS077 Identification of Allosteric Fragments to Rigidify Dynamic Conformation Zhengding Su 1, Rong Chen1, Jingjing Zhou1, Lingyun Qin1, Huili Liu2 1Institute of Biomedical and Pharmaceutical Sciences, Hubei University of Technology, China, 2National Center for Magnetic Resonance in Wuhan, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, China Aberrant protein‐protein interaction (PPI) networks and nodes have been identified to be highly correlated with the proliferation and growth acquisition of cancer cells, rendering PPIs to become promising anti‐cancer therapeutic targets. Using the p53/MdmX interaction as a model system, in this work, we demonstrate a peptide‐directed strategy for the Identification of allosteric fragments to rigidify protein dynamic conformation. In nearly half of cancers, the anticancer activity of p53 protein is often impaired the overexpressed oncoprotein Mdm2 and its homolog MdmX, demanding efficient therapeutics to disrupt this aberrant p53‐MdmX/Mdm2 interactions to restore the p53 activity. Considering that the intrinsic fluorescence residue Trp23 in the p53 transaction domain (p53p) plays an important role in determining in the p53‐MdmX/Mdm2 interactions, we constructed a fusion protein to make use of the intrinsic fluorescence signal of Trp23 to monitor high‐throughput screening of compound library, aiming to identify novel scaffolds of MdmX inhibitors. The fusion protein was composed of the p53p followed by the N‐terminal domain of MdmX (N‐MdmX) through a flexible amino acid linker, where the whole fusion protein contained a sole intrinsic fluorescence probe. The fusion protein was then evaluated using fluorescence spectroscopy against an Mdm2 inhibitor library, identifying few allosteric fragments which enabled to rigidify dynamic conformation. Furthermore, the allosteric fragments were utilized to design MdmX inhibitors. Thus, our work provides a rationale for optimizing MdmX inhibitors. The fusion protein strategy described in this work is also applicable for other protein targets for drug discovery. POS095 Quantifying dynamic blebbing in mammalian cell lines to predict migratory behaviour Netra Unni 1, Anam Qudrat2 1University of Toronto Faculty of Applied Sciences and Engineering, Canada, 2Institute of Biomaterials and Biomedical Engineering, Canada Predicting cell migration requires careful analysis of changes in cell morphology on a spatiotemporal scale. One such morphological change is the formation of a bleb – a protrusion of the cell's plasma membrane that is subsequently filled by cytoplasmic fluid. Recent studies have shown that membrane blebbing may be a useful mechanism in predicting a cell's migratory pathway. Through the analysis of HEK293 mammalian synthetic cell lines, characteristics of bleb formation were quantified using the ADAPT software. Metrics such as bleb size, growth rate, directionality and persistence were quantified. We hypothesize that dynamic, persistent blebbing potentiates a cell's migration in a defined direction. POS113 Distal residues may modulate dynamics of Ornithine transcarbamoylase according to small angle x‐ray solution scattering Jenifer Winters 1, Lisa Ngu1, Dr. Lee Makowski1, Dr. Penny J. Beuning1, Dr. Mary Ondrechen1 1Northeastern University, Massachusetts, USA Studies of enzyme catalytic mechanisms have typically focused on the residues in direct contact with the reacting substrate molecule(s). However, some enzymes utilize residues that are not obviously important for catalysis because they are not in direct contact with the reacting species. Partial Order Optimum Likelihood (POOL) is a machine learning method that predicts catalytically important residues based on the protein's tertiary structure and electrostatic properties. POOL has predicted spatially extended active sites, where residues that are not in direct contact with the substrate are important for catalysis, including for ornithine transcarbamoylase (OTC). Enzyme variants were created through single‐site directed mutagenesis and subsequently assayed for their kinetic activities, showing that predicted residues contribute to catalysis while non‐POOL‐predicted residues do not. Small‐angle x‐ray solution scattering (SAXS) was used to determine if the predicted distal residues play a role in dynamical structural changes, thus affecting catalysis from a distance. Three‐dimensional reconstructions of the solution scattering data for wild‐type OTC and variants were generated using reconstruction programs from the ATSAS suite. In most cases, reconstructions of variants with mutations in distal positions suggest a structural rearrangement that reverses upon the addition of substrates. We conclude that the functional impact of the POOL‐predicted electrostatic effects are realized at least partially through conformational rearrangements of secondary structural elements. Supported by NSF MCB‐1517290. POS164 Leveraging Reciprocity to Identify Unknown Allosteric Sites in PTP1B James Lipchock 1, Patrick Loria2, Danica Cui2, Patrick Ginther1 1Washington College, Maryland, USA, 2Yale University, Connecticut, USA Protein tyrosine phosphatase 1B (PTP1B) is a known regulator of insulin and leptin signaling pathways and is an active target for the treatment of type II diabetes and obesity. Given the importance of PTP1B as a therapeutic target, we have sought to identify unknown sites of allosteric regulation on the surface of this enzyme. To achieve this, we created a series of alanine point mutations in the active site of PTP1B and monitored 1H15N composite chemical shift perturbations across the protein. Specifically, these mutations spanned the acid‐loop (Y176‐P188), which was shown previously to move at the rate of enzyme catalysis. Structural mapping of the chemical shift perturbations revealed a network of residues that connects the active site and the opposite side of the enzyme, including three distal clusters of residues on the surface of PTP1B. One of these clusters includes the benzbromarone analog binding site, which is a known allosteric inhibitor of PTP1B. To confirm the importance of the other two clusters, six residues in these sites were mutated and analyzed with kinetic assays and NMR spectroscopy. The resulting mutants exhibited decreases in catalytic efficiency ranging from 1.5‐3.8 and yielded significant chemical shifts changes along the allosteric network and in the active site, confirming their role in the allosteric regulation of PTP1B. We believe this work will not only help to advance drug discovery efforts for PTP1B, which have proven difficult to date, but also other enzymes with unknown allosteric networks. POS174 Allosteric Destabilization of NBD1 Subdomains and CFTR by Cystic Fibrosis Mutations Naoto Soya 1, Gergely Lukacs1, Ariel Roldan1, Haijin Xu1, Ryosuke Fukuda1, Tamas Hegedus2 1McGill University, Montreal, Canada, 2Semmelweis University, Hungary Cystic fibrosis (CF), one of the most common autosomal recessive diseases, is caused by the functional expression defect of the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. Deletion of F508 residue (ΔF508) in nucleotide binding domain 1 (NBD1), the most prevalent mutation, leads to the misfolding and ER degradation of CFTR. The F508 residue is pivotal for folding/stability of NBD1 and CFTR coupled domain folding by stabilizing a hydrophobic pocket at the NBD1 cytosolic loop 4 (CL4) interface. Hyperstabilization of NBD1 alone or in combination with that of the NBD1‐CL4 interface are pursued to correct the ΔF508‐CFTR folding defect. Despite extensive efforts, we still lack pharmacological chaperones that robustly rescue to the mutant NBD1 and CFTR stability defects. Here we used hydrogen/deuterium exchange mass spectrometry (HDX‐MS) with continuous labeling technique and molecular dynamic simulation to monitor the spatiotemporal unfolding of mutant NBD1 and gain insights into their conformational defects. The NBD1 unfolding was accelerated 10–20‐fold by the ΔF508 at 37°C but not at 25°C. The unfolding starts at the a‐subdomain, and spreads to the F1‐like ATP‐binding and ß‐subdomains. In contrast, second‐site suppressor mutations restored the subdomains’ stability of NBD1 containing ΔF508 or other CF‐causing mutations. The results, in concert with molecular dynamic analysis, help to identify a network of residues that renders NBD1 subdomains sensitive to allosteric unfolding and may serve for rational selection of pharmacological chaperones to alleviate the mutants folding defect. POS194 Conservation of Conformational Motions Impacting Function in an Enzyme Superfamily Chitra Narayanan 1, David N Bernard1, Khushboo Bafna2, Pratul K Agarwal3, Nicolas Doucet1 1INRS ‐ University of Quebec, Canada, 2University of Tennessee, Knoxville, USA, 3Oak Ridge National Laboratory, Tennessee, USA Correlation between enzyme function and conformational motions of amino acid networks >10 Å from the active site has been well established for discrete enzyme systems. However, approaches for characterizing dynamical properties across diverse sequence homologs within a family and their correlation with enzyme activity remain challenging. Members of the pancreatic‐type ribonuclease (ptRNase) superfamily share similarities in structure and fold, but display large variations in conformational dynamics, catalytic efficiencies, and tissue specific biological activities, making them ideal model systems for probing the relationship between conformational motions and function. As a step towards determining this relationship between dynamics and catalytic efficiency for various members of this broad vertebrate family, we performed the systematic characterization of the intrinsic dynamics of >20 RNases, with experimentally solved structures, over a wide range of time‐scales by integrating molecular dynamics simulations and NMR relaxation dispersion experiments. Our results show distinct patterns of dynamical variations between canonical RNases clustered into taxonomic groups, henceforth referred to as subfamilies. We show that conformational motions on the catalytically relevant micro‐ to milli‐second timescale are significantly different for RNases sharing the common fold. Interestingly, sequences sharing similar conformational exchange on this timescale also share similar biological functions. Further, quantitative characterization of pair‐wise correlations of dynamical properties between the RNase members showed strong correlations within subfamilies that share similar functions. These results suggest that selective pressure for conservation of specific atomic‐scale dynamical behaviors, among other factors, may potentially impact distinct biological functions of enzymes sharing the same fold. Further experiments are required to characterize the correlation between conserved dynamical properties and biological function. POS199 CaMKII biophysics and its role in long‐term potentiation Margaret Stratton 1, Ana Torres1, Yasunori Hayashi2, Takeo Saneyoshi2, Emily Agnello1, Rory O'connell1, Brendan Page1, Megan West1 1UMass Amherst, USA, 2Kyoto University, Japan Ca2+‐calmodulin dependent protein kinase II (CaMKII) assembles into an oligomeric ring in which the kinase domains are organized around a central hub. Notably, the stimulated activity of CaMKII persists even after the withdrawal of a calcium stimulus. CaMKII acquires this Ca2+‐independent activity at a threshold frequency and this property is implicated in long‐term potentiation (LTP). Indeed, transgenic mice expressing mutant versions of CaMKII have limited LTP and defects in learning and memory. We have previously shown that CaMKII has a remarkable property, which is that activation of CaMKII triggers the exchange of subunits between holoenzymes, including inactive ones, enabling the Ca2+‐independent activation of neighboring subunits. Our results have implications for an earlier idea that subunit exchange in CaMKII may have relevance for long‐term memory formation. We hypothesize that subunit exchange, along with a more recent finding that specific substrate binding may also potentiate activity, play a role in the importance of CaMKII in LTP. Here, we present a substrate interaction with CaMKII that may play a role in neuronal signaling. Additionally, we present findings that subunit exchange occurs between multiple isoforms of CaMKII, which may be important for changes in gene expression (a hallmark for LTP). POS230 Effects of Evolutionary Distance on Protein Dynamics, Antibacterial Activity, and Cytotoxicity in Members of the Ribonuclease 3 Subfamily David Bernard 1, Myriam Létourneau1, Donald Gagné1, Marie‐Christine Groleau1, Éric Déziel1, Nicolas Doucet1 1INRS ‐ Université du Québec, Canada Recent experimental evidence suggests that conformational exchange may affect catalytic function in a number of enzyme systems. However, the underlying mechanism(s) linking flexibility with biological function remains elusive. For instance, it is unknown whether protein sequence and/or structure are evolutionarily conserved to promote conformational exchange among functional enzyme homologs. Herein, we used a combination of functional assays and NMR relaxation experiments to characterize the catalytically relevant millisecond time frame in various members of the pancreatic‐like ribonuclease superfamily. To provide information on evolutionary conservation, dynamics, and biological function, we characterized mammalian homologs of human ribonuclease 3 (also known as Eosinophil Cationic Protein, or ECP), specifically focusing on monkey ECP homologs from Macaca fascicularis, Pongo pygmaeus, Pongo abelii, and Aotus trivirgatus. Our findings show that conformational exchange in the monkey enzymes strongly resembles that of their human counterpart, with subtle changes in exchange rates and/or structural localization, thus providing insights into the effects of sequence and phylogenetic diversity on protein dynamics. In parallel, antibacterial assays against E. coli and S. aureus illustrate that antimicrobial activity correlates with evolutionary distance from the common ancestor. Finally, cytotoxicity assays performed on HeLa cells highlight a stark difference in potency between human ECP, monkey enzymes, and the prototypical RNase A homolog. Altogether, these experiments provide further details on the potential interdependence between protein function and atomic‐scale flexibility. POS262 Distinct Binding Dynamics of HCV Helicase Mutants Provides Insight into the Mechanism of the Interaction with the Nucleic Acid Christopher Ablenas 1, Christopher Ablenas1, Hsiao‐Wei Liu2, Gonzalo Cosa1, Matthias Götte2 McGill University, McGill University, University of Alberta, McGill University, University of Alberta The dynamics of the interaction between the hepatitis C virus (HCV) NS3 helicase (NS3h) and the nucleic acid have been the subject of great interest given the key role of this enzyme in viral replication. To better characterize these dynamics in the steps preceding ATP‐fueled unwinding, here we describe a single‐molecule protein induced fluorescence enhancement (smPIFE) assay to monitor in real‐time the fluorescence enhancement induced by binding of purified recombinant NS3h to individual surface‐immobilized DNA substrates labelled with the cyanine dye Cy3. The smPIFE experiments revealed three enhancement levels that correspond to three discrete binding sites at adjacent bases on the ssDNA overhang of model substrates with no preference for the single‐stranded/double‐stranded junction. WT NS3h transitioned between binding sites in both directions without dissociation, while the R393A, V423A, and W501A mutants which display poor ATP‐dependent unwinding activity were severely compromised in this switching activity. In the presence of a non‐hydrolyzable ATP analogue, WT NS3h adopted a single binding site with no transitioning and the reduced affinity for the nucleic acid corresponded to a large decrease in the rate of association with little change in the rate of dissociation. Together, our data are consistent with a model that favors ATP‐independent sliding of NS3h on the single‐stranded overhang via a Brownian motor model. ATP binding and hydrolysis would then fuel the more processive and directional proposed inchworm‐like active unwinding process. POS265 Deconstruction of the Ras switching cycle through saturation mutagenesis reveals hot‐spots of allosteric activation Pradeep Bandaru 1, John Kuriyan1, Rama Ranganathan2 1UC Berkeley, USA, 2UT Southwestern Medical Center, USA The sequence of Ras is nearly invariant in vertebrates, despite the general tolerance of proteins to mutation. To define the functional constraints on the sequence of Ras, we developed a bacterial two‐hybrid selection system for Ras activity, and screened libraries containing all single‐site mutants of human Ras. Wild‐type Ras, regulated by a GTPase activating protein and a guanine nucleotide exchange factor, is relatively tolerant of mutation, except in the hydrophobic core and the binding elements for nucleotide, effectors, and regulators. Removal of the regulators reveals hot‐spots of activating mutations in wild‐type Ras, identifying residues that appear to act as latches on Ras dynamics. The activating effect of latch mutations is muted in an oncogenic variant of Ras (G12V), indicative of widely‐distributed epistasis within the structure. Evolutionary analysis, combined with the mutational analysis, suggests that vertebrate Ras has acquired additional allosteric control that distinguishes it from invertebrate Ras and close relatives such as Rap. POS290 Direct and Allosteric Mechanisms of KLK4 Inhibition Revealed by Structural and Dynamical Characterization Blake Riley 1, Olga Ilyichova1, Benjamin Porebski2, Joakim Swedberg3, Simon de Veer3, Kei Sit4, Itamar Kass1, Jonathan Harris4, David Hoke 1Monash University, Australia, 2University of Cambridge, UK, 3University of Queensland, Australia, 4Queensland University of Technology, Australia Kallikreins (KLKs) are a family of serine proteases important in development and normal physiology, and many have similar substrate specificities at P1 in the active site. Kallikrein‐4 (KLK4) is predominantly involved in normal prostatic physiology, but has also been implicated in development and metastasis of some cancers. Determining how KLK4's activity is regulated is critical to developing selective inhibitors, and hence potential cancer therapeutics. Atomic resolution (∼1.0 Å) X‐ray crystal structures were determined for KLK4 in complex with cyclic peptides based on sunflower trypsin inhibitor‐1 (SFTI‐1), and with nickel (Ni). Additionally, molecular dynamics (MD) simulations of these complexes and of selected SFTIs were performed to identify differential motions between the structures. In the Ni‐KLK4 structure, an alternate conformation of the Asn192‐Gly193 peptide bond in the oxyanion hole is observed, suggesting inhibition through destabilization of the oxyanion hole. An unstructured 70–80 loop region adjacent to the inhibitory metal binding site was also seen. MD simulations show that differential motions are seen upon metal binding and are transmitted to the active site. Our results provide a molecular rationalization for the potency and selectivity of these selected inhibitors, as well as an insight into indirect mechanisms of inhibition for KLK4 that are applicable to the serine protease superfamily. POS293 Mechanism of Myristoyl Switching by NMR measurements of local stability and conformational heterogeneity Duncan MacKenzie 1, Elizabeth Meiering1, Travis Ko1, Purnank Shah1, Mikaela Ney1 1University of Waterloo, Canada Hisactophilin is a myristoylated, histidine‐rich, pH‐dependent actin‐ and membrane‐binding protein. In response to cellular changes in pH, hisactophilin reversibly interconverts between cytosolic and membrane‐bound forms whereby the covalently linked N‐terminal C14 myristoyl group escapes the hydrophobic core of the ß‐trefoil fold to insert into a lipid bilayer. Despite the prevalence of myristoyl switches, the details and mechanism of communication between sites of protonation and the buried myristoyl moiety remain elusive. Mutational studies of the hisactophilin thermodynamics hinted that myristoyl switching is enabled by strain arising from an over‐packed hydrophobic core. In pursuit of strain, we sought atomistic insight with the temperature dependence of amide group chemical shifts by NMR, a tool to elucidate protein folding and functional mechanisms, which are readily measured and bountiful proxies for local structural stability and conformational heterogeneity. Some hydrophobic residues, notable for packing against the myristoyl group, exhibited nonlinear chemical shift temperature dependences which may indicate access to functionally relevant low‐energy alternative states. Complementary measures of local stability by analyzing linear temperature dependences highlighted areas throughout the protein, including those myristoyl‐adjacent, complicit in the strain in switching behavior. Mutating myristoyl‐proximate I85L, or a combination of F6L/I85L/I93L, ablates switching behavior, and measured temperature dependences further support the importance of local stability, strain, and conformational heterogeneity in driving hisactophilin's myristoyl switch. POS411 Probing allosteric communication with long‐range rigidity propagation across protein networks Adnan Sljoka1 1Kwansei Gakuin University, Japan Allostery can be viewed as an effect of binding at one site of the protein to a second, often significantly distant functional site, enabling regulation of the protein function. The molecular mechanisms and networks mediating distant communications that give rise to allostery are poorly understood. We have developed a rigidity‐transmission allostery (RTA) algorithm, a computational method based on mathematical rigidity theory. Starting with an X‐ray crystal structure or an ensemble of snapshots, we model the protein as a constraint network consisting of vertices (atoms) and edges (i.e., covalent bonds, electrostatic bonds, hydrogen bonds, and hydrophobic contacts). RTA algorithm provides a mechanical interpretation of allosteric signaling and is designed to predict if perturbation of rigidity (mimicking ligand binding) at one site of the protein can transmit and propagate across a protein network and in turn cause a transmission and change in conformational degrees of freedom at a second distant site, resulting in allosteric transmission. Presence of rigidity‐based allostery means that a change in shape (conformation) at one site (i.e. mechanically change the shape as binding might) would lead to change of shape and conformation of the second site. We will describe the RTA method and illustrate predictions of allosteric interactions on several PDB structures including GPCRs, an enzyme and others, and show how these predictions are in agreement to NMR chemical shift changes caused by allosteric propagations. RTA algorithm is computational very efficient (takes minutes of computational time on standard PC) and is useful in quantification of allosteric signals, mapping out of allosteric pathways and identification of novel allosteric sites. POS420 Structural Dynamics of SufS Cysteine Desulfurase Investigated by Backbone Amide Hydrogen/Deuterium Exchange Mass Spectrometry Patrick Frantom 1, Dokyong Kim1, Harsimran Singh1, Yuyuan Dai2, Guanchao Dong2, Laura Busenlehner1, Wayne Outten1 The University of Alabama, USA, 2University of South Carolina, USA Fe‐S clusters are essential cofactors required for cellular functions. Biogenesis of Fe‐S clusters in Escherichia coli under oxidative stress and iron depletion is carried out by the Suf system. Proteins involved in Suf pathway work together to mobilize sulfide and iron, form Fe‐S clusters, and regulate the assembly pathway by protein‐protein interactions. The E. coli cysteine desulfurase SufS and its accessory protein SufE work together to mobilize persulfide from L‐cysteine and transfer it to the scaffold protein SufB as part of the SufBC2D complex. The overall SufS reaction occurs in two half reactions: (1) transfer of sulfide from cysteine to form an enzyme bound persulfide intermediate along with release of alanine followed by (2) transfer of the persulfide to the accessory protein SufE. To better understand structural changes involved in promoting each half reaction, Apo‐SufS was treated with excess cysteine to create the persulfide intermediate (SufSper) and amide hydrogen deuterium exchange mass spectrometry (HDX‐MS) is used to investigate dynamic changes that occur upon persulfuration of SufS. HDX‐MS analysis shows that conformational changes occur at the SufS dimer interface (peptides 88–100 and 243–255) in addition to changes at the active site predicted by the SufSper crystal structure (peptides 225–236 and 356–366) (Figure 1A,B). Superposition of a structure containing a PLP‐bound product analog also indicates changes in the interactions of R92, E96, and E250 along the dimer interface of SufS, correlating with the HDX‐MS results (Figure 1C). Site‐directed mutagenesis of these residues is currently underway to better elucidate their role in structural changes regulating catalysis in the SufS/SufE system. POS437 On the dynamics of interleukin‐36RA; a key player in psoriasis Nicholas Tiee 1, Patricia Jennings1, Kendra Hailey1 1Department of Chemistry and Biochemistry, University of California San Diego, USA The interleukin‐1 family (IL‐1) of cytokines regulates both the innate and adaptive immune responses, and misregulation manifests in autoimmune disorders and death. The most recent familial addition is the quintet of IL‐36 proteins (IL‐36a, ß,ϒ agonists, IL‐36Ra antagonist and the IL‐36 receptor). Specifically, general pustular psoriasis (GPP), a life threatening form of psoriasis has been linked to point mutations in IL‐36Ra. The IL‐36 family all appear to be processed from the N‐terminus, which drastically affects binding affinities and inflammatory activity. Oddly IL‐36Ra has been reported to have no inhibitory activity without any N‐terminal processing; however as opposed to other members in the family, only the N‐terminal methionine is cleaved. This result is startling as it shows that a single residue cleavage is able to turn off the inhibitory pathway completely. Using NMR structure determination, suites of dynamics studies over an array of timescales, as well as receptor binding experiments we aimed to identify the molecular mechanisms that were responsible for the differences in processed and pre‐processed IL36Ra. Through our studies we have been able to observe allosteric mechanisms that transfer information from the processed N‐terminus to the opposite side of the molecule responsible for receptor interaction. POS453 Contribution of Global Dynamics to Catalysis by Horse Liver Alcohol Dehydrogenase Bryce Plapp 1, Karthik Shanmuganatham1, Rachel Wallace1, Ann Lee1 1The University of Iowa, USA Do fast global protein dynamics participate in catalysis of the hydride transfer by alcohol dehydrogenase? If so, substitution of buried, conserved amino acid residues distal from the active site should alter the stability and dynamics of the protein and affect catalysis. Changes in fast dynamics can be reflected in the X‐ray crystallographic temperature factors and the enzyme kinetics. Five different amino acid residues were substituted by partially random site‐directed mutagenesis, and the structures of the enzyme‐NAD+‐alcohol complexes were determined by X‐ray crystallography at high resolution. The G173A; V197I; I220V, L or F; V222I; and F322L enzymes have almost identical structures, except for local perturbations at the site of substitution, as compared to wild‐type enzyme. These enzymes have very similar kinetic constants for oxidation of benzyl alcohol and reduction of benzaldehyde, and the rates of conformational changes are not apparently altered. Other single substitutions of these amino acid residues decreased expression, and thus it appears that the five residues are in critical locations that can affect protein stability and dynamics. We conclude that alcohol dehydrogenase can tolerate conservative, distal substitutions that do not affect the scaffold of the protein and that fast, global dynamics do not participate significantly to catalysis. Supported by NIH grant GM078446. POS476 Origin of Negative Relationship Between Thermodynamic Stability and Actin Binding Function of Tandem CH Domains Krishna Mallela 1, Surinder Singh1, Swati Bandi1 1University of Colorado Skaggs School of Pharmacy and Pharmaceutical Sciences, USA Tandem calponin‐homology (CH) domains are the most common actin binding domains in proteins, yet their structure‐function relationship is less understood. Such molecular knowledge might help in understanding the disease triggering mechanisms of muscle diseases such as muscular dystrophy. Recent studies on tandem CH domains of dystrophin, utrophin, alpha‐actinin, filamin, and spectrin have shown a surprising inverse relationship between their thermodynamic stability and actin binding function. Many mutants that destabilized the protein structure have increased actin‐binding affinity. We probed the origin for such negative relationship between stability and function by examining the properties of individual CH domains of utrophin and the role of inter‐CH‐domain interactions. The isolated N‐terminal CH1 domain is quite unstable and exists as a molten globule, when compared to the more stable tandem CH domain and the C‐terminal CH2 domain. The CH1 domain binds to F‐actin with higher affinity compared to the tandem CH domain and the CH2 domain. These results suggest that the inverse relationship between the stability and function of tandem CH domains may originate from the nature of individual CH domains. Isolated CH1 is less stable but binds to F‐actin with higher affinity. Isolated CH2 is more stable but binds to F‐actin with lesser affinity. In addition, the tandem CH domains and their mutants that favor closed conformation with stabilizing inter‐CH‐domain interactions bind to F‐actin weaker than those that favor the open conformation, further indicating that the interdomain interactions hinder tandem CH domains binding to actin. POS480 Arginase Of Helicobacter Gastric Pathogens Utilizes A Unique Non‐Catalytic Triad For Catalysis; A Possible Strategy To Develop Specific Inhibitors Apurba Sau 1, Ginto George1, Nikunj Raninga1, Mamata Kombrabail2 1National Institute of Immunology, India, 2Department of Chemical Sciences, Tata Institute of Fundamental Research, India Helicobacter pylori arginase, a bimetallic enzyme is crucial for pathogenesis of the bacterium in human stomach. Despite conservation of the signature motifs in all arginases, the Helicobacter pylori homologue has a non‐conserved motif (153ESEEKAWQKLCSL165), whose role was recently shown to be critical for its stability and function. The sequence analysis also reveals the presence of this motif with critical residues in the homologue of other Helicobacter gastric pathogens. However, the underlying mechanism for its significance in catalytic function is not properly understood. Using H. pylori arginase, we show that the interactions of His122 and Tyr125 with Trp159 are indispensable for tertiary structural intactness through optimal positioning of the motif and thus for the catalytic function. The single and double mutants of His122 and Tyr125 not only enhanced the solvent accessibility and conformational flexibility of Trp159, but also showed complete loss of catalytic activity. An intact bimetallic center and unaltered substrate binding indicate that proper positioning of the motif by aromatic‐aromatic contact is vital for the generation of a catalytically active conformation. Additionally, the metal ions provide higher stability to the holo protein. We also identified the presence of these two residues exclusively in arginase of other Helicobacter gastric pathogens, which may have similar function. Our findings therefore highlight for the first time that arginase of all Helicobacter gastric pathogens utilizes a unique non‐catalytic triad for catalysis, which could be exploited for therapeutics. POS522 The SIS Fold GlcN6P Deaminase: Mechanisms of Cooperativity and Allostery Jorge Angel Marcos Viquez 1, Andrea Celeste Medina García1, Mario L. Calcagno Montans1, Ismael Bustos Jaimes1, Annia Rodríguez Hernández2, Adela Rodríguez Romero2 1Biochemistry Department, Medicine School, National Autonomous University of Mexico, Mexico, 2LANEM, Institute of Chemistry, National Autonomous University of Mexico, Mexico Glucosamine‐6‐phosphate deaminase (GlcN6PD, E.C. 3.5.99.6) catalyzes the isomerization‐deamination of glucosamine 6‐phosphate (GlcN6P) releasing fructose 6‐phosphate (Fru6P) and ammonium ion (NH4+). In E. coli, it is a regulatory metabolic step because of both its positive cooperativity and its allosteric K activation by N‐acetylglucosamine 6‐phosphate (GlcNAc6P). Most of the studied GlcN6PD enzymes share the same Rossmann‐like fold. Nevertheless, some GlcN6PD with Sugar‐Isomerase fold (SIS fold) have been found, displaying similar regulatory properties based on entirely different mechanisms. We have worked on the structural and functional characterization of SIS fold deaminases (SIS‐GlcN6PD) from Shewanella oneidensis and Shewanella denitrificans. The location of the protein binding sites was identified by molecular docking and subsequently verified by site‐directed mutagenesis. The expected binding stoichiometry was confirmed by direct binding experiments using H3‐GlcNAc6P, and by Isothermal Titration Calorimetry (ITC). Positive cooperativity and allosteric activation of these enzymes depend on different molecular mechanisms since both functions can be dissociated by site‐directed mutagenesis. Herein we present the studies of the molecular quaternary transition of SIS‐GlcN6PDs associated with the allosteric activator binding and the communication between the substrate binding sites, both by X‐ray crystallography and molecular dynamics. In conclusion, the enzymes show a single allosteric site per dimer located in the intersubunit space. In contrast, each subunit presents two binding sites for the substrate: the conserved catalytic one and an emergent regulatory one, which modulates the cooperative behavior. 08. ENZYMOLOGY POS065 Comparative study of degradation efficiencies of various emerging pollutants by different peroxidases Syed Salman Ashraf1 1UAE University The ever‐increasing presence of the “emerging pollutants” in the environment is becoming a serious environmental and health issue. One of the major classes of these very dangerous pollutants is “pharmaceuticals and personal care products” (PPCPs), a growing family of chemicals that include antibiotics, anti‐inflammatory drugs, oral contraceptives, etc. and are found in high concentrations in water supplies (e.g. field concentration of Ibuprofen can be as high as 12 ug/L). Amongst the new and novel approaches to degrade these PPCPs in water bodies are the use of enzymes called Peroxidases, which have recently been shown to hold promise in bioremediation applications. However, peroxidases are a large family of enzymes and different types of peroxidases may behave very differently towards different PPCPs. The current study focused on first of its kind comparative study to examine the efficiencies of three different peroxidases (Soybean Peroxidase, Chloroperoxidase, and Lactoperoxidase) to degrade 10 different classes of PPCPs (e.g. antibiotics (Sulfamethoxazole) and anti‐inflammatory (Ibuprofen)). Our results show that these peroxidases showed remarkably different degradative abilities, such that some (like Chloroperoxidase) seemed to be very promiscuous and could degrade most of the PPCPs. Additionally, we show that the use of redox mediators were able to dramatically increase the degradation of some otherwise recalcitrant PPCPs. These results are the first study in which three different peroxidases are directly compared for their abilities to degrade 10 different emerging pollutants. POS080 Characterization of Intra‐Melanosomal Domain of the Recombinant Human Tyrosinase Related Protein 1 Monika Dolinska 1, Yuri Sergeev1 1OGVFB, NEI/NIH, USA Tyrosinase Related Protein 1 (Tyrp1), transmembrane glycoenzyme involved in melanin synthesis pathway exhibits significant homology with tyrosinase and dopachrome tautomerase, enzymes played an important role in pigment formation. Although Tyrp1 is the most abundant among them in melanosomes, its specific role in human melanogenesis is not fully known. Up to date efforts to decipher Tyrp1 function have been mostly limited to melanoma cells and murine model studies. Here, we study in vitro the recombinant intra‐melanosomal domains of human tyrosinase related protein 1 (hTyrp1Ctr) and human tyrosinase (hTyrCtr), which were expressed in baculovirus and produced in whole T.ni larvae, then purified in milligrams quantities using the IMAC and size exclusion chromatography (SEC). Both proteins contain Cu2+ atoms as shown by inductively coupled plasma‐mass spectroscopy, however, hTyrCtr demonstrates its diphenol oxidase enzymatic activity using L‐DOPA as a substrate, while hTyrp1Ctr do not show same activity and does not oxidizes DHICA, as murine Tyrp1 does. Using SEC and sedimentation equilibrium, we show that both, hTyrCtr and hTyrp1Ctr behave as monomeric proteins and there do not form stable hetero‐dimers or any other hetero‐oligomers among themselves, in contrast to in vivo studies in mouse cells, which proved Tyrp1/Tyr hetero‐dimeric complexes formation. Nonetheless, 43°C incubation of excessive amount of hTyrp1Ctr with hTyrCtr increases protection of tyrosinase stability over the time. Those observations suggests that although hTyrp1Ctr is important for tyrosinase stabilization under above conditions, its mechanism is not based on formation of stable hetero‐oligomers between those two proteins. POS083 Kinetic And Structural Characterization of KabA, Glutamate Aminotransferase Involved in The Production of Kanosamine from Bacillus Cereus Theerawat Prasertanan1, David Sanders1 1Department of Chemistry, University of Saskatchewan, Canada Our laboratory recently discovered a new biosynthetic pathway in Bacillus spp that generates kanosamine, a key component of many antibiotics. We have studied the structure and function of the enzymes of this pathway, which make kanosamine from glucose 6‐phosphate. KabA is a pyridoxal 5’‐phosphate (PLP)‐dependent aminotransferase that catalyzes the conversion of 3‐keto‐glucose‐6‐phosphate and L‐glutamate to kanosamine‐6‐phosphate (K6P) and 2‐oxoglutarate. Here we report the first steady‐state kinetic study of KabA. Using a fluorescence‐based coupled‐enzyme assay, a kinetic constants for the ping‐pong kinetic mechanism were determined. High‐resolution X‐ray crystal structures in the presence and absence of K6P show the internal and external aldimines, establishing Lys‐254 as the key active site residue required for Schiff‐base ligation of PLP, and indicating all important interactions between KabA and the aminoglycoside substrate. POS086 The Thioredoxin System from the Thermophilic Bacterium Thermosipho africanus: Structure and Function Naheda Sahtout 1, David A. R. Sanders1, Jijin Raj Ayanath Kuttiyatveetil1 1University of Saskatchewan, Canada The thioredoxin system is a ubiquitous oxidoreductase system that consists of the enzyme thioredoxin reductase (TrxR), the protein thioredoxin (Trx) and the cofactor NADPH. The system has been comprehensively studied from many organisms, such as Escherichia coli (E. coli); however, structural and functional analysis of this system from thermophilic bacteria has not been as extensive. Thermosipho africanus (T. africanus) is a thermophilic bacterium; strain TCF52B was isolated from a high‐temperature oil reservoir in the North Sea. Analysis of the complete genome sequence of T. africanus strain TCF52B, suggested the presence of two putative Trxs (TaTrx1 and TaTrx2) and a TrxR (TaTrxR) as components of its thioredoxin system. In this study, TaTrx1 and TaTrxR have been successfully cloned, overexpressed and purified and characterized using biophysical techniques, biochemical assays and X‐ray crystallography. Our studies have indicated, not surprisingly, that TaTrx1 and TaTrxR are far more stable than the thioredoxin system components of E. coli. Consistent with these results, kinetic assays indicated that TaTrxR had a higher optimal temperature (70°C) for activity, compared to E. coli TrxR (EcTrxR, 55°C). Furthermore, TaTrxR was found to be catalytically more efficient at its optimal temperature than at room temperature (7 X) or at 10°C (255 X); a trend not observed with EcTrxR. To understand and identify the differences that may contribute to these results, X‐ray crystallography was used to determine the structure of TaTrx1 and TaTrxR. The phase problem for TaTrx1 was effectively solved using S‐SAD (Sahtout et al. 2016). The crystal structure of TaTrxR was successfully solved using molecular replacement. POS089 Recognition and cleavage of corn defense chitinases by fungal polyglycine hydrolases Todd Naumann 1, Neil Price1, Marcia Chaudet2, David Rose2 1ARS‐NCAUR, USA, 2Department of Biology, University of Waterloo, Canada Polyglycine hydrolases are secreted fungal endoproteases that cleave peptide bonds in the polyglycine interdomain linker of ChitA chitinase, an antifungal protein from domesticated corn. Polyglycine hydrolases are novel proteins in terms of activity and sequence. The objective of the study is to understand how they recognize their substrates and cleave featureless polyglycine sequences. Multiple proteases, plant chitinase substrates, and mutant forms of each were produced recombinantly. SDS‐PAGE and MALDI‐TOF/MS based in vitro protease assays were performed. We identified the catalytic serine within an SXXK motif, indicating that the active site is related to that of bacterial beta‐lactamases. Analysis of reaction products from assays with plant chitinases and analog peptides showed that this active site plays a limited role in substrate specificity. Through mutagenesis studies we have found that seven invariant tryptophans are crucial for polyglycine hydrolase folding and stability. We also found protease mutations that change cleavage site preference. In summary, the activity of these novel proteases has been characterized and we are mapping these activities to the amino acid sequence. POS102 Role of cystathionine ß synthase module in Trypanosoma brucei GMP reductase Akira Imamura 1, Takuya Otani1, Takuya Otani1, Manatsu Tamura1, Tomoka Kobayashi1, Asami Shibata1, Tetsuya Okada1, Shigenori Nishimura1, Takashi Inui1 1Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Japan GMP reductase (GMPR) is involved in purine salvage pathway, and catalyzes the reductive deamination of GMP to IMP. Our recent study revealed that TbGMPR possesses cystathionine ß synthase (CBS) module, which is absent in mammalian GMPRs. In this study, we evaluated the effect of purine nucleotides on TbGMPR activity and their binding affinity to the CBS module of TbGMPR. Furthermore, we prepared a CBS module‐deletion mutant of TbGMPR (TbGMPRΔCBS), and characterized its enzymatic properties to investigate the contribution of the CBS module to TbGMPR activity. The fluorescence quenching assays were performed to evaluate the binding affinity of purine nucleotides to TbGMPR by utilizing the tryptophan residue (Trp120) uniquely localized in the CBS module. Each of GTP, ATP and AMP induced the fluorescence quenching, showing that they bound to the vicinity of Trp120 in the CBS module. In the absence of these nucleotides, Hill coefficient (nHill) of TbGMPR for GMP was 3.1, demonstrating that GMP induced a positive cooperativity effect on the enzymatic activity. In contrast, nHill of TbGMPRΔCBS for GMP in the absence of other purine nucleotides was 1.2, indicating that the deletion of CBS module in TbGMPR caused a loss of positive cooperativity effect. Addition of GTP decreased Km GMP and increased kcat, while addition of ATP or AMP increased Km GMP without altering nHill and kcat. These results suggest that GTP, ATP and AMP bind to the vicinity of Trp120 in the CBS module of TbGMPR and alter the affinity between GMP and TbGMPR to regulate the enzymatic activity. POS122 Anion Inhibition of PEPCK Manifested as Substrate Inhibition; Using Crystallographic Methods to Determine Thermodynamic Data Matt McLeod 1, Todd Holyoak1 1University of Waterloo, Canada Phosphoenolpyruvate carboxykinase (PEPCK) has traditionally been labelled as the enzyme responsible for the first committed step towards gluconeogenesis and is responsible for the reversible conversion of oxaloacetic acid (OAA) to phosphoenolpyruvate (PEP). Recently, this metabolic enzymes role has been greatly expanded as it has been implicated in cancer, Mycobacterium tuberculosis infection, glucose stimulated insulin secretion, aging, and general TCA cycle flux regulation. Many biochemical characterizations have been completed on various isozymes of PEPCK, and one kinetic phenomena has been identified as substrate inhibition. Our study has shown that this substrate inhibition is actually a manifestation of the kinetic assay conditions, in which bicarbonate (as a source of CO2) is used. High concentrations of bicarbonate seemed to inhibit the enzyme. Upon further investigation, it was shown that the inhibition is a more general inhibition by anionic species. This information can give context regarding the preferred direction of catalysis in vivo, towards the production of PEP. Using macromolecular crystallographic techniques, we have determined inhibition constants of some anions that correspond with experimentally derived parameters. POS137 X‐ray crystallography reveals mechanism for two gain‐of‐function clinical variant carbapenemases from Acinetobacter baumannii Cynthia June 1, Christopher Russell1, Emma Schroder1, Rachel Powers1, Agnieszka Szarecka1, David Leonard1 1Grand Valley State University, Michigan, USA Evolution of carbapenemases can increase the level of antibiotic resistance of dangerous Gram negative bacteria such as Acinetobacter baumannii. OXA‐66 is a carbapenemase with weak hydrolytic activity against ß‐lactam substrates, and thus low potential for harm. The presence of single substitutions in OXA‐66 such as P130Q (OXA‐109) and L167V (OXA‐82) has been shown to increase affinity for carbapenem substrates such as doripenem, and is associated with increased levels of resistance against those potent drugs. Using X‐ray crystallography, we have solved the structures of OXA‐109 and OXA‐82 with doripenem bound as an acyl‐intermediate. In both cases, the substitution results in the rotation of a key active site isoleucine side‐chain that normally hinders the binding of doripenem, and thus provides an explanation for the gain‐of‐function kinetic results observed. Molecular dynamics simulations confirm that interactions between L167, P130 and I129 are sufficient to affect the orientation of I129 in this manner. POS138 A class D ß‐lactamase clinical variant from Acinetobacter baumannii that possesses an unusually high turnover rate for cephalosporins Jonika Forbes‐Benjamin 1, Cynthia June1, Joshua Mitchell1, Rachel Powers1, David Leonard1 1Grand Valley State University, Michigan, USA Class D ß‐lactamases pose a serious threat to the treatment of infections caused by Gram negative bacteria. We investigated the biochemical properties of OXA‐139, a class D clinical variant that differs from A. baumannii OXA‐24/40 carbapenemase by a single substitution (N87I). After expressing and purifying the variant, we used steady‐state kinetic analysis to compare the kinetic properties of OXA‐139 and OXA‐24/40. We discovered that the N87I substitution has a different effect on the turnover rate (kcat) depending on the substrate used. Increases in kcat values were observed for the cephalosporins CENTA (∼100‐fold above OXA‐24/40), ceftriaxone (∼60‐fold) and the carbapenem doripenem (∼ 5‐fold). The carbapenem imipenem saw a decrease in kcat of ∼ 20‐fold, while the kcat for aztreonam was unchanged. Km values were only modestly changed compared to OXA‐24/40. To explore a potential mechanism for the differential effect of N87I on the turnover rates, we used x‐ray crystallography to determine the structure of OXA‐139 and OXA‐24/40 in the absence and presence of various substrates. The results show that N87 is normally involved in stabilizing an unusual N‐carboxylated lysine that acts as a general base in the catalytic mechanism. Thus, the N87I substitution has the potential to disrupt the carboxylation state of this important catalytic residue. This direct modulation of the stability of the carboxylysine is a novel mechanism by which clinical substitutions increase the activity (and thus the potential threat) of these enzymes. POS173 Refining the Sequence Signature of Bacterial Laccases Robert Collins1 1Eastern Connecticut State University, USA Laccases are copper oxidases that directly oxidize phenolic compounds, and indirectly oxidize other substrates through redox mediators. Laccases are used industrially and have shown promise in pilot applications including wastewater treatment, enzymatic synthesis, and enzymatic fuel cells. Fungal laccases have the highest redox potential and broadest range of substrates, but have not been recombinantly expressed in bacteria. To tap the potential of this class of enzymes in a cost‐effective manner, focus has turned on microbial laccase‐like proteins. Microbial copper oxidases include laccase‐like multi copper oxidases (LMCO), but also include other oxidases with lower redox potentials and limited substrate ranges (e.g. bilirubin oxidase, catechol oxidase). As the microbial enzymes share low sequence identity (less than 20%) with fungal laccases, the signature motifs that distinguish enzymes with industrial potential from less desirable enzymes remain elusive. Most databases include low redox and non‐laccase copper oxidases in sequence alignments, eroding sequence signatures of laccases. To remedy this, I aligned curated sequences of characterized bacterial and fungal laccases. Conserved motifs were used to search archaeal and bacterial genomes. Diverse putative laccases were selected for gene synthesis and expression in E. coli. The successful expression of bacterial enzymes with laccase‐like activity indicates the sequence motifs identified can discern laccases. Each ‘hit’ helps further refine the search. Biochemical and biophysical characterization indicates stable enzymes with broad specificities on model substrates have been isolated. This project was designed for an undergraduate biochemistry lab course. POS198 The role of residues C301 and C303 in the active site of human ALDH2 in the inactivation by lipid peroxidtion products Luis Francisco Calleja Castañeda 1, José Rodríguez Zavala1 1Instituto Nacional de Cardiología, Mexico Aldehyde dehydrogenases (ALDHs) are responsible for detoxifying aldehydes generated during lipid peroxidation by their oxidation into their corresponding carboxylic acid. Though, lipid aldehydes inactivate these enzymes. ALDH3A1 is 50–100 times more resistant to inactivation by lipid aldehydes compared with ALDH1A1 and ALDH2. Analysis of the amino acids sequence of the aldehyde binding site showed that enzymes which are more susceptible to lipid peroxidation products (ALDH1 and ALDH2) have cysteine residues near the reactive Cys. Based on these criteria and considering that these aldehydes react preferentially with cysteine residues, we generate two mutated enzymes of ALDH2 by changing the Cys residues adjacent to the reactive Cys. In the first one, C303V residue was changed, while in the second one the residues C301T and C303V were changed. Both mutant enzymes were resistant to the inactivating effect of acrolein, 2‐hexenal, 4‐HNE, and 2‐nonenal; and their catalytic efficiency for lipid aldehydes did not change significantly compared to the wild‐type enzyme. However, the double mutant presents a modification of the rate‐limiting step of the reaction. Finally, we evaluated the effect of double mutant expression on Escherichia coli cells exposed to hydrogen peroxide. Cells expressing the mutant enzyme were more resistant than those expressing the wild‐type enzyme; indicating that the expression of the mutant ALDH2C3031T‐C303V protects the cell from toxic aldehydes generated from lipid peroxidation promoted by exposure to oxidizing agents. These data revealed that Cys residues near to the reactive Cys in ALDH2 are important in the inactivation process induced by lipid aldehydes POS235 Cardioprotective effect of piperlonguminin through the activation and stabilization of the mitochondrial aldehyde dehydrogenase ALDH2 Belem Yoval Sánchez 1, José Rodríguez Zavala1 1Instituto Nacional de Cardiología, Mexico Excessive generation of ROS during the ischemia‐reperfusion events in different cardiovascular diseases exacerbates the peroxidation of polyunsaturated fatty acids present in biological membranes. These lipid hydroperoxides subsequently form secondary toxic product such as 4‐HNE (4‐hydroxy‐2‐nonenal). When these aldehydes are accumulated in the cell, the cardiac function is compromised due to its reaction with different biomolecules that progressively damage the mitochondrial function. Thus, the participation of aldehyde dehydrogenases (ALDHs) in the detoxification of aldehydes is crucial to maintain cell function. Therefore, the diminution of aldehydes content generated in the heart during an ischemia‐reperfusion event, through the activation and stabilization of ALDH2, may contribute to the preservation of cell integrity. To date, only Tamoxifen and ALDA‐1 have been characterized as activators of ALDHs; however these compounds may have potential toxic effects. Currently, we are characterizing the effect of piperlonguminine (PPLG), a compound structurally similar to ALDA‐1 as activator of ALDH2. Studies in vitro showed that this compound increased the activity of ALDH2 and protected to the enzyme against inactivation by the toxic aldehydes as 4‐HNE. Based on these results, we evaluated the protective effect of this compound in a model of myocardial infarction. In this regard, it was observed that the damage by ischemia‐reperfusion periods is prevented by administering PPLG, restoring blood pressure and heart rate, which correlated with a lower content of lipid aldehydes and the preservation of the activity of ALDH in the tissue. Therefore, we suggest that activation of ALDH2 by PPLG may help to diminish the damage generated in other pathologies involving high levels of oxidative stress POS277 Automated High‐throughput Fucosyltransferase Inhibition Assay on a Chip Laura Leclerc 1, David Kwan1, Steve Shih1 1Concordia University, Canada The structure and abundance of different cell‐surface carbohydrates— or glycans— heavily influence cellular signalling mechanisms that control cell behavior, growth, and death. In many cancers, modification of these glycans by fucosylation— the addition of a fucose sugar residue— results from the upregulation of fucosyltransferase enzymes. Fucosylation of cell‐surface glycans can have many downstream effects in cancer development. For example, one such fucosylated glycan, called sialyl‐Lewis X, promotes higher metastatic potential and malignancy. In order to screen for potential inhibitors of the fucosyltransferases involved in the assembly of sialyl‐LewisX, we have developed a fluorescence‐based inhibition assay for the fucosylation of a labeled synthetic oligosaccharide. Upon treatment with specific glycosidase enzymes, hydrolysis of this oligosaccharide releases fluorescent 4‐methylumbelliferone. However, fucosylation of the labeled oligosaccharide prior to this treatment results in a structure that is not recognized by the glycosidases, preventing hydrolysis and its associated fluorescent signal. We demonstrate that this assay can be used to detect the inhibition of a fucosyltransferase, since blocking fucosylation will allow glycosidase‐catalyzed hydrolysis of the labeled oligosaccharide to produce a fluorescent signal. We aim to harness this inhibition assay on a microfluidic platform which combines digital and droplet microfluidics to achieve precise, automated, high‐throughput, low‐cost drug discovery. POS281 Catalytically Important Remote Residues of E. coli Ornithine Transcarbamoylase that Contribute to Electrostatic and Dynamic Properties of the Enzyme Lisa Ngu 1, Jenifer Winters1, Kien Nguyen1, Kevin Ramos1, Nicholas DeLateur1, Paul Whitford1, Lee Makowski1, Penny Beuning1, Mary Jo Ondrechen1 1Northeastern University, Massachusetts, USA Ornithine transcarbamoylase (OTC) is an important enzyme of the urea cycle that converts excess ammonium to urea and is important in arginine biosynthesis. OTC catalyzes the reaction of carbamoyl phosphate (CP) and ornithine (ORN) to produce citrulline and inorganic phosphate. We have applied Partial Order Optimum Likelihood (POOL), a machine learning computational tool developed at Northeastern University, to predict catalytically important residues. Unlike purely informatics‐based approaches, POOL uses the 3D structure of a protein and computed electrostatic properties to accurately predict residues important for enzyme activity, including those remote to the substrate. POOL predicts a spatially extended active site for E. coli OTC, for which an induced‐fit conformational change upon binding of CP is believed to play a role in its catalytic mechanism. Conserved mutations of POOL‐predicted residues Arg57, Asp140, Tyr160, His272 and Glu299 and kinetics assays of these variants show significant loss of catalytic efficiency relative to wild‐type OTC. In order to further understand how these residues play a role in OTC catalysis, MD simulations of WT and variants have been performed to determine if mutations at these positions affect the conformational dynamics of the enzyme. POS286 Structural and functional studies of PINK1: The First ubiquitin kinase Shafqat Rasool 1, Jean‐Franois Trempe1, Kalle Gehring1 1McGill University, Montreal, Canada Mutations in PINK1 (PARK6 gene) and Parkin (PARK2 gene) are associated with the autosomal recessive form of Parkinson's disease. PINK1 is a protein kinase, best known for its role in signaling mitochondrial damage and consequently initiating mitochondrial repair or autophagy mechanisms. Upon mitochondrial damage, PINK1 accumulates on the outer membrane (OMM) of the mitochondria as an active kinase, and recruits the E3 ubiquitin ligase, Parkin, which ubiquitinates multiple OMM proteins and signals mitochondria for autophagic destruction. In recent years, the mechanism for Parkin's recruitment and activation has been a subject of extensive study in the context of PINK1's kinase activity. The most recent evidence suggests the role of PINK1 in directly phosphorylating both Parkin (on its ubiquitin‐like domain) and ubiquitin for a complete activation of Parkin, making PINK1 the first known ubiquitin kinase. However the underlying molecular mechanisms underlying Parkin's activation are unknown. We have used PINK1 from the insect species Tribolium castaneum (TcPINK1) to characterize the kinetics of Parkin and ubiquitin phosphorylation and its consequences for the activation of Parkin. Our recent work elucidates the role of phosphorylated ubiquitin (pUb) as an enhancer for Parkin phosphorylation by PINK1, hence establishing a role of pUb in the mitochondrial quality control pathway. NMR studies and phosphorylation assays also reveal residues in Parkin that are critical for the interactions with PINK1. POS291 Inhibition of Pancreatic Lipase by Phenolic Compounds from Mango: Enzyme Kinetics and Molecular Interactions Elena Moreno‐Cordova 1, Aldo Arvizu‐Flores1, Karina Garcia‐Orozco1, Elisa Valenzuela‐Soto1, Fernando Ayala‐Zavala1, Abraham Wall‐Medrano2, Emilio Alvarez‐Padilla2, Gustavo González‐Aguilar1 1Centro de Investigación en Alimentación y Desarrollo, Universidad de Sonora, Mexico, 2Universidad Autónoma de Ciudad Juárez, Mexico Pancreatic triacylglycerol lipase (PTL, EC 3.1.1.3) is crucial for dietary triglyceride digestion, considered a therapeutic target in obesity treatment since its inhibition can reduce the intestinal absorption of fats. Recent studies showed that phenolic compounds (PC) are potential natural PTL inhibitors; however, the molecular mechanisms supporting this effect are not well understood. In this work, we evaluated the ability of PC from mango (Mangifera indica L.) to inhibit PTL activity, through enzyme kinetics, fluorescence quenching and molecular docking. Seven PC from mango were assessed against porcine PTL as a model enzyme due to its high similarity to human PTL. The results showed that tannic acid (TA) and penta‐O‐galloyl‐ß‐D‐glucose (PGG) were the most effective PTL inhibitors (IC50 values 22.4 µM and 64.6 µM, respectively). Moreover, inhibition kinetic assays indicated that both compounds were uncompetitive inhibitors of PTL, showing low Ki values (PGG 0.0094 µM and TA 0.018 µM), which suggests a high affinity towards the enzyme. Furthermore, PGG and TA quenched PTL intrinsic fluorescence at a concentration dependent manner and caused red shifts on the enzyme's emission maxima, implying more tryptophan residues exposed to a more polar environment. Finally, molecular docking analysis showed that TA and PGG may bind to PTL‐colipase complex in its open‐ active conformation at the interface formed by PTL C‐terminal and lid domains, and colipase, which is supported by the experimental findings. This research highlights the potential of TA and PGG found in mango peel to be used as nutraceuticals for PTL inhibition and obesity treatment. POS295 Binding of (‐)‐epigallocatechin‐gallate to porcine trypsin followed by isothermal titration calorimetry and enzyme kinetics Aldo Arvizu‐Flores 1, Manuel Carretas‐Valdez2, Elena Moreno‐Cordova11, María Moreno‐Vásquez2, Abril Graciano‐Verdugo1 1Departamente de Ciencias Químico Biológicas, Universidad de Sonora, Mexico, 2Departamento de Investigación y Posgrado en Alimentos, Unviersidad de Sonora, Mexico Trypsin, as a member of the serine‐protease family, is a widely studied enzyme from the structure and function relationship. Since the increased interest in health from the functional polyphenolic compounds like (‐)‐epigallocatechin‐gallate (EGCG), many researchers looked for the molecular target of these compounds. In this work, we used isothermal titration calorimetry (ITC) and enzyme kinetics to assess the binding of EGCG to porcine trypsin as a model enzyme. ITC was conducted on a VP‐ITC microcalorimeter at 25°C, where trypsin was titrated with several aliquots of EGCG. Binding of EGCG to porcine trypsin was enthalpically favored, whereas the entropy change was negligible. EGCG showed high affinity for trypsin binding with a Kd of 17 µM. Inhibition kinetics showed a non‐competitive inhibition mechanism using Na‐benzoyl‐L‐Arg ethyl ester (BAEE) as substrate. The kinetic data indicate that EGCG binds to an alternate site that blocks the enzyme for catalysis. These data suggest that multiple interactions are formed between porcine trypsin and EGCG, but the conformational change required for catalysis is affected. ITC and enzyme kinetics were correlated and give insight to the molecular basis of EGCG inhibition to digestive enzymes that can be extrapolated to the human trypsin. POS299 Structural And Functional Characterization Of Polysaccharide Lyase Family Enzymes (PL24 & PL25) ThirumalaiSelvi Ulaganathan 1, Miroslaw Cyglef1, Ehud Banin2, William Helbert3 1University of Saskatchewan, Canada, 2Bar‐Ilan University, Israel, 3Institut de Chimie Moléculaire de Grenoble, France Ulvan is a complex sulfated polysaccharide biosynthesized by marine green algae and constitutes one of the two major polysaccharides of their cell wall. This water‐soluble polysaccharide comprises of predominantly 3‐sulfated rhamnose (R3S), glucuronic acid (GluA), iduronic acid (IdoA) and xylose. The physicochemical and biological properties of ulvan make it of interest for numerous industrial applications. Bacteria cohabiting with the green algae contain enzymes able to degrade ulvan by a lytic ß‐elimination mechanism. Genes coding such lyases have been discovered in the genomes of several bacteria. Pseudoalteromonas sp. strain PLSV gene PLSV_3936 encodes an ulvan lyase that cleaves the glycosidic bond between 3‐sulfated rhamnose (R3S) and glucuronic acid (GluA) or iduronic acid (IdoA). Another ulvan lyase, discovered in Alteromonadales and encoded by the gene LOR_107, degrades ulvan endolyticaly cleaving the bond between the rhamnose‐3‐sulfate and glucuronic acid. We have characterized biochemically these two lyases and determined their three‐dimensional structures. They represent the first structures of lyases capable of degrading ulvan. In spite of only 17% sequence identity, these two enzymes share the same 7‐bladed ß propeller fold. The putative active site was identified from structure conservation and confirmed by mutagenesis and structures of these enzymes with bound tetrasaccharide substrates. The catalytic residues are histidine and tyrosine while the substrate acidic group is neutralized by an arginine. Metal ions were detected in both lyases but they play only structural roles and are not involved directly in the catalysis. POS318 Monitoring Enzyme Activity at the Nanoscale with DNA Probes Scott Harroun 1, Alexis Vallée‐Bélisle1, Arnaud Desrosiers2 1Département de Chimie, Université de Montréal, Canada, 2Département de Biochimie et Médecine Moléculaire, Université de Montréal, Canada Several studies have recently reported enhanced diffusion of enzymes during exothermic catalysis, but explaining this phenomenon remains controversial. How does heat produced at the active site affect the enzyme and surrounding medium? Is the enzyme's structure destabilized? These questions are a matter of debate. Here, we employ programmable DNA switches to measure structural destabilization, and possibly local temperature rise, in the vicinity of an enzyme. The unfolding temperature of DNA stem‐loops can be readily tuned by varying their nucleobase composition. By attaching a fluorophore/quencher pair at the extremities of these stem‐loops, we obtain a library of fluorescent switches that can act as nanothermometers. We selected a DNA switch with optimal signal sensitivity around 37°C, and anchored it onto an enzyme via the strong biotin‐streptavidin interaction. We then measure the effects of heat released during enzyme catalysis by monitoring fluorescence variation. Alkaline phosphatase was chosen because its conversion of para‐nitrophenylphosphate to para‐nitrophenol is highly exothermic, and this enzyme undergoes enhanced diffusion during this reaction. We find that the DNA switches attached to the enzyme are destabilized during enzyme catalysis, while control DNA switches not attached to the enzyme (i.e. free in solution) do not undergo destabilization. Along with distance‐dependent destabilization, these results suggest that enzyme activity may destabilize structures located in their near vicinity. POS332 Thermodynamic Analysis of Enzyme Reaction: Lactate dehydrogenase Shogo Furuya 1, Ai Higashiyama1, Noriko Nakagawa2 1SEEDS Program, Osaka University, Japan, 2Department of Biological Sciences, Graduate School of Science, Osaka University, Japan The thermodynamic parameters of enzyme reaction were determined for lactate dehydrogenase from an extreme thermophile, Thermus thrmophilus HB8. The steady‐state kinetic parameters, kcat and Km, were determined at 14, 25, and 35°C. The thermodynamic parameters for the substrate (pyruvate) binding step (Km) at 25°C were ΔG = ‐ 13.9 kJ/mol, ΔH = + 12.7 kJ/mol, and TΔS = + 26.6 kJ/mol, and those for rate‐limiting step (kcat) were ΔG‡ = + 41.1 kJ/mol, ΔH‡ = + 45.9 kJ/mol, and TΔS‡ = + 4.8 kJ/mol. The contribution of the water molecule was reflected in the compensation between ΔH and TΔS in the substrate‐binding step and in the small TΔS‡ value. POS333 Functional analysis of new proteases from an extremely thermophilic organism, Thermus theromphilus HB8 Yumi Kimura 1, Daisuke Sasaki2, Naoya Fujimura2, Tadashi Ono2, Chinami Sako3, Suzuka Yamasaki3, Ryoji Masui3, Noriko Nakagawa4 1SEEDS Program, Osaka University, Japan, 2Osaka Prefectural Kozu Senior High School, Japan, 3Department of Biology, Faculty of Science, Osaka City Universit, Japan, 4Department of Biological Sciences, Graduate School of Science, Osaka University, Japan We challenged to discover the new functional proteins. The target proteins were zinc proteases A and B of different families from an extremely thermophilic organism, Thermus thermophilus HB8. Substrate proteins were digested with proteases A or B. The resultant peptides were separated by liquid chromatography, and analyzed by a mass spectrometer to determine their sequences. The N‐ and C‐terminals of the peptides gave us information on the cleaved peptide bonds, except for those of the substrate proteins and proteases themselves. From these results, we analyzed the substrate specificity of each subsite in proteases A and B. Both proteases showed broad substrate specificity. POS360 Identification and characterization of a laccase activity from native fungi Dictyopanus pusillus Andres Rueda 1, Yossef Lopez de los Santos1, Clara Sánchez2, Daniel Molina2, Sonia Ospina3, Nicolas Doucet1 1INRS‐Institut Armand‐Frappier University of Quebec, Canada, 2Universidad Industrial de Santander, Colombia, 3Universidad Nacional de Colombia, Colombia Laccases are multi‐purpose biocatalysts that reduce and oxidize several hazardous chemical compounds due to their broad substrate specificity. Among other applications, laccases are employed for the degradation of lignin, chemical dyes, and phenolic compounds, acting as an eco‐friendly alternative for pollutant waste management. However, the lack of industrial interest for the massive use of laccases in bioremediation is primarily due to high production costs. The objective of this research was to identify and characterize a laccase activity from the native Colombian fungi Dictyopanus pusillus. Enzymatic extracts from D. pusillus showing laccase activity were obtained by fermentation using lignocellulose as substrate, and further purified by liquid chromatography to isolate putative new laccases. Enzyme activity was assessed for dye degradation and delignification of lignocellulose from oil palm, showing higher stability at high temperatures and low pH relative to a commercial laccase. Peptides identified by mass spectrometry were used to design degenerate oligonucleotide primers to isolate the coding sequences, and cDNA was synthesized to identify laccase isoenzymes. Further experiments are underway to improve laccase overexpression in a Pichia pastoris heterologous system, which will provide means to adapt this system for low cost industrial production. POS391 Timolol and pentose phosphate pathway enzymes N. Nuray Ulusu1 , Muslum Gok2, Belma Turan3 1Koç University Faculty of Medicine, Turkey, 2Department of Biochemistry, Hacettepe University, Turkey, 3Ankara University We aimed to investigate whether timolol treatment has a beneficial effect on pentose‐phosphate pathway enzyme activities such as glucose‐6‐phosphate dehydrogenase (G6PD) and 6‐phosphogluconate dehydrogenase (6PGDH). Diabetes was induced by streptozotocin in 3‐month old male Wistar rats and confirmed by measuring high blood‐glucose level. The diabetic rats were treated with timolol (5 mg/kg body weight, for 12 weeks) while the control group received saline. Timolol‐treatment of STZ‐induced diabetic rats had no noteworthy effect on high blood‐glucose levels. However, this treatment induced activities of G6PD and 6PGDH in the diabetic rats. Since cAMP is one of the negative regulators of G6PD and directly associated with ß‐AR signaling, we measured the cAMP level in pancreatic tissues. Interestingly, timolol treatment significantly increased cAMP level in diabetic pancreatic tissue. For further support, using Protein Data Bank structures for G6PD and 6PGD, we performed in silico calculations and showed that timolol cannot bind strong enough to either G6PD or 6PGD but its binding affinity to adenylyl cyclase (AC), responsible for cAMP production serving as a regulatory signal via specific cAMP‐binding proteins, is relatively high in comparison to the others. Our data points out that timolol treatment has a beneficial action of antioxidant defense mechanism enzymes on the pancreas of STZ‐induced diabetic rats. POS430 Improving RNA Modification Mapping Sequence Coverage through a Nonspecific RNase U2‐E49A Variant Beulah Solivio 1, Balasubrahmanyam Addepalli1, Patrick Limbach1 1University of Cincinnati, USA The location of RNA post‐transcriptional modifications gives a clue on the potential function of a certain modification. The method used to determine the sequence location of a modification is referred to as post‐transcriptional modification mapping. This method makes use of ribonucleases that cleave long RNA sequences into fragments that can be detected by the mass spectrometer. This method involves the use of multiple enzymes that have different cleavage specificities to characterize as much sequence coverage as possible. Currently, RNase T1 is the only base‐specific enzyme available in the market. The goal of this study is to explore the potential of RNase U2, a purine specific enzyme with a preference to cleave adenosine at the 3′‐end, as a tool for RNA modification mapping. Here we demonstrate the use of RNase U2‐E49A, a nonspecific variant of RNase U2 that generates random long sequences, allowing RNA modification mapping. We will discuss the advantages and setbacks of using this enzymes. Our current work also explores enhancing the adenosine specificity of RNase U2 by phage display mutagenesis. POS433 Phosphate Affects The Quaternary Structure of Alanine Racemase from Mycobacterium Tuberculosis John C Ford 1, Shannon A. Stirling1, Jaeju Ko1, Sudipta Majumdar1 1Indiana University of Pennsylvania, USA In Mycobacterium tuberculosis (MT), the causative agent of tuberculosis, active alanine racemase (ALR) exists as a dimer, with two active sites, each formed on the interface between the individual monomers. A complete understanding of the interaction between the two monomers could provide insight into approaches to inactivate the dimeric ALR. We investigated the degree of association of ALR in by means of high performance size exclusion liquid chromatography (HPSEC), In buffers containing phosphate over the pH range of 5–12, MT ALR eluted with a retention volume consistent with a molar mass of approximately 70 kDa, i.e., as a monomer‐dimer equilibrium mixture. However, in pH 8.0 borate buffer, MT ALR eluted with a retention volume consistent with a molar mass of 80 kDa, i.e., the expected dimer. Further, when phosphate was added to the borate buffer, the HPSEC retention volume of MT ALR decreased. These results are consistent with MT ALR possessing a phosphate binding‐site that inhibits the dimerization necessary for its activity. POS441 Mechanisms of activation and substrate recognition by PINK1, a ubiquitin kinase implicated in mitochondrial quality control and Parkinson's disease Jean‐François Trempe 1 1McGill University, Canada Mutations in Parkin and PINK1 cause an early‐onset autosomal recessive form of Parkinson's disease (PD). PINK1 is a kinase that acts as a sensor of mitochondrial damage and initiates Parkin‐mediated quality control. Recruitment and activation of Parkin by PINK1 leads to the ubiquitination of outer mitochondrial membrane proteins and subsequent autophagic clearance of the damaged organelle. PINK1 then phosphorylates ubiquitin at Ser65 on the mitochondrial outer membrane (Tang et al. Nature Commun 2017). Phospho‐Ub binds to Parkin with high affinity and releases the Ubl through an allosteric mechanism (Sauvé et al., EMBO J, 2015). This enables PINK1 to phosphorylate the ubiquitin‐like (Ubl) domain at Ser65, which in turn increases the affinity of Parkin for E2 ubiquitin‐conjugating enzymes. Here, we use biophysical methods to characterize substrate recognition by PINK1. Using hydrogen‐deuterium exchange mass spectrometry, we find that the C‐helix, a canonical feature of active kinases, is disordered in the catalytically‐dead state. The C‐helix is preceded by an invariant serine residue (Ser205), which is the exclusive target of auto‐phosphorylation in trans. Mutants of Ser205 retain catalytic activity, but fail to phosphorylate ubiquitin. NMR studies show that only active PINK1 phosphorylated at Ser205 can bind to the Parkin Ubl (Kd ∼ 30 µM) or ubiquitin (Kd ∼ 400 µM). Finally, we use targeted proteomics methods to show that PINK1 phosphorylates ubiquitin chains foremost on Mfn1/2, which explain the substrate specificity of the E3 ligase Parkin. Our findings suggest that PINK1 must auto‐phosphorylate first prior to engaging ubiquitin chains in its immediate vicinity on the mitochondrial outer membrane. POS447 Development of a high‐throughput assay to detect fatty acid decarboxylase activity Jama Hagi‐Yusuf1, David Kwan1 1Centre for Applied Synthetic Biology, Concordia University, Canada Biofuels have the potential to move our society's dependence away from fossil‐fuel systems towards cleaner and more renewable energy sources. The technology has moved from focusing on an ethanol‐dependent fuel source, to more complex and energy rich hydrocarbons, similar to those found in petroleum. Recently, a heme‐dependent P450 decarboxylase enzyme, OleTJE, was discovered in the bacterium Jeotgalicoccus sp, which was determined via in vitro and in vivo studies to be capable of catalyzing the decarboxylation of long‐chain fatty acids, producing terminal alkenes and CO2, thus making OleTJE an ideal candidate for biofuel production. The development of a high‐throughput assay is important in engineering and studying the protein. OleTJE activity can be determined by detecting CO2, a byproduct of the OleTJE‐catalyzed conversion of fatty acids into terminal alkenes. Here we developed a high‐throughput coupled enzyme assay to detect OleTJE activity. In this assay, CO2 is converted to bicarbonate, which is then consumed along with phosphoenol pyruvate in a phosphoenolpyruvate carboxylase‐catalyzed reaction generating oxaloacetate and releasing phosphate. Coupling this with an existing phosphate‐dependent assays results in a highly sensitive and high‐throughput fluorescence‐based assay to detect OleTJE activity. POS452 Biochemical Characterization of the A233N Mutant of Trypsin III from Monterey Sardine (Sardinops sagax caerulea) Manuel Carretas‐Valdez 1, Aldo Arvizu‐Flores2, Francisco Cinco‐Moroyoqui1, Marina Ezquerra‐Brauer1, Enrique Marquez‐Rios1, Rogerio Sotelo‐Mundo3, Francisco Castillo‐Yañez2 1Departamento de Investigación y Porsgrado en Alimentos, Universidad de Sonora, Mexico, 2Departamento de Ciencias Químico Biológicas, Universidad de Sonora, Mexico, 3Laboratorio de Estructura Biomolecular, Centro de Investigación en Alimentación y Desarrollo, A.C., Mexico Trypsin (EC 3.4.21.4) is a well‐known member of the serine protease family, and catalyzes the hydrolysis of proteins and peptides specifically at the carboxyl group of lysine and arginine residues. The Trypsin III studied from Monterey sardine presented high catalytic efficiency at low temperatures as described for enzymes adapted to extreme cold. In this work, kinetic constants and optimum temperature for the mutant A233N of Trypsin III from Monterey sardine were determined. In a previous work, we first established the experimental conditions to perform the recombinant expression of the Trypsin III mutant in Escherichia coli and its purification, which was obtained in soluble and active form. The kinetic constants for the A233N mutant of Trypsin III were determined by isothermal titration calorimetry using BAEE as substrate, while the optimum temperature determined by the spectrophometric assay using BApNA as substrate. The A233N mutant showed an optimum temperature of 35°C, whereas its kinetic parameters were of 0.041 s‐1 and 0.084 mM for kcat and KM, respectively. A molecular model of A233N did not predict an altered structure compared to the wild‐type Trypsin III that could explain the lower optimum temperature. Also, the A233N mutant showed a higher specific activity than wild‐type Trypsin III. The mutant A233N of Trypsin III from Monterey sardine could have potential applications in the food industry. The trypsin III from Monterey sardine is a feasible model for structure‐function studies. POS472 Steady‐State Kinetics of the Degradation of Insulin by Insulin‐Degrading Enzyme using Circular Dichroism Spectroscopy Valerie Ivancic1, Claire Krasinski1, Donald Spratt1, Noel Lazo1 1Clark University, Massachusetts, USA Insulin‐degrading enzyme (IDE) is a highly conserved metalloprotease and is the major protease for the degradation of insulin. However, despite previous investigations, the mechanism for the degradation of insulin is still not well understood. Here, we probed the steady‐state kinetics of the IDE‐catalyzed degradation of insulin by circular dichroism spectroscopy. Our results show that the catalytic efficiency of the enzyme towards insulin, measured by kcat/Km, is not affected by high concentrations of salt, suggesting that electrostatic interactions do not play a significant role in the specificity of IDE towards insulin. POS484 Unique cleavage specificity of novel cold adaptive trypsins from North Atlantic cod (Gadus morhua) based on multiplex substrate profiling Gunnar Sandholt1, Ágústa Guðmundsdottir1, Bjarki Stefansson2 1University of Iceland, Reykjavík, Iceland, 2Zymetech The North Atlantic cod (Gadus morhua) is cold adapted and contains numerous different enzymes for digestion of its prey under extreme conditions. Understanding the biochemical properties of cod enzymes gives insight to their function, evolution and possible use in research, industry and for therapeutic purposes. Previously, two cod trypsin isoenzymes (trypsin I and CTX‐V7) were characterized after isolation from a benzamidine purified cod trypsin isolate using anion exchange chromatography. This study describes characterization of novel trypsins ZT, purified from the isolate, with unique cleavage properties relative to other trypsins. A fraction resulting from an anion exchange chromatography containing the novel trypsins was analyzed using MALDI‐TOF mass spectrometry. Four closely related trypsin amino acid sequences (trypsin ZT‐1, trypsin ZT‐2, trypsin ZT‐3 and trypsin ZT‐4) deduced from expressed cod sequence tags (ESTs) gave the best match. To compare the substrate specificity of the cod trypsin ZT isoenzymes and cod trypsin I, multiplex substrate profiling by mass spectrometry (MSP‐MS) was used. The MSP‐MS method uses MS to characterize protease cleavage patterns within a library of 14‐mer peptides (124 defined peptides, total of 1612 peptide bonds). Also, it provides data on the effect of different amino acids at both sides of the scissile amide bond. MSP‐MS on trypsin ZT revealed that amino acids surrounding arginine or lysine in a substrate have a different effect on its cleavage compared to trypsin I. Unexpectedly, cleavage sites were identified that are unique to trypsin ZT compared trypsin I. Continuing studies are exploring other biochemical properties of cod trypsin ZT compared to those of cod trypsin I. POS518 Characterization of the C‐Glycosyltransferase IroB Utilizing a Novel Coupled Enzyme Assay Employing E. coli UDP‐glucose Dehydrogenase Cory Campbell1, Peter Pawelek1, Peter Pawelek1 1Department of Chemistry & Biochemistry, Concordia University, Canada In certain E. coli strains, the C‐glycosyltransferase IroB glycosylates enterobactin prior to its secretion. Glycosylated enterobactin (aka salmochelin) facilitates bacterial iron uptake in a host while evading the mammalian immune system. To characterize its enzymatic properties, recombinant hexahistidine‐tagged IroB was expressed and purified to near‐homogeneity. We developed a coupled enzyme assay utilizing UDP‐glucose dehydrogenase (Ugd) to quantitate UDP‐glucose consumption, and thus IroB activity, more rapidly and efficiently than a previously reported HPLC‐based assay. Purified IroB and recombinant E. coli H6‐Ugd were then employed in a coupled assay where the glycosylation of enterobactin, catalyzed by IroB using the substrate UDP‐glucose, was quenched at appropriate time points. Upon NAD+ addition, H6‐Ugd was used to convert all remaining UDP‐glucose to UDP‐glucuronate and NADH. The consumption of UDP‐glucose by IroB was then quantitated from NADH production, measured spectrophotometrically. Initial rates were obtained and Michaelis‐Menten steady‐state kinetic parameters were determined for wild type IroB, which agreed with previously reported values. The kinetic parameters of the mutant variant W264L, hypothesized to be impaired in UDP‐glucose binding, were then determined. An equilibrium‐binding assay employing spin columns was used to determine equilibrium‐binding parameters of IroB co‐substrates. As expected, saturation of the W264L variant with UDP‐glucose was not observed, confirming the role of W264 in UDP‐glucose binding. Furthermore, the binding assay demonstrated that the KD for UDP‐glucose binding to wild‐type IroB in the absence of Mg2+ and enterobactin was higher than the observed Km value for UDP‐glucose obtained from the coupled assay. POS525 Catalytic mechanism of the Salmonella typhimurium effector acetyltransferase AvrA. Jonathan Labriola, 1 Bhushan Nagar1 1McGill University, Montreal, Canada Bacterial effector proteins play an essential role in the infection and proliferation of pathogenic bacteria within their targets through manipulation of the host immune response pathways. AvrA is a bacterial effector acetyltransferase from Salmonella typhimurium which suppresses JNK signalling in intestinal epithelial cells. This occurs through acetylation of the JNK pathway specific and essential mitogen activated receptor kinase kinases (MKKs) 4 and 7. AvrA belongs to the YopJ family of acetyltransferases, which are most closely related to the CE peptidase clan, suggesting that the acetyltransferase activity of these enzymes proceed through a novel mechanism. Through biophysical and kinetic analysis we determined that AvrA acetylates MKK 4 via a ping‐pong mechanism. We also identify residues of AvrA that may play a role in substrate binding and/or recognition. 09. EVOLUTION POS045 Engineering Cal‐A Towards Discriination in the Hydrolysis of Short vs Long‐chain Fatty Esters Daniela Quaglia1 1Universite de Montreal, Canada We target the development of the enzyme Candida Antarctica lipase A (Cal‐A) into an improved biocatalyst for the food industry. Studies suggest that milk‐fat products rich in diglycerides composed of short‐chain saturated fatty acids might have health benefits. Cal‐A is already intrinsically selective for the SN2 hydrolysis of triglycerides: our goal is to evolve the enzyme further to make it able to discriminate between the hydrolysis of short‐chain vs long‐chain fatty acid esters, and thus develop strategies to enrich the content of short‐chain lipids. [1] A combinatorial approach based on the synthetic biology Golden Gate cloning strategy was adopted for the creation of ‘smart’ mutant libraries: Cal‐A was disassembled into three constitutive domains, which were separately mutated and flexibly re‐assembled at a later stage. [2] The libraries of variants were screened on agar plates containing model triglycerides, namely tributyrin (C4), and olive oil (mainly C18), using a high‐throughput screening method that we developed. The most promising variants, which showed the ability to discriminate between the short and long‐chain substrates, were quantitatively characterized through a standard p‐NO2‐phenyl ester assay with substrates of different chain lengths. References: 1. Nyyssölä, A. et al. International Dairy Journal 41, 16–22 (2015). 2. Quaglia D, et al. PLoS ONE. 2017;12(2):e0171741. doi: 10.1371/journal.pone.0171741. PubMed PMID: 28178357. POS092 The mitoribosome of a primitive eukaryote Andalucia godoyi Jose Gonzalez 1, Gertraud Burger1, Matus Valach1 1Robert‐Cedergren Centre of Bioinformatics and Genomics, Biochemistry, Université de Montréal Canada Mitochondria originate from an internalized alpha‐proteobacterium that developed a permanent endosymbiotic relation with its host. During eukaryotic evolution, nearly 99% of the endosymbiont's genes were lost. The ribosome in particular underwent drastic changes, featuring novel mito‐ribosomal proteins (mt‐r‐proteins) and a shortened mito‐ribosomal RNAs (mt‐rRNAs). But how exactly this evolutionary remodeling took place remains speculative due to the small number of studies in primitive eukaryotes. To get insight into this process, we are analyzing the composition of the mitoribosome of Andalucia godoyi, a free‐living heterotrophic flagellated jakobid that contains the largest and least derived mitochondrial gene set currently known (1, 2). Our in silico analysis of the nucleus‐encoded portion of the Andalucia's mitoribosome predicts 41 genes, adding up together with the mitochondrion‐encoded portion to a total of 69 mt‐r‐proteins. This number is slightly higher than in bacteria (58) and much smaller than e.g. in human (82). We will present data that will show how far Andalucia's mt‐r‐proteins resemble bacterial homologs or counterparts from derived eukaryotes such as human and yeast. In order to validate our findings experimentally, we are currently purifying Andalucia's mitoribosome to identify whether the predicted proteins are indeed part of the mitochondrial translation machinery. This will provide convincing evidence that Andalucia's mitoribosome represents an intermediate –frozen in time– in the evolutionary process of mt‐r‐proteins recruitment. Acknowledgements: We thank Michael W. Gray for help in annotating nucleus‐encoded proteins. References: 1. Burger G, Gray MW, Forget L, Lang BF. Strikingly bacteria‐like and gene‐rich mitochondrial genomes throughout jakobid protists. Genome Biol Evol. 2013;5(2):418‐38. POS104 Cryptic genetic variation determines the adaptive evolutionary potential of enzymes Nobuhiko Tokuriki1, Florian Baier1, Colin Jackson2 1University of British Columbia, Canada, 2Australian National University, Canberra, Australia Genetic variation across orthologous proteins is generally neutral in respect to their native, physiological function, but can cause “cryptic genetic variation”, i.e., phenotypic variation in other non‐physiological properties. Such cryptic genetic variation may also define the adaptive, evolutionary potential, i.e., evolvability, of proteins. In other words, orthologous enzymes may evolve significantly different evolutionary outcomes when these enzymes are subjected to the same selection pressure. We address this question using laboratory evolution of enzymes, combining with genetic, molecular (catalytic activity, protein soluble expression, thermostability, oligomeric state and crystal structure) changes of the evolutionary trajectories. We evolved two orthologous metallo‐beta‐lactamases (MBLs), NDM1 and VIM2, towards a promiscuous phosphonate hydrolase (PMH) activity. We found that seemingly neutral genetic variation between the orthologous enzymes can cause significant difference in evolutionary outcomes for the level of PMH activity, i.e., 70‐fold difference in enzyme fitness increase between the two enzymes. Interestingly, mutational analysis revealed that the two trajectory followed completely different mutational pathways to increase the enzyme fitness. Moreover, the two enzymes increased enzyme fitness through substantially different adaptive strategies, one adapted though increasing catalytic efficiency, and the other though the combination of catalytic efficiency, protein expression and oligomerizations. Using structural analysis and MD simulations, we also unveiled molecular basis underlying such variation in evolvability, i.e., cryptic and subtle structural properties, such as the orientation of a key active site residue, Trp93, can cause large difference in mutational responses. Thus, genetic variation among orthologous enzymes define evolutionary outcomes, proposing important implications for evolution in nature and protein engineering strategies to generate new functions in the laboratory. POS114 Ancestral Reconstruction of Ligand Binding Proteins: Clues to Understand Protein Specificity Evolution Saira Maldonado‐Puga 1, Alejandro Sosa‐Peinado1, Jesús Banda‐Vázquez1 1Laboratory of Physicochemistry and Protein Engineering, Biochemistry Department, School of Medicine, UNAM, Mexico The attempts to design proteins with specific ligand binding properties are still difficult to achieve. Most of the times, the computational design of ligand binding does not behave as expected at wet lab, since more details are needed to succeed. Ancestral reconstruction could provide information about sequence changes that led to modify the affinity or specificity for their ligands and further clues to understand the combinatorial events that led to such diversity. Methods. 637 phylogenetically diverse bacterial homologs of LAOBP (PDB: 1LAF), GlnBP (PDB: 1WDN), HisBP (PDB: 1HSL), CysBP (PDB: 1XT8), CisBP (PDB: 2YLN), EctBP (PDB: 2Q88), NopBP (PDB: 4POX), Glu‐AspBP (PDB: 2VHA), SulfBP (PDB: 1SBP) and PhosBP (PDB: 1IXH) were selected from the UniRef90 database using the UniProt BLAST server. Final alignments were constructed by dividing sequences into subfamilies, aligning sequences within their subfamilies using MAFFT (Katoh et al., 2002) and combining these alignments with the merge tool of MAFFT. CheY protein (PDB: 3CHY) was used as outgroup. Phylogenetic trees were computed using the maximum‐likelihood method implemented in PhyML (Guindon et al.,2010) and bootstrapped with 100 replicates. The alignment and tree will be used for ancestral reconstruction with PAML (Yang, 2007). Results. The evolutionary relationships between the subfamilies were well resolved. The proteins separated according to their reported ligand specificity: positive charged ligands, negative and those which bind non‐charged amines. Conclusions. These results could lead to obtain ancestral sequences with good posterior probabilities (>0.8) at the nodes of interest. POS127 Multi‐Scale Model of Microbial Evolution to Predict the Emergence of Drug Resistance Anh‐Tien Ton 1, Adrian Serohijos1, Pouria Dasmeh1 1Université de Montréal, Canada Currently, our ability to predict pathways in bacterial evolution to drug resistance is limited. It entails bridging several constraints on various levels of biological organization—from molecular properties of proteins, to organismal fitness, and to microbial population dynamics. To address this issue, we develop a new multi‐scale framework for microbial evolution that integrates protein biophysics with population genetics by relating the biochemical effects of mutation and population demography and structure to predict pathways to drug resistance. Additionally, we utilize previously published deep mutational scanning (DMS) data to determine the mutational fitness landscape of ß‐lactamase. This experimental data is integrated into a computational model of evolution that replicates bacterial population dynamics. From the population dynamics simulations, we find that the quantitative dependence of the time to emergence of resistance on drug concentration and microbial population size. This dependence is surprisingly non‐linear due to the complexity of the fitness landscape measured from experimental DMS. Broadly, we are able to trace the evolution of the minimal inhibitor concentration (MIC) and IC50 of ß‐lactamase. This framework can be used to study the emergence of antibiotic resistance under more complex selection scenarios, such as oscillating drug concentration dosage. POS129 Exploring the innovability potential of a primitive enzyme that confers antibiotic resistance Lorea Alejaldre 1, Joelle Pelletier1 1Université de Montréal, Canada The rise of antibiotic resistance is an emergent health crisis due to the speed at which it is developing and its economical and clinical repercussions. The development of novel enzymatic activities within bacterial cells is one of the most common causes of antibiotic resistance. Therefore, assessing the capacity of enzymes to evolve towards novel activities is important to understand and counteract this issue. In the current study, we explore the capacity of the primitive enzyme R67 dihydrofolate reductase (R67 DHFR), which confers resistance to the commonly prescribed antibiotic trimethoprim, to develop new antibiotic resistance activities. And whether R67 DHFR could evolve to render a bacterial cell multi‐drug resistant by preserving its trimethoprim resistance while evolving an enzymatic activity that confers a different antibiotic resistance. A site‐directed saturation mutagenesis library in the residues involved in binding and catalysis in the enzyme R67 DHFR was screened against several types of antibiotics. Previous studies have shown that the active site could be thoroughly modified while maintaining its native activity. The antibiotics chosen allowed us to screen for most of the known strategies of enzymatic inactivation of antibiotics (hydrolysis, group transfer and redox reaction). Differential survival of clones has allowed to identify a variant that confers a mild resistance to an antibiotic that is chemically unrelated to trimethoprim: tetracycline. Further work to understand the resistance mechanisms to tetracycline are ongoing. The results obtained will help to understand the evolution of this primitive enzyme and its potential as a multi‐drug resistance source. POS171 Tuning the course of viral evolution on a protein fitness landscape using droplet microfluidics Adrian Serohijos 1, Assaf Rotem2, Eugene Shakhnovich2, David Weitz2, Christiane Wobus3, James Pipas4, Andrew Feldman5 1University of Montreal, Canada, 2Harvard University, USA, 3University of Michigan at Ann Arbor, USA,4University of Pittsburg, USA, 5Johns Hopkins University, USA Evolution is a unifying theme in the urgent medical and public health problems we face today including cancer, the rise of antibiotic resistance, and the spread of pathogens. But the ability to predict evolution remains a major challenge because it requires bridging several scales of biological organization. Potential evolutionary pathways are determined by the “fitness landscape” (the genotype‐phenotype relationship), but how this landscape is explored depends on microbial population dynamics. I will describe our recent work where we showed that the fitness landscape of norovirus escaping a neutralizing antibody can be projected onto two traits, the capsid folding stability and its binding affinity to the antibody. We then developed a theory based on protein biophysics and population genetics to predict how the fitness landscape might be explored. Using a droplet‐based microfluidics “Evolution Chip”, we propagated millions of independent viral sub‐populations, and showed that by tuning viral population size per drop, we could control the direction of viral evolution. Additionally, I will describe how this combined framework of biophysics and evolutionary biology also applies to bacterial evolution due to horizontal gene transfer. Altogether, these stories demonstrate the broad applicability of the techniques and concepts from protein engineering to fundamental problems in evolution and genetics. POS196 Evolution of the molecular mechanisms of dependency between paralogous genes within the protein interaction network Axelle Marchant 1, Christian. R Landry1, Axelle Marchant2, Isabelle Gagnon‐Arsenault2, Christian. R Landry2 1Département de Biologie; The Quebec Network for Research on Protein Function, Engineering, and Applications; Institut de Biologie Intégrative et des Systèmes ‐ Université Laval, 2Département de Biologie, University of Laval, Canada Mutational robustness is the ability of cells to perform normal functions in spite of genetic perturbations. Paralogous proteins (derived from gene duplication) that have retained redundant functions can compensate for each other when faced with deleterious mutations and thus contribute to mutational robustness. For instance, deleterious mutations in paralogous genes lead to less severe effects than the mutation of single copy genes in yeast as in humans. It was recently shown that this could be achieved directly at the level of protein‐protein interactions due to the ability of paralogs to compensate for each other's losses. However, some human genetic diseases are, on the contrary, associated with paralogs. The mechanisms underlying these contradictory observations are still poorly understood. It was recently suggested that paralogs often cannot compensate for each other's loss but rather depend on each other in the protein‐protein interaction network. Our working hypothesis is that the duplication of ancestral genes forming homodimers leads to the formation of heterodimers of paralogs that evolve in a way that makes them dependent on each other. We are studying the mechanisms underlying the emergence of this constraint by reconstructing the evolutionary steps leading this phenomenon. Using Protein Complementation Assays (PCA) and the yeast protein interaction network, we examined the evolution of homodimer and heterodimer interactions after gene duplication to understand how the duplication of homodimers leads to physically dependent paralogs. Our results will help understand how protein interaction networks increase in complexity without necessarily gaining in mutational robustness. POS205 Estimating the contribution of selection for folding stability to epistasis in protein evolution Pouria Dasmeh 1, Adrian Serohijos1 1Departement de Biochimie, Centre Robert Cedergren en Bioinformatique et Génomique, Université de Montréal, Canada The extent of non‐additive interaction among mutations or epistasis reflects the ruggedness of the fitness landscape, the mapping of genotype to reproductive fitness. In protein evolution, there is a strong support for the importance and prevalence of epistasis, but whether there is a universal mechanism behind epistasis remains unclear. It is also not established which of the biophysical properties of proteins—folding stability, activity, binding affinity, and dynamics—have the strongest contribution to epistasis in proteins. Here, we determine the contribution of selection for folding stability to epistasis in protein evolution. By combining theoretical estimates of the rates of molecular evolution and protein folding thermodynamics, using numerical integration, Monte Carlo and sequence‐explicit simulations, we show that stability can account for ∼30% to ∼60% epistasis among substitutions. Our model predicts substantial epistasis at marginal stabilities therefore linking epistasis to the strength of selection. Since the strength of selection for thermodynamic stability is substantially higher in genes with higher expression levels, epistasis is predicted to be stronger in highly expressed genes. In line with theoretical prediction, we estimated the extent of epistasis for 2382 genes in E. coli and found stronger epistasis in highly expressed genes. Taking these altogether, our results show that selection for protein folding stability to minimize costs of unfolding and aggregation would account for significant fraction of epistasis in protein evolution. Estimating the contribution of governing factors in molecular evolution such as protein folding stability and expression level to epistasis would provide a better understanding of epistasis and hence insight to predictability of molecular evolution. POS206 Highly expressed proteins evolve with stronger epistasis in E.coli Pouria Dasmeh 1, Eric Girard1, Adrian Serohijos1 1Departement de Biochimie, Centre Robert Cedergren en Bioinformatique et Génomique, Université de Montréal, Canada Despite the critical role of epistasis in molecular evolution, most computational methods in protein evolution assume additivity of mutational effects and independent evolution of residues, thus accounting for epistasis remains a challenge for these approaches. One reason for such shortcomings is the lack of a universal mechanism for evolution at molecular level. Among the prime candidates for mechanisms behind epistasis at the molecular level is selection for protein folding stability. Additionally, a consistent observation from comparative genomics is that proteins that are highly expressed and abundant in the cytoplasm evolve slowly. This result is observed in genomes across all kingdoms of life—in bacteria, yeast, worm, and mammals. The link between expression level, as the strongest predictor of evolutionary rate of proteins, and epistasis is still unclear. To elucidate the extent of epistasis in genes with different expression levels, we estimated the extent of epistasis for 2382 genes in E. coli using several hundreds of orthologues for each gene. We find that highly expressed proteins experience stronger epistasis. Using a combination of numerical integration and forward evolutionary simulations of protein evolution, we show that positive correlation between epistasis and expression level can be explained by selection for protein folding stability to minimize costs of unfolding and aggregation. POS255 Biochemical and structural insights into the evolution of the E3 ubiquitin ligase Casitas B‐lineage Lymphoma (Cbl) and its highly conserved mechanism of action for ubiquitylation of tyrosine kinase targets Jeanine Amacher 1, John Kuriyan1, Helen Hobbs1, Sarah Mulchand1, Deepti Karandur1, Aaron Cantor1 1UC Berkeley, California, USA Tyrosine phosphorylation is a hallmark of metazoan signal transduction pathways, directly regulating critical cellular processes such as cell growth. Until recently, it was believed that tyrosine kinase signaling emerged with multicellularity; however, the full complement of necessary proteins for tyrosine phosphorylation and dephosphorylation exists in choanoflagellates, our closest non‐metazoan ancestor. The presence of these signaling components in such distantly related organisms is intriguing, suggesting that there remains a lot about these pathways we do not fully understand. Here, we focus our attention on an important mediator of tyrosine kinase signaling, the E3 ubiquitin ligase Casitas B‐lineage Lymphoma (Cbl), which targets many extant tyrosine kinases for lysosomal degradation. Biochemical and structural characterization of a number of Cbl homologues with varying degrees of sequence identity: including proteins from the choanoflagellate species Salpingoeca rosetta, as well as Caenorhabditis elegans and Drosophila melanogaster, reveals highly conserved phospho‐activation and substrate recognition mechanisms. In addition, we solved the crystal structure of choanoflagellate Cbl (sCbl) in its inactive state, revealing remarkable similarity to human Cbl and performed molecular dynamics simulations on both sCbl and human Cbl, confirming substrate interactions outside the SH2 domain of the tyrosine kinase binding domain. This work provides important insight into the evolution of Cbl, revealing additional nuances of its mechanism of action. POS258 Characterization of a Curious CaMKII Isoform Ethan McSpadden 1, John Kuriyan1 1UC Berkeley, California, USA Ca2+/calmodulin‐dependent protein kinase II (CaMKII) is a protein kinase important in learning and memory. CaMKII functions as a homomeric holoenzyme. N‐terminal kinase domains, each tethered by an unstructured linker, extend out from a central ring assembly of C‐terminal association or “hub” domains. The characterization of divergent CaMKII orthologs has helped us begin to understand the surprising structural plasticity of CaMKII hub domains. We chose to study a predicted CaMKII hub domain from the single celled green algae Chlamydomonas reinhardtii. This ortholog, 48% identical to the human hub, appeared particularly interesting because no CaMKII kinase domain is present in the open reading frame of this gene. Biochemical and structural characterization of the purported Chlamydomonas CaMKII hub shows that it is a true CaMKII hub domain. Chlamydomonas hub domains form an 18‐ subunit ring assembly with 9‐fold radial symmetry. This is in contrast to human CaMKII where hub domains form, with roughly equal propensity, only 12‐ or 14‐subunit ring assemblies. A subtle compaction of the Chlamydomonas hub domain fold relative to human may allow for this difference in stoichiometry. The more compact fold and lack of stoichiometric variability suggest that this hub domain is less flexible relative to its human counterpart. Mutation of core residues in human CaMKII hub to those present in Chlamydomonas heavily disfavors formation of 14‐subunit assemblies, suggesting that the domain has been rigidified. Understanding hub domain plasticity will help us understand how the disassembly and reassembly of CaMKII holoenzymes facilitates signaling by this kinase. POS280 Diversity and evolutionary analysis of iron‐containing (Type‐III) alcohol dehydrogenases (FeADHs). Adriana Julián‐Sánchez 1, Héctor Riveros‐Rosas1 1Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico Currently, there are three non‐homologous NAD(P)+‐dependent alcohol dehydrogenase (ADH) families reported: Type I ADH comprises Zn‐dependent ADHs; type II ADH comprises short‐chain ADHs described first in Drosophila; and, type III ADH comprises iron‐containing ADHs (FeADHs). These three families arose independently throughout evolution and possess different structures and mechanisms of reaction. While types I and II ADHs have been extensively studied, type III ADHs have been scarcely analyzed. Therefore in this work, an evolutionary analysis of FeADHs was performed. Results showed that FeADHs are distributed in twenty two protein subfamilies, eight of them exhibit a universal distribution. Protein sequences from bacteria are present in all FeADH subfamilies, but protein sequences from archaea and eukarya are present in fourteen and thirteen subfamilies, respectively. Interestingly, none of these protein subfamilies possess protein sequences found simultaneously in the three main eukaryotic kingdoms (animals, plants and fungi). Animal FeADHs are found in just one protein subfamily, the hydroxyacid‐oxoacid transhydrogenase (HOT) subfamily, which includes protein sequences widely distributed in fungi, but not in plants), and in several taxa from lower eukaryotes, bacteria and archaea. Fungi FeADHs are found mainly in two subfamilies: HOT and maleylacetate reductase (MAR), but some can be found also in other three different protein subfamilies. Plant FeADHs are found only in chlorophyta but not in higher plants, and are distributed in three different protein subfamilies. In conclusion, FeADH is an ancient protein family that probably was present in the last common ancestor, and comprises a diversity of subfamilies that shares a common 3D scaffold that resulted with a patchy distribution in eukaryotes. POS429 Cold‐Adapted ADP‐Dependent Sugar Kinase: Biophysical and Evolutionary Study of its Flexibility. Victoria Guixe 1, Ricardo A Zamora1, Cesar A Ramirez‐Sarmiento2, Victor Castro‐Fernandez1, Pablo Villalobos1, Elizabeth Komives3 1Universidad de Chile, Santiago, Chile, 2P. Universidad Católica de Chile, Santiago, Chile, 3University of California, San Diego, USA The general mechanism adopted by psychrophilic enzymes to perform catalysis at low temperature was to reduce the free energy of the transition state rather than the Michaelis constant (Km). This was achieved by relaxing the structures of these enzymes by structural modifications that comprise the absence of stabilizing ionic interactions. The increased structural flexibility and decreased affinity for its substrates has been shown to be compensating by an increase in the catalytic rate (kcat). Few psychrophilic enzymes have been reported to optimize their catalytic efficiency (kcat/Km) by decreasing their Km values. We use the psychrophilic phosphofructokinase/glucokinase from Methanococcoides burtonii (MbPFK‐GK) and the mesophilic phosphofructokinase/glucokinase from Methanococcus maripaludis (MmPFK‐GK) to identify functional and structural features of a psychrophilic enzyme that would make this enzyme more flexible than their thermostable homologues. Enzymes were characterized by spectroscopic, biophysical and computational techniques. By H/D exchange coupled to mass spectrometry we showed that the psychrophilic enzyme presents increased structural flexibility, particularly in segments flanking the metal‐nucleotide binding motif, mainly due to the absence of two ion pairs present in MmPFK‐GK. This increase in structural flexibility is reflected in the exponential increase in the Km values with temperature. Reconstruction of the phylogenetic tree of this enzyme family and the inference of all ancestral sequences between psychrophilic and mesophilic enzymes, allowed us to establish that the absence of these ionic interactions in the psychrophilic branch is an ancestral trait. Insertion of these two ionic interactions in the psychrophilic enzyme showed in silico as well as experimentally, that they alter active site flexibility and enzyme dynamic (Fondecyt 1150460). POS432 Evolution of Multi‐Domain Conformational Ensembles from the Tyrosine Kinase Family Helena Gomes Dos Santos 1, Jessica Siltberg‐Liberles1 1Florida International University, USA We recently found clade‐specific variation in predicted intrinsic disorder for 543 tyrosine kinases across 17 paralogous clades and 45 species. Here, we explicitly characterize the protein conformational dynamics and free energy from 3801 3D models. Protein flexibility was addressed with normal modes analysis on the modeled conformational ensembles and their intrinsic dynamics compared across same‐protein conformations, orthologs and paralogs. Further, molecular dynamic simulations were performed on two representative subsets. Subset 1 contained the 17 human paralogs from each of the 17 clades, while subset 2 contained the same 7 species from 4 closely related paralogs. From these results we infer (i) the different conformations are accessible to all proteins but some are preferred for different clades, (ii) extensive variation in the amount of intrinsic dynamics persists within and between different paralogs, (iii) clade‐specific shifts in stability, partly due to shifts in the electrostatic contribution to the free energy, are apparent and consistent for all conformations, and (iv) large shifts in electrostatic potential surfaces for different clades regardless of the conformation. Remarkably, one paralogous clade has become significantly more dynamic and divergent in multiple measures. After gene duplication, this clade appears to have evolved to perform its function via a unique mechanism of binding to and stabilizing its paralogs, as confirmed by previous experimental studies. Overall, the divergence of protein dynamics after gene duplication is supported by these results, implying the importance of altered dynamics on functional divergence. POS456 Functional Evolution of Pentameric Ligand‐Gated Ion Channels in Helminths Jennifer Noonan 1, Robin N. Beech1 1Institute of Parasitology, MacDonald Campus, McGill University, Canada Gene duplication in early animal evolution was instrumental in creating the diversity of neurotransmitter signalling found throughout the animal kingdom today. Recent duplications of two subunits of the filarial parasitic nematode acetylcholine receptor provide a unique, tractable model of the mechanisms involved. Our objectives are to: 1) reconstruct the ancestral subunits present when the duplications occurred 2) estimate selective pressure acting on duplicated subunits and 3) determine amino acid sites under positive selection during subsequent evolution. Predicted subunits were identified for 50 nematode species from the Helminth Genome Initiative. Ancestral gene complement, loss and duplication events were inferred by Dollo parsimony from an ML phylogeny (PhyML). Relative substitution rates were evaluated using CodeML (PAML), positively selected sites using 2‐Rate FEL (HyPhy) which were mapped onto a predicted 3D structure (YASARA). Three site classes were identified: 70% very highly conserved, likely responsible for conserved core 3D structure, 20% less conserved, possibly sites at subunit interfaces, 10% the least conserved, possibly non‐conserved loops. This last rate class was elevated for duplicated subunits, consistent with functional adaptation. Strongly selected sites were inferred, adjacent to the characteristic cys‐loop and the TM2‐TM3 linker. The different elevated substitution rates and positively selected sites suggest the duplicate subunits of the two genes are evolving under different functional constraints; providing a possible mechanism of subunit functional change. POS497 How Mutational Epistasis Impairs Predictability in Protein Evolution and Design Charlotte Miton 1, Nobuhiko Tokuriki1 1Michael Smith Laboratories, University of British Columbia, Canada There has been much debate about the extent to which mutational epistasis, i.e. the dependence of the outcome of a mutation on a particular genetic background, constrains evolutionary trajectories. The degree of unpredictability introduced by epistasis, due to the non‐additivity of phenotypic effects, strongly hinders the strategies developed in protein engineering. While several studies have tackled this issue through systematic characterization of evolutionary trajectories within individual model enzymes, the field lacks a consensus view of its scope To address this need, we performed a comprehensive survey of mutational epistasis based on nine previous studies that examined the evolution of novel functions in natural and laboratory systems. We quantified epistasis by comparing the effect of mutations occurring between two genetic backgrounds: the starting enzyme (typically, wild type) and the intermediate variant on which the mutation occurred during the trajectory. We found that most trajectories exhibit positive epistasis, in which the mutational effect is more beneficial when it occurs later in the evolutionary trajectory. Approximately half (49%) of functional mutations were neutral or negative on the wild type background, but became beneficial at a later stage in the trajectory, indicating that these functional mutations were not predictable from the initial starting point. While some cases of strong epistasis were associated with direct interaction between residues, many others were caused by long‐range indirect interactions between mutations. Our findings highlight the prevalence of epistasis in enzyme adaptive evolution and have implications for the engineering and design of functionally optimized catalysts, for which the identification and understanding of epistatic interactions are pivotal. 10. FOLDING POS033 Organoselenium compounds: A new class of oxidative folding reagent Kenta Arai 1, Haruhito Ueno1, Yuki Asano1, Michio Iwaoka1 1Department of Chemistry, School of Science, Tokai University, Japan Chemically or biologically prepared polypeptide chains generally need to fold into the bioactive native state through two chemical reactions, which are random disulfide (SS) formation and subsequent SH‐SS isomerization for searching the native SS pairings. Whereas this oxidative folding is greatly promoted by assistant of protein disulfide isomerase (PDI), such enzymatic systems are not applicable practically to the oxidative folding in a test tube because of expensiveness of the enzymes. In contrast, use of synthetic additives, which imitate a function of foldases, is one of a useful strategy to achieve an oxidative folding efficiently. In this study, therefore, we attempted to synthesize organoselenium compounds (Figure 1) as a foldases mimic, which are derived from inexpensive starting materials, and to demonstrate their applications as folding additives. When dihydoroxy selenolaneoxide 1, which has a much higher redox potential (375 mV) than cysteine/cystine (−238 mV), was reacted with reduced ribonuclease A (RNase A) with eight thiol groups, the fully oxidized species (4SS) having non‐native SS bonds was produced quickly (< 1 min). In addition, when the obtained 4SS species was subsequently reacted with GSH (1 mM) in the presence of a catalytic amount of a cyclic diselenide (2–6), the native state was efficiently recovered through SH‐SS isomerization catalyzed by working of a reduced diselenide (i.e., selenol moieties), which should be generated in the reaction solution. It was also found that the pKa value of selenols and diselenide‐reduction potential, which are the factors affecting the PDI‐like catalytic activity, can be controlled by changing the substituents and ring sizes of the compounds. POS040 Variant Structural Intermediates of a Novel Human Calcium‐Binding Protein, Calnuc Vignesh Ravichandran 1, Gopala Krishna Aradhyam1 1Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, India Nearly two‐thirds of the human proteome is comprised of multi‐domain proteins. The intricate folding/unfolding behavior of multi‐domain proteins is highlighted by the presence of non‐native structures or folding intermediates. These intermediates engage in cellular events like its misfolding/aggregation and interactions with other proteins. Calnuc, a multi‐domain, multi‐functional, calcium‐binding protein is reported to interact with numerous proteins with pleiotropic functions. Unfolding of calcium‐free Calnuc/Nucleobindin1 proceeds through two structural intermediates, as detected by circular dichroism despite their fluorescence spectral signatures are similar. These two intermediate states are trapped at 1 M and 1.6 M guanidinium chloride (GdmCl) before being completely unfolded at >2.5 M GdmCl. The unfolding of calcium‐bound Calnuc proceeds through a single intermediate state with a gradual loss in secondary structure. Calcium‐binding confers a marginal increase in stability (ΔG unfolding, Ca = ∼ 3 kCal mol‐1) as inferred from thermodynamic analysis by far‐UV, near‐UV and DSC experiments. To demonstrate the local conformational changes around EF‐hands (calcium‐binding domains), fluorescence studies on single tryptophan mutants (W232A/W333A) reported W333 is more stable than W232. As the nature of intermediates differs between calcium‐free and calcium‐bound form, we hypothesize that calcium‐binding might not alter the conformational stability of Calnuc, but regulate its protein‐protein interaction. POS058 Incorporating a functional mutation into a symmetric scaffold as proxy for functional adaptation via rearrangement of its folding nucleus. Connie Tenorio1 1Florida State University, USA In proteins the conserved heritable unit of folding is known as the folding nucleus (FN), which is described by a subset of amino acid positions with favorable local contacts that induce, or nucleate, the formation of a folded structure. The FN has previously been identified in FGF‐1 and Symfoil‐4T, a hyper stable functionless derivative of FGF‐1. Both proteins adopt the ß‐trefoil fold, which has three‐fold symmetry. Using the known location of the folding nucleus in Symfoil‐4T the hypotheses concerning the tradeoff between folding and function, as well as the tradeoff between stability and function, can be probed in terms of FN perturbation. Using Symfoil‐4T as a purely symmetric scaffold, the design and insertion of a functional cassette into each symmetrically related domain will elucidate the plasticity of the FN upon acquisition of function. The functional cassette utilized is an amalgamation of multiple heparin sulfate binding motifs within the fibroblast growth family. This study will report the effects of functional mutation upon thermostability and folding. Mapping the rearranging of a protein's unique FN within a proxy for functional adaptation can be invaluable for future endeavors in de novo protein design efforts and protein evolution studies. POS073 Role of An Intramolecular Disulfide Bond in Stability of Lipocalin‐Type Prostaglandin D Synthase Yoshiaki Teraoka 1, Yoshiaki Teraoka1, Shogo Atsuji1, Young‐Ho Lee2, Yuji Goto2, Takashi Inui1 1Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Japan, 2Institute for Protein Research, Osaka University, Japan Lipocalin‐type prostaglandin D synthase (L‐PGDS) is a unique multi‐functional protein, acting as a PGD2 synthase and an extracellular transporter for small lipophilic molecules. By using its binding capability for various hydrophobic ligands, we recently proposed a novel drug delivery system of L‐PGDS as a delivery vehicle for poorly water‐soluble compounds. L‐PGDS has a classical lipocalin fold, which consists of an eight‐stranded anti‐parallel ß‐barrel and an internal disulfide (SS) bond. The SS bond links the C‐terminus of L‐PGDS to its ß‐hairpin between C and D strands (ßC–ßD hairpin) and is highly conserved in other lipocalins (see Figure). The purpose of this study is to reveal the effects of SS bond on the stability of L‐PGDS. We constructed a disulfide bond mutant (C89A/C186A) by site‐directed mutagenesis and investigated its conformational and thermal stability using circular dichroism (CD) spectroscopy. CD measurements showed that the removal of SS bond slightly changed the tertiary structure of L‐PGDS and decreased the Tm values from 68.8 to 57.8°C in PBS. Next, we performed two dimensional 1H‐15N heteronuclear single quantum coherence experiments with molecular dynamics (MD) simulations. The cross‐peaks of 11 residues disappeared without the SS bond and were mainly located in the ßC–ßD hairpin of L‐PGDS. Furthermore, the ßC–ßD hairpin of C89/C186A was significantly fluctuated in MD simulations for 500 ns. Experimental and theoretical results revealed that the lack of SS bond affected the chemical environment and the dynamics of ßC–ßD hairpin in L‐PGDS. Taken together, the highly conserved SS bond in L‐PGDS plays an important role in the maintenance of its structure in physiological conditions. POS106 Expression, purification and functional reconstitution of the ligand‐domain of umami taste receptor for the development of umami taste sensor Sae Ryun Ahn 1, Ji Hyun An1, Il Ha Jang1, Jyongsik Jang1, Tai Hyun Park1 1School of Chemical and Biological Engineering, Seoul National University, South Korea Human can taste five basic taste qualities; sweet, umami, bitter, salty and sour. Ikeda discovered the umami taste in 1908 and named from “UMAI”. Recently interest in food culture has been raised, thereby seasoning market has been growing up. Most seasonings are containing umami tastants such as MSG, IMP, GMP and etc. Therefore, many efforts of making taste detection tools have been developed in last decade. However, some established detection tools have low sensitivity and selectivity. Also, they need any amount of information before detection, thereby it is not possible to detect unknown compound. Herein, our group developed high‐performance bioelectronic sensor for umami taste using human G‐protein coupled receptor (GPCR). Umami taste receptor is heterodimeric class C GPCRs; T1R1/T1R3. According to recent research, extracellular N‐terminal domain of T1R1 is ligand domain for umami tastants. Therefore, we expressed the extracellular N‐terminal domain of T1R1 from Escherichia coli and then purified and functional reconstituted. Graphene field‐effect transistor (FET) was functionalized with the reconstituted protein. The well‐established bioelectronic sensor for umami taste using extracellular N‐terminus domain of T1R1 was able to detect MSG with very low concentration (ca. 1 nM) and selectively detect the target ligand. Also, this bioelectronic sensor detect enhancing effect using IMP as human sensory system. This bioelectronic tongue will be a useful tool for food and beverage industry and for the study of class C GPCRs. POS112 Entropically‐Challenged Tandem‐repeat Proteins: Breakdown of Nearest‐Neighbour Cooperativity Albert Perez‐Riba 1, Alan Lowe2, Ewan Main3, Elizabeth Komives4, Laura Itzhaki1 1University of Cambridge, UK, 2University College London, UK, 3University of London, Queen Mary, UK, 4University of California San Diego, USA Folding cooperativity in tandem‐repeat proteins follows a different paradigm from that of globular proteins with significant repercussions for stability and function. Repeats are coupled to their nearest‐neighbours, and the stability of the tandem‐repeat arrays follows a 1‐D Ising model. Despite their structural differences, all repeat proteins studied to date follow this paradigm, including for example the helical ankyrin repeats and tetratricopeptide repeats (TPRs) which differ greatly in inter‐repeat loop length. TPRs have short loops with little apparent impact on the folded structure. As TPRs are promising building blocks for biomaterials research, we herein explored the possibilities of loop extension to enable further material diversification. For this purpose, we designed a series of consensus TPR proteins with different numbers and lengths of loop extensions. Interestingly, we found that loop extensions are conservative with respect to the folded structure, but the effect on stability does not follow the additive rule of the Ising model. The TPR arrays are destabilized not only due to the entropic penalty but also due to decoupling of the folded repeats. Furthermore, the stability of these repeat arrays no longer increases linearly with the number of repeats. Our findings provide insights into the relationship between structure and folding of repeat proteins, which will be important for predicting the biophysical and functional properties of natural repeat‐protein sequences and with implication for exploitation in the design of protein‐based biomaterials. POS144 Quantifying Microscopic Pathway Heterogeneity in Protein Folding Soundhararajan Gopi 1, Sayan Ranu2, Athi N. Naganathan1 1Department of Biotechnology, Bhupat & Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai 600036, India., 2Department of Computer Science and Engineering, Indian Institute of Technology Madras, Chennai 600036, India Quantifying the number of microscopic routes available for a protein molecule to fold has been a challenging venture. This has been challenging to address experimentally as single‐molecule studies are constrained by the number of observed folding events while atomistic simulations are restricted by sampling and the inability to reproduce thermodynamic observables directly. We work around these constraints to provide a comprehensive picture of the intricate network of folding routes by borrowing concepts from statistical mechanics, physical kinetics and graph theory. We study the folding behavior of five single‐domain proteins that folds at different time‐scales and possess varying secondary structure content and topologies, at two thermodynamic conditions, from an analysis of 100,000 folding events generated from a statistical mechanical model. The resulting microstate energetics, from more than a million conformational states, predicts various ensemble based measurements including the results of protein engineering experiments, thermodynamic stabilities of secondary‐structure segments, and the end‐to‐end distance estimates from single‐molecule studies. Graph theoretical networks reveal the presence of rich ensembles of folding mechanisms that are generally masked in reduced representations (e.g. a one‐dimensional free energy profile). It is shown that the relative flux order through the numerous folding routes depend on the stability conditions, topological complexity and the resolution at which the folding events are observed. Our predictive methodology thus reconciles the contradictory observations from experiments and simulations and provides an experimentally consistent avenue to quantify folding heterogeneity. POS145 Understanding the Molecular Origins of Temperature‐Induced Collapse Transition in Disordered Proteins Sneha Munshi 1, Athi N Naganathan1 1Department of Biotechnology, Bhupat and Jyoyti Mehta School of Bio Sciences Indian Institute of technology Madras, India The molecular origins of temperature‐induced collapse in intrinsically disordered proteins (IDPs) are currently unclear. To explore this phenomenon, we perform a multi‐probe spectroscopic study of the intrinsically disordered Cytidine Repressor‐DNA Binding Domain (CytR‐DBD) that includes dynamic light scattering, circular dichroism, fluorescence, NMR and scanning calorimetry measurements. We find that CytR loses its secondary structure with increasing temperature, but with significant local and global structural rearrangements preceding the actual collapse transition. Remarkably, the hydrophobic surface burial happens thermodynamically much earlier than the collapse transition. The collapse transition regime that spans ∼20 K is characterized by a continuous increase in enthalpic fluctuations, the first such measurement for an IDP, arguing against a specifically collapsed state. We also observe little changes in the binding affinity of CytR to the cognate DNA in transition regime despite the disordered molecular dimensions reducing by ∼7 Å. Our experimental results together with implicit solvent simulations suggest that the CytR collapse transition is second‐order like. Furthermore, they seem to go hand‐in‐hand with an increase in the number of non‐native hydrogen bonds but with only minimal contributions from hydrophobic surface burial. POS147 Tuning the Continuum of Structural States in the Native Ensemble of a Regulatory Protein Abhishek Narayan 1, Athi N Naganathan1 1Department of Biotechnology, Bhupat and Jyoyti Mehta School of Bio Sciences Indian Institute of technology Madras, India The mesoscale nature of proteins allows for an efficient coupling between environmental cues and conformational changes enabling their function as molecular transducers. Delineating the precise structural origins of such a connection and the expected spectroscopic response has however been challenging. In this work, we perform a combination of urea‐temperature double perturbation experiments and theoretical modeling to probe the native conformational landscape of Cnu, a natural thermosensor protein belonging to the Hha‐family. Proteins of the Hha‐family, conserved among enterobacteriaceae, have been implicated in dynamically regulating the expression of pathogenic genes upon temperature shifts. Cnu displays probe dependent unfolding, graded increase in structural fluctuations and temperature‐dependent swelling of native ensemble within the physiological range of temperatures. In addition, we observe unique ensemble signatures that point to a continuum of conformational sub‐states in the native ensemble that respond intricately to perturbations upon monitoring secondary‐, tertiary‐structures, distances between an intrinsic FRET pair and hydrodynamic volumes. Binding assays further reveal a weakening of the Cnu functional complex with temperature, highlighting the molecular origins of signal transduction critical for pathogenic response in enterobacteriaceae. POS150 Structural Insights Gained into SCCH Domain of Yeast Ubiquitin Activating Enzyme E1 Brinda Panchamia 1, Ratna Prabha Chivukula1, Mukesh Kumar2, Vishal Prashar2 1The Maharaja Sayajirao University of Baroda, India, 2Bhabha Atomic Research Centre, India Conjugation of ubiquitin (Ub) to proteins is a post‐translational modification found in eukaryotic cells, which is responsible for maintaining intracellular protein levels and regulating protein activity. Ubiquitination of proteins is catalyzed by a set of three enzymes. The first enzyme, ubiquitin activating enzyme (E1) activates and transfers Ub to conjugating enzyme (E2). Yeast E1 is monomeric. It is a multidomain protein of 110kDa size. Deletion of the gene for E1 results in lethality. E1 adenylates ubiquitin and forms thioester bond. The crystal structure of yeast E1‐Ub complex has already been resolved by x‐ray crystallography. However, the detailed mechanism of folding of domains is unknown. The Second Catalytic Cysteine Half‐domain (SCCH) is one of six domains of E1 spanning 598–860 residues. It has catalytic cysteine at 600th position which forms thioester bond with C‐terminal glycine residue of adenylated ubiquitin. SCCH has core motif of ∼80 residues around catalytic cysteine. Moreover, SCCH is linked with its neighbouring domain by 18‐residue linker, which suggests conformational changes in the domain are necessary for its function. Here, to understand the structural features of SCCH we have cloned, expressed and purified it and carried out structural studies using circular dichroism (CD) and fluorescence spectroscopy. Gradual unfolding of SCCH observed in guanidine hydrochloride established that the peptide has intact structure. CD spectra of SCCH peptide showed that it mainly consists of a‐helices similar to SCCH domain present in crystallized E1‐Ub. This suggests SCCH domain can fold into native like structure independent of rest of the protein. POS152 Unfolding Simulations and structural analysis provide clues for the role of alpha‐beta loops in the stability of the TIM Barrel fold Ramakrishna Vadrevu 1, Rajashekar Kadumuri1 1BITS Pilani, India TIM barrel fold is formed by the repetition of the basic ßaß building motifs in which the ß‐strands are followed by a‐helices eight times, alternating in sequence and structure. The architecture imposes that alpha‐beta and beta‐alpha loops connecting alpha‐helices to the adjacent beta‐strands and the beta‐strands to the alpha‐helices contribute to stability and function respectively. The barrel architecture provides a unique opportunity to address if properties such as reduced loop flexibility, increased inter residue interactions arising from within the loops to their flanking and distant secondary structural elements are dominating in the alpha‐beta loops. Molecular dynamics simulations coupled with structural analysis on the alpha subunit of tryptophan synthase (aTS) reveal a clear distinction between alpha‐beta and beta‐alpha loops in their flexibility derived from the thermal factors and root mean square fluctuations. A clear distinction in their resistance to thermal and chemical unfolding is also observed from unfolding simulations. The first four beta strands (beta‐strands1–4) along with a couple of alpha‐helices show more stability than the other four strands (beta5‐beta8) and that the alpha‐beta loops are more rigid and are stable to unfolding than their beta‐alpha counterparts. Increased number of inter residue non‐covalent interactions including hydrogen bonds, ionic and hydrophobic contacts from within the loops and their flanking secondary structural elements are observed in alpha‐beta loops. Therefore, non‐covalent interactions dominant in the alpha‐beta loops could contribute to the overall fold stability of aTS. Intriguingly, the features observed in the prototypic aTS, can be recapitulated in the entire set of TIM barrels. POS155 Multidomain Protein Folding Pathways: Deciphering the Complexity of Folding Reactions in Large Proteins Vipul Kumar 1, Tapan K. Chaudhuri1 1Indian Institute Of Technology, Delhi, India Large multidomain proteins of loosely dependent/independent domains can fold to their native states despite of slower refolding rates and possible domain interactions. To understand this seemingly simple yet complex phenomena, studies on Malate Synthase G, an 82kDa enzyme have been performed using classical equilibrium and kinetics techniques. Previously GdmCl‐mediated denaturation established, a folding mechanism through native like on‐pathway species that aggregates at higher concentrations. Here we explore details of un/folding mechanism with urea‐assisted denaturation. The equilibrium studies suggest at least two intermediates, IN (native like helix content) and IO, in the system which are explored using kinetic methods. During single jump refolding, conversion of entire population to IO is observed within mixing dead time (∼100ms) irrespective of initial unfolding conditions. Subsequent refolding from this obligatory intermediate is a biphasic phenomenon that exhibits off pathway like rollover in two refolding arms. Additional Viscosity dependent refolding experiments demonstrate diffusion controlled nature of the slower phase. Interrupted refolding experiments confirm that IN accumulation is the fast phase (t=51s), that produces native state, probably via slow domain rearrangement step (t=227s). Triple phasic unfolding reactions revealed although intersecting but linear unfolding arms in chevron which excludes additional unfolding intermediates. Thus minimal model for the protein's folding is essentially through a compulsory off pathway intermediate that folds to native state in a sequential manner. POS168 Deep convolutional networks for protein structure quality assessment Georgy Derevyanko 1, Guillaume Lamoureux1, Sergei Grudinin2 1Concordia University, Canada, 2INRIA Grenoble – Rhone‐Alpes, France Structure quality assessment (QA) is a crucial step during computational protein structure prediction. During this step one predicts the quality of the candidate structures and selects those that are likely to be closest to the native one. Current QA models typically rely on a predefined list of structural features such as contact distances, secondary structure, or solvent exposure. While many of these features are useful in defining the quality of a structure, it is expected that an approach based on deep learning, in which the features themselves are learned from the data, would perform better. Deep convolutional networks have recently achieved outstanding performance in computer vision. In this work we explore the application of 3D deep convolutional networks for the quality assessment of individual structures. Figure 1 shows the architecture of the network. The input of the model is the 3D atomic density broken down by atom types and the output is a real number that ranks the structure. The model is trained to rank a set of decoy structures according to their proximity to the corresponding native structure. We trained our model on the CASP7 to 10 datasets and assessed the quality of the predictions on the CASP11 dataset. The datasets were pre‐processed with SCWRL. Table 1 shows that this approach (3DCNN) achieves results comparable to state‐of‐the‐art QA methods without the need for any feature engineering. The application of deep learning algorithms to protein model QA is still sparsely explored and we believe that this work will facilitate the use of similar techniques to the protein folding problem. POS266 Refolding and Purification of Unmodified Human Elongation Factor 2 Brian Grossman 1, Jed Weldon1, Nirja Patel1, Joshua Ostovitz1, Nathaniel Donahue1 1Department of Biological Sciences, Towson University, 8000 York Road, Towson, Maryland, USA Translation elongation factor 2 (EF2) is an essential component of protein synthesis that advances ribosomes along mRNA in eukaryotes and archaea. EF2 contains a unique post‐translationally modified histidine amino acid called diphthamide. Diphthamide has been found only in EF2. Its bacterial homolog (elongation factor G) lacks the modification. Previous work suggests that diphthamide is important for translation fidelity, but its precise function remains elusive. The diphthamide synthesis pathway is also complex, with a mechanism divided into three stages that requires seven different proteins in yeast (DPH 1–7). The synthesis of diphthamide has yet to be reconstituted in vitro. In order to study both the function of diphthamide and its synthesis, we are developing a protocol to purify human EF2 (hEF2) without the diphthamide modification. We chose to express hEF2 in E. coli due to the lack of diphthamide synthesis in bacteria. When overexpressed, hEF2 partitioned into the insoluble fraction of E. coli lysates, which was subsequently purified. We then evaluated refolding conditions to increase hEF2 solubility. Our results suggest a strategy for the preparation of hEF2 lacking diphthamide. Future directions include the use of mass spectrometry to confirm the absence of diphthamide from hEF2 and further modification of refolding conditions to maximize hEF2 solubility. POS274 Comparing the folding dynamics of prion proteins from species with different disease susceptibility at the single‐molecule level Michael Woodside 1, Uttam Anand1, Craig Garen1 1University of Alberta, Canada Misfolding of the prion protein PrP causes the prion diseases, including BSE in cattle, scrapie in sheep, chronic wasting disease in cervids, and CJD in humans. PrP is highly conserved across species, yet disease susceptibility varies widely: deer and bank voles are very susceptible, for example, whereas rabbits and horses are very resistant. These differences result from just a few amino‐acid changes, but how these changes alter the misfolding remains unclear. We explored the effects of species‐related sequence differences using force spectroscopy to observe the folding dynamics of single PrP molecules held by optical tweezers. We compared the behavior of hamster PrP (HaPrP) to that of rabbit PrP (RbPrP) and bank vole PrP (bvPrP): hamsters are disease‐susceptible, rabbits are resistant, and bank voles are extremely susceptible. Unfolding and refolding trajectories were measured while ramping the applied force up/down. The resulting force‐extension curves (FECs) revealed the existence of any on‐pathway intermediates or misfolded (off‐pathway) states, reflecting also energetic and kinetic properties. In contrast to HaPrP, which exhibited two‐state folding, RbPrP folded via multiple on‐pathway intermediates; in both cases, native folding was rapid and misfolded states were not detected in FECs. FECs of bvPrP showed 0–1 on‐pathway intermediates, with much slower kinetics; notably, misfolded states with second‐scale lifetimes were also observed. By relating the observed contour‐length changes to structural features in PrP, we propose possible intermediates in the native folding pathways for RbPrP and bvPrP. These results show that the subtle sequence differences between PrP from different species produce important differences in the folding dynamics that may be relevant to disease susceptibility. POS289 Breaking Down Protein Metamorphosis: Primary Drivers Behind the Structural Transformation of the Bacterial Virulence Factor RfaH Cesar Antonio Ramirez‐Sarmiento 1, Pablo Galaz‐Davison1, Elizabeth A Komives2, Irina Artsimovitch3 1Institute for Biological and Medical Engineering, Schools of Engineering, Medicine and Biological Sciences, Pontificia Universidad Catolica de Chile, Chile, 2Department of Chemistry and Biochemistry, University of California San Diego, USA, 3Department of Microbiology and The Center for RNA Biology, The Ohio State University, USA Proteins commonly fold into a single native state, a basin on the energy landscape low enough to be stable in solution and broad enough to allow motions associated with protein function. However, a group of so‐called metamorphic proteins challenge this paradigm by folding into at least two structurally different yet functionally relevant configurations. The bacterial transcriptional regulator RfaH constitutes a dramatic example of this behavior, where its C‐terminal domain (CTD) refolds from a stable a‐helical hairpin bound to and occluding the RNA polymerase binding site of the N‐terminal domain (NTD) into a small ß‐barrel. Here we explore in detail the metamorphic process of RfaH using a combination of multiscale molecular dynamics and biophysical experiments. Dual‐funneled coarse‐grained simulations highlight the role of specific NTD‐CTD interactions in stabilizing the a‐state, with the CTD residue F130 having a dual role in stabilizing both folds, and the impact of RNA polymerase in facilitating the metamorphic process. Moreover, per‐residue free‐energy decomposition from all‐atom simulations using the confine‐convert‐release method (CCR) unveils local structural preferences towards either native state within the CTD, with residues contributing to stabilize the a‐state localized in the upper part of the a‐helical hairpin (residues 135–140) in strong consistency with previous NMR evidence of the effect of mutations in this region. Comparison between computational simulations and hydrogen deuterium exchange mass spectrometry experiments on RfaH exhibit a remarkably high correlation, demonstrating that the combination of these strategies unveils the determinants of the structural transformation of RfaH and reverse this information back to its primary sequence. FUNDING: Fondecyt 11140601. POS402 Combining Differential Scanning Calorimetry and Isothermal Titration Calorimetry to Characterize Reduced Zinc bound Superoxide Dismutase 1 Harmeen Deol 1, Elizabeth Meiering1 1University of Waterloo, Canada Superoxide dismutase 1 (SOD1) is a ubiquitously expressed metalloenzyme that reduces oxidative stress in cells by catalyzing the dismutation of superoxide radical. Mutations in the gene encoding for SOD1 have been linked to the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS). The prevalent hypothesis for how mutant SOD1 causes disease is the formation of toxic intracellular protein aggregates. SOD1 undergoes posttranslational modifications in vivo, including metal binding, disulphide bond formation and dimerization, to reach its final maturation state, a stable homodimer. Recent studies support the theory that more immature states may play a key role in the disease pathology, and the monomeric species may be linked to toxicity. The zinc‐bound form of SOD1 with a reduced disulphide bond (E,Zn SH SOD1) has been the centre of debate with contradictory literature published regarding whether it exists as a monomer or dimer. Although this key state has been in widely discussed, relatively little has been characterized compared to other forms of this protein. Here we investigate E,Zn SH SOD1 using a combination of isothermal titration calorimetry (ITC), to measure dimer dissociation, and differential scanning calorimetry (DSC), to measure global unfolding of the protein, in order to dissect the contribution of mutations to protein stability. Results are compared to the effects of mutations in other forms of SOD1 to better understand protein maturation and different roles of SOD1 species in misfolding and disease. POS439 Characterization of the domain swapping mechanism of the forkhead domain of human FoxP1 at a single‐molecule level Jorge Babul 1, Exequiel Medina2, Exequiel Medina2, Hugo Sanabria3, César A. Ramírez‐Sarmiento, Jorge Babul4 1Facultad de Ciencias, Universidad de Chile, 2Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Departamento de Biología, Facultad de Ciencias, Universidad de Chile, 3Department of Physics and Astronomy, School of Health Research, Clemson University, South Carolina, USA, 4Institute for Biological and Medical Engineering. Pontificia Universidad Católica de Chile, Chile Domain swapping is a relevant folding and oligomerization process, described in several proteins, in which two or more subunits exchange specific secondary structure segments to obtain an intertwined species. The functional relevance of this mechanism has been described in widely studied proteins such as p13suc1 and diphtheria toxin, where the thermodynamic and kinetic behaviors of this mechanism have shown that the unfolded state is a requirement to obtain the dimeric (or oligomeric) conformation. Crystallographic and in solution experiments show that the DNA‐binding domain of human FoxP proteins reaches its dimeric structure via domain swapping. Specifically, we have obtained relevant biophysical details related to this process using FoxP1 as a model, which presents a dissociation constant two or three orders of magnitude lower than most of the domain‐swapped proteins. To obtain a detailed structural information of the domain‐swapped dimer of FoxP1, fluorophores were attached to cysteines that were introduced at specific regions of the protein to study the dynamic of possible sub‐ensembles conformations via single‐molecule FRET, using the multiparameter fluorescence detection (MFD) approach. Our data showed that the timescale of events is region‐dependent and is in accordance with differences in flexibility observed in the wild‐type protein. On the other hand, two conformations of the dimeric protein were observed: a closed sub‐ensemble consistent with the domain‐swapped FoxP1, and an open sub‐ensemble whose distances and dynamics possibly correspond to an unstructured and flexible protein. These findings will be relevant to understand the kinetic and thermodynamic properties of this protein studied in multiplo. Funding Fondecyt 1130510, 11140601 and doctoral fellowship 21130478. POS442 On the understanding of the low catalytic activity of TIM monomeric mutants. Janet Garduño 1, Edgar Vázquez‐Contreras1, María Elena Chánez‐Cárdenas2 1UAM‐C, 2Laboratorio de Patologi´a Vascular Cerebral, Instituto Nacional de Neurologi´a y Neurocirugi´a Manuel Velasco Sua´rez Janet GArduño#, María Elena Chanez‐Cardenas2 and Edgar Vazquez‐Contreras3. 1. Natural Sciences, Universidad Autonoma Metropolitana Unidad Cuajimalpa, Ciudad de Mexico, Mexico. 2. Vascular Cerebral Pathology, Instituto Nacional de Neurología y Neurocirugía, Ciudad de Mexico, Mexico. In this work we describe the investigation of the differences in the structural and functional properties of the reported in the PDB Triosephosphate Isomerase monomeric mutants (monoTIMs). In particular regarding MonoTcTIM, a monomeric mutant derived from Trypanosome cruzi TIM (TcTIM) that was designed, synthetized, purified and characterized by our team (Zárate‐Pérez et al. 2009; Biochem Biophys Res Comm. 382: 626–630). The structural, functional and thermodynamic properties of monoTcTIM were compared with those of the available monomeric TIM mutants. This comparisons shown a like properties between those mutants like the main structural characteristics of the TIM barrel folding as well as differences in the three dimensional structures, many of them were derived of the differences found in the origin of the mutations performed in the amino acid sequence. No in all cases thermodynamic analysis were available, however some differences in the stability of the mutants were found. Finally we discuss on the origin if the low catalytic activity of TIM monomeric mutants. POS466 An evolutionary trend towards kinetic stability in the folding trajectory of RNases H Shion Lim 1, Susan Marqusee1, Eric Bolin1, Kathryn Hart2, Michael Harms3, Susan Marqusee1 1University of California Berkeley, USA, 2Washington University in St. Louis, USA, 3University of Oregon, USA Proper folding of proteins is critical to producing the biological machinery essential for cellular function. Over the course of evolution, the rates and energetics of a protein's folding landscape must be maintained such that the protein folds and remains folded over its biological lifetime. Developing a comprehensive understanding of how a protein's folding process is modulated during evolution is critical to our understanding and engineering of protein biophysical properties. In this study, we characterized the folding trajectories of ancestral proteins of the ribonuclease H (RNase H) family by using ancestral sequence reconstruction to access the evolutionary history between RNases H from mesophilic and thermophilic bacteria. We find that the overall folding pathway of RNase H is preserved over billions of years of evolution. Although thermodynamic stabilities diverge between the mesophilic and thermophilic lineages, kinetic stability increases along both, with the last common ancestor folding and unfolding faster than the modern descendants. The conserved folding intermediate permits this paradoxical uncoupling of thermodynamics and kinetics, and allows for the folding landscape to independently respond to different selective pressures on global stability and kinetic barriers. Additionally, further characterization of the RNase H folding pathway by fragment models and hydrogen‐exchange mass spectrometry (HX‐MS) reveals a distinctly changing folding pathway for the RNase H family, highlighting how subtle sequence changes can alter the structure of partially folded intermediates over the course of evolution. POS498 Examining the effect of ubiquitination on the energetics of substrate proteins Emma Carroll 1, Susan Marqusee1, Susan Marqusee1 1UC Berkeley, California, USA Ubiquitination is a common protein posttranslational modification in which the protein ubiquitin is attached to the primary amine of lysine (K) residues on the target protein. Ubiquitination is canonically associated with targeting proteins to the proteasome for degradation; however, ubiquitination is also involved in many other cellular processes, and the cell must carefully regulate which ubiquitinated proteins should be degraded. While proteasomal degradation is dependent on ubiquitin chain length and topology, it has also been shown that the conformational features of the tagged protein can play a role in proteasomal engagement. However, these biophysical factors driving the in vivo proteasomal degradation code remain largely unexplored. These types of studies are hampered by the difficulty of characterizing the energetics and dynamics associated with probing macromolecular complexes such as ubiquitinated proteins. I will present our approach and results on a model system for characterizing the stability and dynamics of proteins with and without defined ubiquitin modifications. POS508 Insights into Protein Unfolding Transition States from Modeling Protein Denaturation John Strahan 1, Sheila Jaswal1, Paul Cohen2 1Amherst College Department of Chemistry, Massachusetts, USA, 2Brown University Department of Emergency Medicine, Massachusetts, USA Understanding the energetic landscape of a protein's conformational space is key to understanding the mechanisms which underlie protein folding and denaturation. One of the most common ways to probe the free energy landscape is to destabilize the protein using a high concentration of a denaturant such as urea or guanidine hydrochloride so that the folding kinetics and thermodynamics can be measured and then extrapolated back to zero denaturant concentration. Several models have been proposed to carry out this extrapolation, the most common of which is a simple linear fit. This method works well for many proteins, but in several cases curvature is observed in the unfolding rates as a function of denaturant, leading to errors in extrapolated rate constants. In this work, we develop several models to understand the unfolding kinetics of kinetically stable proteases, for which the denaturant dependence of the unfolding rate can be measured over a large range of denaturant concentrations and temperatures. In order to develop our models, we make use of denaturant dependent transfer free energy data for several polar amino acids and the model hydrophobic amino acid analogue N‐acetyltryptophanamide. We have found that the curvature in activation free energy plots can be well modelled by scaling contributions from the solvation of a model nonpolar and polar amino acid. Our results further suggest that the solvation of polar surface area makes a large contribution to the denaturant's stabilizing effect on the transition state. POS510 From Folding to Function: Investigating the Structural Dynamics Underlying the Evolved Plasticity of Folding‐Assisted Landscapes Max Paul 1, Sheila Jaswal2, Katie Ventre2, Abel Samanez2, Catherine Amaya3, Kendall Melvin2, Kimberly Burnett2 1Amherst College Biochemistry and Biophysics Program, Massachusetts, USA, 2Amherst College Department of Chemistry, Massachusetts, USA, 3Yale University Molecular Biophysics & Biochemistry Program, Connecticut, USA The amino acid sequence for spontaneously folding proteins must balance requirements of both folding and function in a single landscape. Proteins that fold with the assistance of a chaperone or pro region are freed from this limitation. One hypothesis holds that functional features of a sequence can become more extreme when the requirement to spontaneously fold is removed. For example, the bacterial a‐lytic protease (aLP), which folds as a precursor, demonstrates extreme functional longevity once its 166‐residue pro‐region is removed. This extended lifetime is proposed to result from extreme tuning of its structural dynamics to minimize unfolding on any scale. To probe this hypothesis, we compared aLP dynamics to its mammalian homologue trypsin, which also folds as a precursor but functions once a hexapeptide is removed. Hydrogen Exchange Mass Spectrometry, fluorescence and loss of enzymatic activity were used to monitor structural dynamics ranging from local native‐state fluctuations to global unfolding. While both aLP and trypsin display a high barrier to activity loss (>25kcal/mol), aLP preserves its barrier up to higher temperatures. In the case of trypsin, small‐scale native state dynamics appear to render trypsin sensitive to proteolytic cleavage before actually inactivating the enzyme. aLP, however, only becomes sensitive to proteolysis after global unfolding. Initial experiments with the trypsin precursor demonstrate even more enhanced native state dynamics, which may be correlated with trypsinogen's significant thermodynamic stability. Additional experiments comparing the distinct folding and functional landscapes of aLP, trypsin and trypsinogen will illuminate the interplay between native‐state dynamics, stability, and function. POS512 N‐HXMS: A New Method to Map Protein Folding Landscapes Under Native Conditions Minjee Kim 1, Sheila Jaswal1, Jacob Witten2 1Amherst College Department of Chemistry, Massachusetts, USA, 2MIT Computational and Systems Biology Program, Massachusetts, USA Protein function depends on the proper calibration of stability, dynamics and structure of the native state. Defining a protein's landscape in terms of the conformations sampled, the differences in their thermodynamic stabilities, and the rates of transition between them has been an essential approach to investigate the link between function and energetics. Traditionally, landscape parameters are measured using equilibrium denaturation and kinetic chevron analysis monitored by spectroscopic methods. In addition to requiring large numbers of measurements as a function of denaturant and/or temperature to induce bulk unfolding under conditions far from native, such studies are limited to proteins that unfold and refold reversibly on a short timescale without misfolding or aggregating. We have developed an approach using native‐state hydrogen exchange coupled with mass spectrometry (N‐HXMS) that addresses these challenges. Fitting the mass change of N‐HXMS time course data on intact proteins, our method extracts landscape parameters from limited measurements under non‐denaturing conditions. We have validated our approach by verifying landscape parameters measured by N‐HXMS across HX regimes with those determined by traditional denaturation experiments for the model two‐state protein, Protein L. In addition, we have explored the potential of N‐HXMS under certain conditions to allow simultaneous determination of the unfolding and folding rate constants from a small number of HXMS time courses. This work helps establish N‐HXMS as a possible alternative to chevron analysis and equilibrium denaturation that requires less time and material for two‐state proteins, and as a milder method to probe landscapes of proteins in general. POS513 Probing the Folding Landscape of Alpha 1‐Antitrypsin Upneet Kaur 1, Weiwei Kuo1, Daniel Deredge3, Haiping Ke1, Eugenia Clerico1, Lizz Bartlett3, Patrick Wintrode2, Lila Gierasch1, Anne Gershenson1 1Department of Biochemistry and Molecular Biology, University of Massachusetts Amherst, USA, 2University of Maryland School of Pharmacy, USA, 3Institute of Applied Life Sciences, University of Massachusetts Amherst, USA The secreted serpin a1‐antitrypsin (a1AT) regulates serine proteases associated with inflammation. How a1AT folds in the endoplasmic reticulum (ER) is not well understood, and misfolding can result in a1AT deficiency and lung disease. The topologically complex serpin, fold consists of two domains, an aß and mainly ß domain, both of which contain regions from near the N‐ and the C‐termini. This complicated fold along with serpin misfolding diseases raise the question of how large proteins like a1AT (394 amino acids) with non‐sequential domains fold in the cell. One can hypothesize that relatively long proteins like a1AT may fold co‐translationally. To mimic co‐translational folding in vitro, a1AT N‐terminal fragments predicted to fold autonomously were purified and characterized. Analytical ultracentrifugation experiments show that the 1–190 fragment, which consists of the N‐terminal piece of the a/ß domain, is the only monomeric fragment. Protein denaturation monitored by far UV circular dichroism reveal that this N‐terminal fragment also displays significant amounts of secondary and likely tertiary structure suggesting that this fragment could fold co‐transitionally. The longer fragments 1–290 and 1–323 adopt multiple oligomeric states. Both of the oligomeric fragments contain an incomplete ß rich domain that may induce aggregation, and in cells these regions of a1AT likely need to interact with chaperones and/or to form stable hydrogen bonds by folding quickly in order to avoid aggregation and disease‐associated polymerization. 11. INTRINSICALLY DISORDERED PROTEINS POS068 Tuning the Growth and Maturation of Phase‐Separated Elastin‐Based Droplets Through Amino Acid Sequence Mutations Lisa Muiznieks 1, Fred Keeley1, Régis Pomès1 1Hospital for Sick Children, Toronto, Canada Liquid‐liquid phase separation underlies the spontaneous formation of protein‐rich droplets in a protein‐poor solution phase, concomitant with a sharp increase in solution turbidity. Phase separation is typically triggered in response to a stimulus such as change in protein concentration, ionic strength, or temperature, and may be reversible or precede gelation, fibre formation, or aggregation. Phase separation is a well‐known property of synthetic polymers, and is exploited for material design, for example, capsules for drug delivery and fast‐setting hydrogels, but is not well understood for proteins. Some examples of phase‐separating protein systems include lens gamma crystallin (implicated in cataract formation), ‘membraneless organelles’ (transiently formed intracellular compartments that regulate molecular interactions), and self‐assembling elastic proteins, including some spider silks, insect resilin and vertebrate elastin, for which phase separation is on‐pathway for the formation of elastic materials. Here we studied the phase separation of model polypeptides based on the protein elastin. These polypeptides contain both regions of well‐defined secondary structure and intrinsic disorder. Droplet growth and biophysical properties were monitored using a variety of microscopy and optical spectroscopy techniques. Amino acid sequence mutations, particularly those affecting fraction secondary structure, modulated the size, stability, reversibility and interactions of phase‐separated droplets. These data indicate strategies for the functional tunability of droplets for application such as the rational loading of small molecules. POS151 Promiscuous but selective: how intrinsically disordered BH3‐only proteins regulate apoptosis through binding to BCL‐2 like proteins Liza Dahal1, Jane Clarke1 1University of Cambridge, UK The BCL‐2 family of proteins plays a central role in regulating cell survival and apoptosis. To date, six pro‐survival and at least two pro‐apoptotic BCL‐2 proteins have been identified. A third group of ten pro‐apoptotic proteins (termed BH3‐only) are intrinsically disordered, but form a contiguous helical segment upon binding to their partner BCL‐2 proteins. The disordered nature of these proteins might be the source of promiscuous but selective binding, thus increasing the diversity and flexibility of this intricate network. Here, we use kinetic and thermodynamic analyses to understand the biophysical nature of these interactions, providing insights into the underlying molecular mechanisms regulating apoptosis. POS167 Effect of Environmental Stress on Genomic Instability in the C‐Terminal Domain of RNA Polymerase II in S. cerevisiae Taylor Stewart1, Tokio Sano1, Stephen M. Fuchs2 1Tufts University, Department of Biology, USA, 2Tufts University, Department of Biology, Allen Discovery Center at Tufts University The carboxyl‐terminal domain (CTD) is an essential domain of the largest subunit of RNA polymerase II, Rpb1p, and is composed of 26 tandem repeats of a seven‐amino acid sequence, YSPTSPS. At least 8 repeats are required for survival, but we have recently shown that yeast with a suboptimal CTD length promote spontaneous expansion and contraction of the coding region to improve fitness (Morrill et al. 2016). In this work, we make use of a tet‐off reporter system for RPB1 to monitor expansion and contractions of the repetitive CTD. The effects of various environmental stresses on the rate of CTD expansion and contraction, including temperature, salt, and osmotic stress, were investigated using the reporter system and fluctuation assays. Our results suggest that several of these stresses, especially hyperosmotic stresses such as NaCl and KCl, increase the rate of mutation in the CTD. Stress‐induced instability in a repetitive protein domain may therefore lead to phenotypic diversity that enables the population to better cope with the environmental stress. POS170 PolyQ tracts as efficient C‐capping elements for coiled‐coils Albert Escobedo 1, Busra Topal1, Jesús García1, Oscar Reina1, Camille Stephan‐Otto Attolini1, Xavier Salvatella1 1Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Spain Poly‐glutamine (polyQ) tract expansions have been linked to a variety of neurodegenerative diseases. The conservation of such sequences despite evolutionary pressure points to a relevant role, which is suggested to involve their organization into secondary structure elements. For the particular case of the androgen receptor (AR) we recently reported that the Leu‐rich segment N‐terminal to the polyQ tract acts as a helical N‐capping sequence that propagates helicity into the tract itself [1]. Based on that, we have acquired in vitro CD and NMR as well as in silico MD data on a battery of peptides showing that the helicity of the sequence positively correlates with the number of glutamines in the tract up to the values found in the average population (16–25 residues, depending on ethnicity), and that helix stabilization depends on glutamine sidechain‐mediated hydrogen bonds. This supports a C‐capping role for the polyQ tract, as a minimum number of glutamine residues are required to stabilize the helicity while further growth of the tract is detrimental because of increased aggregation rates. Proteome analysis shows that regions predicted to fold into coiled‐coils are highly enriched in adjacent sequences N‐terminal to polyQ tracts, thus providing the grounds for a general role of such tracts as C‐caps for these helical elements. [1] Eftekharzadeh, Bahareh et al. Sequence Context Influences the Structure and Aggregation Behavior of a PolyQ Tract. (2016) Biophysical Journal, Volume 110, Issue 11, 2361 – 2366. POS202 The transactivation domain of the androgen receptor drives its phase separation Elzbieta Szulc1, Jill Bouchard2, Tanja Mittag2, Xavier Salvatella1 1IRB Barcelona, Spain, 2St. Jude Children's Research Hospital, Memphis, USA Intrinsically disordered proteins and intrinsically disordered regions are especially common in eukaryotic proteomes [1]. Many significant biological functions, like cellular signal transduction, transcription and translation, have been associated with protein disorder, underlying the importance of this phenomenon [2]. Recently, intrinsic disorder has been proposed to play a key role in liquid‐liquid phase separation (LLPS) [3]. LLPS in cell nuclei allows the organization of this organelle by the generation of membrane‐less organelles such nucleoli, splicing speckles, Cajal bodies, gems, and PML bodies [4]. The androgen receptor (AR) is a transcription factor that regulates the expression of a specific subset of genes by association with androgen response elements [5] and its dysfunction is associated with prostate cancer [6] and the rare neuromuscular disease spinal bulbar muscular atrophy [7]. The intrinsically disordered N‐terminal domain of AR is the largest in the family of nuclear receptors and constitutes 60% of the protein [8]. Here, we show that AR undergoes LLPS and present a preliminary characterization of this phenomenon. References: [1] Peng, Z. et al. Life Sci. (2015) [2] Dyson, H.J., Wright P.E. Nat Rev Mol Cell Biol (2005) [3] Uversky, V.N. et al. FEBS Letters (2015) [4] Mitrea, D.M., Kriwacki R.W. Cell Commun Signal. (2016) [5] Cato, A. C. et al. Embo J. (1987) [6] Huggins, C., Hodges, C.V. Cancer Res. (1941) [7] Beitel, L.K. et al. Front Neurol. (2013) [8] McEwan, I.J. et al. Nucl R Sig (2007) POS204 Structure and Self‐Assembly of Elastin‐Like Peptides: A Joint Molecular Dynamics and NMR Study Régis Pomès 1, Quang Huynh1, Sean Reichheld1, Sarah Rauscher2, Zhen Hao Wu1, Simon Sharpe1 1Hospital for Sick Children, Toronto, Canada, 2Max Planck Institute for Biophysical Chemistry, Germany Elastin endows tissues such as skin, arterial walls, lung alveoli, and the uterus with extensibility and elasticity. Elastin and elastin‐like peptides are structurally disordered and self‐aggregate via a liquid‐liquid phase separation process. Although elastin has been the object of biophysical investigation for over eighty years, the structural basis for the self‐assembly and the mechanical properties of elastin remains controversial. As an essential step towards elucidating the structural ensemble of elastin, we combine molecular dynamics simulations and NMR spectroscopy to study an elastin‐like peptide modelled after the sequence of alternating hydrophobic and cross‐linking domains of elastin. We perform extensive all‐atom molecular dynamics simulations totalling hundreds of microseconds and obtain detailed structural and dynamic data by NMR on the same peptide sequences. Simulation and spectroscopic results are in excellent agreement and show that although the peptide is highly disordered, it possesses a significant propensity for local secondary structure. The cross‐linking domains are characterized by fluctuating helical structure, whereas the hydrophobic domains adopt sparse and local hydrogen‐bonded turns. As a result, the individual domains do not form extensive interactions; they are collapsed but not compact, and they remain disordered and hydrated despite their predominantly hydrophobic character. These findings resolve long‐standing discrepancies between previous models of the structure and function of elastin and afford unprecedented insight into the physical and structural basis for the liquid‐liquid phase separation of disordered proteins. POS234 Peptide Maps Of Blood Plasma Proteins (Fibrinogen And Plasma Fibrin‐Stabilizing Factor) With Oxidative Modifications Anna Bychkova1, Alexandra Vasilyeva1, Lyubov' Yurina1, Anna Bugrova1, Maria Indeykina1, Alexey Kononikhin1, Eugene Nikolaev1, Mark Rosenfeld1 1N. M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Russia The blood plasma proteins being targets for reactive oxygen species (ROS) can serve as biomarkers of oxidative stress. Despite the significant amount of issues on oxidative modifications of proteins, there is still a general lack of studies aimed at the identification of oxidation sites in the molecules. A number of amino acid residues susceptible to oxidant in different structural elements of the two key blood clotting factors (fibrinogen and plasma fibrin‐stabilizing factor) were identified in the study by mass spectrometry method. Among the modified amino acids detected in the catalytic subunit of pFXIII, Tyr442 and Tyr481 are in calcium binding site of the molecule possibly bringing about the enzymatic activity loss. In respect to fibrinogen, location of the oxidized residue AaTrp302, nearby AaLys303 covalently cross‐linking with a2‐antiplasmin, may have an impact on the resistance of fibrin clot to plasmin hydrolysis. Oxidative alterations both of the residue AaMet476 and other sites revealed at aC‐domain can inhibit lateral aggregation of protofibrils. Oxidation of the residue BßMet367 along with other residues within the ß‐module structure, can affect the conformation of the fragment ß330–375 responsible for the lateral aggregation of protofibrils by reacting two ß‐modules. The information gained can play a crucial role to generating accurate protein profiles for quick analysis of modifications by brand new test systems. The study was supported by RFBR, research project Na. 15‐04‐08188a and Na. 16–34‐60244 mol_a_dk. The part of research related to peptides and PTM identification by high resolution mass spectrometry measurements was supported by the Russian Science Foundation Na. 16‐14‐00181. POS238 The Activities Of Patched‐1 Are Regulated By The Interactions Of Distinct Structural Modules Andrew Fleet 1, Jennifer PY Lee1, Paul A. Hamel1 1University of Toronto, Department of Laboratory Medicine and Pathobiology, Canada Patched‐1 (Ptch1) is the principal receptor of the Hedgehog (Hh)‐pathway, a signalling cascade crucial in directing morphogenesis. Based on the closely‐related primary structure of the integral membrane protein, Neimann‐Pick disease, type C1 (NPC1), whose structure was recently solved by cryo‐EM, we predicted that Ptch1 has distinct modular regions that interact to regulate its activities. The cytoplasmic domains of Ptch1 contain highly‐conserved protein‐binding motifs and are predicted to form an intrinsically disordered protein region (IDPR). These motifs bind to specific factors, including c‐src, PIK3R2 and Grb2, and mediate Hh‐signalling through distinct cascades. Subsequently, we demonstrated that the IDRP mediates Ptch1 oligomerization, but that this activity is not required for the principal function of Ptch1, repression of Smoothened (Smo) activity. However, the IDPR acts in a complex manner to control Ptch1‐dependent repression of Smo. Specifically, deletion of the “middle loop” generates a dominant form of Ptch1 that, despite binding to Hh‐ligand, constitutively‐represses Smo. This constitutive activity is reversed by introducing discrete mutations in the C‐terminal domain. We further investigated the activities of the extracellular and transmembrane modules using a series of deletion mutations as well as chimeric proteins that transposed analogous regions of Ptch1 and NPC1. These analyses revealed a complex interaction with the co‐receptor Brother of Cdon (BOC), which interacts with multiple regions of Ptch1. We have also shown that, like in NPC1, the activities of these regions are exquisitely sensitive to changes in the primary sequence. These data have begun to define the complex interactions between the structural modules of Ptch1 that regulate its many signalling and cell‐specific activities. POS254 Virulent phase separation in Mycobacterium tuberculosis Florian Heinkel 1, Joerg Gsponer1, Mark Okon1, Yossef Av‐Gay1, Lawrence McIntosh1 1University of British Columbia, Canada Protein phase separation or liquid‐liquid demixing is an important mechanism of compartmentalization in cells, creating specialized reaction and signalling environments by assembly of separated protein‐rich phases and clusters. It has been shown to be biologically relevant in eukaryotic cells in a wide variety of processes such as RNA processing, stress response and T‐Cell receptor signalling. Despite the fact, that liquid‐liquid demixing has only been reported in eukaryotes, the regulatory domain of a virulent ABC transporter in Mycobacterium tuberculosis (Mtb) possesses the two main characteristics associated with protein phase separation: weak interactions and multivalency. It consists of two phospho‐interacting Forkhead associated (FHA) domains connected by a disordered linker containing phospho‐acceptor sites shown to associate weakly with both FHA domains. This tandem‐FHA regulatory domain and the activating phosporylation are also critical for the function of the transporter and the virulence of the pathogen. Here, we show that upon phosphorylation by several serine threonine kinases from Mtb, the tandem FHA domain undergoes phase separation into liquid droplets with commonly reported dynamic characteristics and that this process is reversible by the Mtb serine threonine phosphatase PstP. Furthermore, we show that the linker connecting the FHA domains by itself possesses the ability to phase separate at higher concentrations, pointing to a synergy between classical modular interactions and weak self‐association of a disordered region. Our results suggest that protein phase separation plays a role as a mechanism of phospho‐dependent clustering of an ABC transporter in Mtb that is important for its virulence and is the first bacterial system shown to phase separate. POS267 Computational Investigation of Protein Disorder‐Order Transitions Induced by Mutations in Human Proteome Chen Li 1, Benjamin Porebski2, Julia McCoey1, Natalie Borg1, Geoffrey Webb3, Itamar Kass1, Malcolm Buckle4, Jiangning Song1, Adrian Woolfson5 1Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Australia, 2Medical Research Council Laboratory of Molecular Biology, University of Cambridge, UK, 3Faculty of Information Technology, Monash University, Australia, 4LBPA, ENS Cachan, CNRS, Université Paris‐Saclay, France, 5Pfizer Oncology, USA A disordered region (DR) in a protein is defined as a region that lacks a stable, well‐defined 3D structure. DRs are ubiquitous in proteins and highly related to human disease. In this study, we performed a system‐level computational investigation of protein disorder‐order transitions induced by point mutations extracted from the human proteome. Using bioinformatic analysis of predicted disordered regions, we analysed a large‐scale up‐to‐date human disease mutation dataset that contains 12,678 proteins and 31,251 disease mutations. We defined the disorder state changes of regions with mutations by dividing them into four categories: order‐to‐disorder (O‐>D), order‐to‐order (O‐>O), disorder‐to‐order (D‐>O) and disorder‐to‐disorder (D‐>D). In addition, given the variability in the predictions among the predictors tested, we cross‐referenced our human disease mutations and polymorphisms with a currently available database for experimentally validated disordered regions of proteins. The two resulting datasets, based on pure computational prediction and verified disordered regions, are expected to provide experimentally testable candidates for protein disorder‐order transitions and the investigation of microstructure formation induced by point mutations. POS310 Disorder, Evolution and Plasticity: Biophysical Signatures of the Arbitration of Apoptosis Basile Wicky 1, Tristan Kwan1, Jane Clarke1 1Department of Chemistry, University of Cambridge, UK The BCL2 family of proteins arbitrates cellular life or death via the intrinsic pathway of apoptosis. Composed of about twenty proteins, their interaction network allows for the regulation of cellular fate. Dissimilar in function (pro‐survival vs. pro‐apoptotic), and structural properties (folded vs. disordered), they all share a common sequence homology motif, termed BH3. These segments are involved in the binary interactions between BCL2's, but depending on which member, can either be found in disordered regions, or embedded in the protein core. Using a model tripartite system, we interrogate the role, and consequences, of this structural plasticity, and investigate whether unfolding is a pre‐requisite to binding. We use thermodynamics and kinetics to characterize the biophysical signatures of these coupled (un)folding and binding reactions, providing insight into the mechanisms underlying the molecular decision‐making of BCL2 proteins. POS377 Conformational changes and flexibility of the ArkA binding Abp1SH3 domain Kristina Foley 1, Katherine Ball1, Elliot Stollar2 1Skidmore College, New York, USA, 2Eastern New Mexico University, New Mexico, USA SH3 domains are the most common protein interaction domains, however, little is known about how intrinsically disordered proteins (IDPs) bind to these domains. One SH3 domain found in yeast, Abp1SH3, has a binding site for the ArkA IDP. We are using Molecular Dynamics (MD) simulations to model the Abp1SH3 domain and the Abp1SH3‐ArkA complex since IDPs are difficult to model experimentally. These simulations allow us to better understand how Abp1 residues are involved in ArkA binding and how binding contributes to conformational allostery and flexibility. SH3 domains are believed to have a two‐step binding process in which part of the peptide binds to Surface I, then the remaining part binds to Surface II (SII). In SII, the peptide binds to the SH3 domain via specific interactions, therefore, it is necessary for the residues located in this region to fluctuate to form these favorable bonds. MD simulations of the Abp1SH3 unbound structure show that five of the ten residues with the greatest atomic fluctuation are in SII. Gly57 also has a large atomic fluctuation, but is far from the ArkA binding site. Previously a large chemical shift was measured for Gly57 upon binding (Stollar et al., 2012). Of these ten flexible residues, Glu13 and Gly57 have an alternate phi dihedral angle that is frequently populated. This suggests that residue flexibility could enable allosteric conformational changes upon binding. POS390 Elucidating the molecular mechanisms underlying the viral hijacking of human protein‐protein interactions Jae‐Hyun Cho 1, Qingliang Shen1, Danyun Zeng1, Jie Shi2, Baoyu Zhao1, Wonmuk Hwang2, Pingwei Li1 1Department of Biochemistry and Biophysics, Texas A&M University, USA, 2Department of Biomedical Engineering, Texas A&M University, USA The 1918 Spanish influenza A virus (IAV) caused one of the most serious pandemics in human history. Unlike the common seasonal flu strains, the nonstructural protein 1 (NS1) of the 1918 IAV hijacks the interaction of human CrkII with cAbl kinase and c‐Jun‐N‐terminal kinase (JNK1), inhibiting the host antiviral immune response. A single amino acid mutation in the disordered proline‐rich motif of 1918 IAV NS1 enables this unique interaction with the N‐terminal SH3 domain of CrkII. Little is, however, known about its molecular mechanism. Recently, we have discovered that NS1 binds CrkII with strikingly rapid kinetics and high affinity. To our knowledge, this is the strongest binding observed for any known SH3‐ligand mediated protein interactions. Here, we performed X‐ray crystallography, NMR relaxation dispersion experiment, and fluorescence spectroscopy to determine the structural, kinetic, and thermodynamic mechanisms underlying the hijacking of CrkII by 1918 IAV NS1. In addition, our molecular dynamics simulation elucidates that the interplay between long‐range electrostatics and structural disorder of NS1 plays an important role in enhancing the binding affinity and kinetics to CrkII. These results provide unprecedented insights into the mechanism by which 1918 IAV NS1 hijacks CrkII and disrupts its interactions with critical cellular signaling proteins. Moreover, we show that the peptide derived from NS1 has a great potential as a protein‐protein interaction inhibitor between CrkII and cAbl kinase, which plays an important role in many cancers and bacterial infection. POS418 Disorder‐to‐order transitions in the regulation of synaptic vesicle release. David Eliezer1 1Weill Cornell Medicine, New York, USA Neurons communicate primarily via the release of chemical neurotransmitters from presynaptic nerve terminals and their detection post‐synaptically. Thus, the process of synaptic vesicle exocytosis is at the heart of neuronal function and cognition. Intriguingly, the proteins that play the key role in the fusion of vesicles with membranes, the SNARE proteins, are IDPs, and a number of the additional factors that regulate neuronal SNARE function are also intrinsically disordered, including the protein complexin. Complexin functions to inhibit synaptic vesicle fusion in a manner that requires a central region of the protein that binds to SNARE bundles as a helix, but disordered regions on either side of this central helix are required for efficient inhibition of vesicle release. Our work has helped to establish the mechanisms by which disorder‐to‐order transitions in both the C‐terminal domain and the accessory helix of complexin contribute to its function. A novel membrane‐curvature dependent structural transition helps to localize complexin and to gate its activity to the surface of synaptic vesicles, while the independently stable accessory helix acts to nucleate and propagate helical structure into the central helix region in order to facilitate SNARE binding. POS425 NMR Characterization of O‐GlcNAc Modified CKIIa Jacob Brockerman 1, Mark Okon1, Lawrence McIntosh1 1University of British Columbia, Canada O‐linked ß‐N‐acetylglucosamine (O‐GlcNAc) is a dynamic post‐translational modification found in higher eukaryotes. Remarkably, only a single enzyme, O‐GlcNAc transferase (OGT), catalyzes its transfer from UDP‐GlcNAc to serine and threonine residues on numerous cytoplasmic and nuclear proteins, and only a single enzyme, O‐GlcNAc hydrolyase (OGA), catalyzes its removal. Although dysregulated O‐GlcNAcylation has been linked to many diseases spanning cancer to neurodegeneration, there is no clear mechanistic understanding of how this widespread post‐translational modification regulates protein function. To help address this question, we have prepared a 13C/15N‐labeled sample of the C‐terminal tail of casein kinase 2a (CKIIa). The 60 residue polypeptide was O‐GlcNAc modified by co‐expression with OGT in E. coli. Based on NMR chemical shift and relaxation measurements, the CKIIa tail is intrinsically disordered and the presence O‐GlcNAc at Ser347 only minimally perturbs its local structural and dynamic properties. Additional minor sites of O‐GlcNAcylation were also identified. These data indicate that the reported effects of O‐GlcNAc on the activity of CKIIa do not arise by directly altering the conformational properties of its C‐terminal tail. We are now attempting to ligate this polypeptide onto the catalytic domain of the kinase using the Sortase A transpeptidase in order to investigate if O‐GlcNAcylation modulates potential interactions between these two segments of native CKIIa. POS428 Effect of Elongin B C‐terminus on Correlated Motions in HIV complex Lieza Chan 1, Elise Tierney1, Katherine Ball1, John Gross2 1Skidmore College, New York, USA, 2University of California San Francisco, USA HIV‐Vif is an intrinsically disordered protein that gains function through interactions with Elongin B (ELoB), Elongin C (EloC), and CBF‐ß making the VCBC complex and is responsible of the ubiquitination of virus fighting proteins allowing HIV to thrive in host cells. The crystal structure of the VCBC complex with Cullin 5 (Guo et al., 2014) contains a shortened EloB tail and previous NMR data has shown that with the full‐length EloB tail, the NMR signal improves through quenching of dynamics. Through Molecular Dynamics (MD), the conformations of the VCBC complex including the full‐length EloB C‐terminus were simulated. Principal Component Analysis (PCA) discerns global movements in the MD simulations through separation into essential dynamics based on correlated motions of the backbone. The principle components is compared to PCA on the VCBC complex with the shorter EloB tail. The first principle component is the same for both structures, capturing a general breathing motion. The second principle component differs, with less of an opposition twisting motion for the complex with the full length EloB due to increases in secondary structure and interactions between EloB and Vif. MD simulations of the VCBC complex show that the global correlated motions are affected by the presence of the C‐terminal of EloB. POS440 Conformational Flexibility of Intrinsically Disordered HIV‐1 Vif Protein Elise Tierney 1, K. Aurelia Ball1, Lieza Chan1 1Skidmore College, New York, USA Intrinsically disordered proteins (IDPs) are not thought to influence the conformation of folded proteins because, inherently, IDPs lack a fixed secondary structure. However, as is the case with the HIV‐1 viral infectivity factor (Vif), IDPs can gain structure in complex with other proteins to enact a specific function. The IDP HIV‐1 Vif, binds to human proteins Elongin C (EloC), Elongin B (EloB), CBF‐b, and Cullin‐5 (Cul5), also referred to as the VCBC‐Cul5 complex. HIV‐1 Vif utilizes the complex to hijack the ubiquitination mechanism and ubiquitinate the antiviral APOBEC enzyme. The structure of this VCBC‐Cul5 complex has been solved by x‐ray crystallography(Guo et al. 2014). However, the crystal structure only reveals one conformation of the complex. To investigate the dynamics and alternate conformations of the HIV Vif complex, Molecular Dynamic (MD) simulations were performed. We simulated the complex with and without Cul5 to investigate the effect of Cul5 on the complex's motion and flexibility. MD simulations were analyzed using Principle Component Analysis (PCA) to identify the correlated motions of the complex. The results indicate that VCBC exhibits more movement in the absence of Cul5. The more rigid structure of VCBC‐Cul5 compared to VCBC indicates that this complex may be the most structurally defined state of the complex in infected cells. However, better structural characterization of the VCBC complex with and without Cul5 allows us to study the role of these potentially functional alternate conformations before and after Cul5 binding. POS467 Hidden Structural Codes in Protein Intrinsic Disorder Gonzalo de Prat Gay 1, Silvina Borkosky1, Leonardo Alonso1, Ignacio Sanchez2 1Fundacion Instituto Leloir‐Conicet, Argentina, 2Departamento de Química Biológica, Facultad de CIencias Exactas y Naturales, Universidad de Buenos Aires Protein intrinsic disorder is a major structural category in biology yet its definition is often limited to the absence of folding. The explosion of information in the genomic era showed that it may account for over 30% of coding regions across life domains, and it is particularly overrepresented in viruses. Papillomaviruses are are an unparalleled case for sequence to structure correlation analysis because of the existence of hundreds of anciently evolved and stable virus types which are divergent enough in sequence, but conserving the function of each protein. E7, the main transforming oncoprotein from human papillomaviruses, is a paradigmatic example of an intrinsically disordered protein with pathological moonlighting activities evolved for hijacking cell cycle control. Despite of being intrinsically disordered, the N‐terminal domain shows more conserved residues than the globular C‐terminal domain. Mutation of five hyper conserved residues precisely distributed along the sequence lead to a marked increase in both a‐helix and ß‐sheet structural content, reflected by drastic effects on equilibrium propensities and oligomerization kinetics. These results strongly suggest the existence of local nuclei, yet to be defined in structural terms, that oppose to canonical folding as expected for globular proteins. These anti‐folding nuclei acting as folding relays represent a novel concept that must involve hidden structural codes for intrinsic disorder clearly distant from random coil ensembles. POS488 Native Binding Interactions at the Transition State for Association between the TAZ1 Domain of CBP and the Disordered TAD‐STAT2 are not a Requirement Ida Lindström 1, Jakob Dogan1 1Stockholm University, Sweden A significant fraction of the eukaryotic proteome consists of proteins that are either partially or completely disordered in native‐like conditions. Intrinsically disordered proteins (IDPs) are common in protein‐protein interactions and are involved in numerous cellular processes. Although many proteins have been identified as disordered, much less is known about the binding mechanisms of the coupled binding and folding reactions involving IDPs. Here we have analyzed the rate‐limiting transition state for the binding between the TAZ1 domain of CREB binding protein and the intrinsically disordered transactivation domain of STAT2 (TAD‐STAT2) by site‐directed mutagenesis and kinetic experiments (φ‐value analysis), and found that the native protein‐protein binding interface is not formed at the transition state for binding. Instead, native hydrophobic binding interactions form late, after crossing the rate‐limiting barrier. Furthermore, the ionic strength dependence of the binding kinetics suggests that the disordered nature of TAD‐STAT2 results in an increased number of possible collisions that lead to a productive on‐pathway encounter complex compared to folded proteins. The initial complex is thus very nonspecific with few, if any, obligatory contacts present, which is consistent with the α‐value analysis. Also, linear free energy relationships clearly demonstrate that native interactions are cooperatively formed, a scenario that has been usually observed for proteins that fold according to the so‐called nucleation–condensation mechanism. Thus, native binding interactions at the rate‐limiting transition state for association between TAD‐STAT2 and TAZ1 are not a requirement, which could be a common mechanism for IDPs. POS499 Modulation of TDP‐43 Phase Separation Yulong Sun 1, Kevin Hadley1, Jacob Brady1, Avi Chakrabartty1 1University of Toronto, Canada The aggregation and misfolding of proteins is a pathological hallmark of neurodegenerative diseases. Amyotrophic lateral sclerosis (ALS) is a devastating, untreatable neurodegenerative disease that causes fatal paralysis. In recent years, TAR DNA binding protein of 43 kD (TDP‐43) has emerged as a focal point of ALS research, as pathognomonic, neuronal inclusions are found to contain TDP‐43 in 97% of all ALS cases. Mutations to TDP‐43 are also known to cause ALS, suggesting a central role in pathogenesis. However, little is known about the mechanism by which TDP‐43 positive inclusions are produced. One emerging theory suggests that aggregates arise from improperly formed or persistent stress granules (SGs) caused by environmental factors such as chronic stress, which would explain the largely sporadic nature of ALS. SGs are membraneless organelles formed through liquid‐liquid phase separation (LLPS), which TDP‐43 can partake through its intrinsically disordered C‐terminal domain (CTD). Characterizing the conversion of TDP‐43 from a reversible droplet state into irreversible aggregates is therefore important in understanding the underlying cause of ALS, and identifying modulators of this process may provide a basis for treatment of the disease. We have developed an in vitro system to assess the dynamics of TDP‐43 in the droplet state. Diffusion rates of full length protein, CTD fragments, and disease‐causing mutants are measured using fluorescence recovery after photobleaching (FRAP) and the effect of compounds such as single‐strand oligonucleotide on droplet dynamics is assessed. POS528 Characterization of a fungal cutinase/acetyl esterase with a disordered domain Felipe Venegas 1, Justin Powlowski1 1Concordia University, Canada Carbohydrate esterases (CEs) are enzymes that de‐esterify carbohydrates: they have many potential applications in processes that involve biomass conversion, such as new generation biofuels. CEs are grouped in 16 families according to the CAZy (Carbohydrate Active enZYmes) database, and encompass proteins found in organisms ranging from viruses to multicellular eukaryotes. The sequence of a predicted fungal CE5 family enzyme unexpectedly was found to include two distinct predicted regions: a long disordered segment (LDS), as well as the CE5 domain. This protein did not show any esterase activity towards model para‐nitrophenyl‐esters or acetylated carbohydrates; these are two documented activities for CE5s. Interestingly, the CE5 domain shows high sequence similarity to crystallized CE5s, including the catalytic triad residues. Therefore, in order to determine if protein inactivity was related to some grade of disorder of the predicted LDS, biophysical experiments were conducted. Chemically‐denatured protein shows a pH‐dependent refolding with no signs of disorder, contrary to expectations. In order to understand the significance of these observations, as well as the roles of the two domains, expression of the full polypeptide and independent domains in E. coli is being attempted: the progress of these studies will be reported. Based on the sequence of the LDS, we have determined how frequently LDSs are encountered in the CE5 family, and what types exist: from this we have constructed a CE5 disorder‐order landscape. In addition, other families from this superfamily were analyzed. It was found that 33% of the CE5 sequences exhibit some disorder, while this value is 44 and 16% for families CE1 and CE7. 12. MEMBRANE PROTEINS POS028 LURE‐ing the Male Gamete: Tale of Pollen Receptor Kinases and Their Role in Pollen Tube Growth Sayan Chakraborty 1, Haiyun Pan1, Guozhou Xu1 1North Carolina State University, USA During reproduction in flowering plants, the male gametophyte moves to the female gametophyte in pistil by formation of pollen tubes and delivers immotile male gamete. Two synergid cells situated on either side of the egg cell produce cysteine rich chemoattractant peptide LURE that guides the pollen tube to the female gametophyte for sexual reproduction. Recently, in Arabidopsis thaliana, Pollen Receptor Kinase 6 (PRK6), along with PRK3, PRK8 and PRK1 have been identified as the receptors that senses LURE. These receptors belong to Leucine Rich Repeat Receptor Like Kinases (LRR‐RLKs)‐ the largest family of receptor kinases found in Arabidopsis thaliana. These proteins have three domains‐ extracellular domain interacting with the external cues, a membrane spanning region and a kinase domain. How LURE and pollen specific receptor kinases regulate the growth and development of pollen tube remains elusive. To resolve the signaling mechanism of LURE and its cognate receptor kinases in the growth of pollen tube, we have expressed LURE and the extracellular domains of PRK1, 3, 6, 8 in baculovirus‐insect system. The soluble proteins have been purified by nickel‐affinity and gel filtration chromatography in sufficient quantity for biochemical and crystallographic studies. Therefore, structural elucidation of the pollen specific receptor kinases alone and in complex with the cysteine rich peptide LURE can provide insight into their role in pollen tube growth, ligand perception, specific interactions between the LURE and the cognate receptors, and ligand‐induced receptor activation. This research will broaden our overall understanding about the reproduction system of flowering plants. POS035 Structural and Functional Analyses of A Bacterial Quorum‐Sensing Signal Peptide Receptor Protein Yung‐Hua Li 1, Xiao‐Lin Tian1 1Dalhousie University, Canada Quorum sensing activation by signal peptide pheromone (CSP) in Streptococcus mutans depends on the membrane‐associated histidine kinase receptor, ComD, which senses the signal and triggers the signaling cascade for bacteriocin production and other cell density‐dependent activities. However, the mechanism of the signal recognition via the ComD receptor in this species remains elusive. Here, we report the results of structural and functional analyses of the ComD receptor using a dual phoA‐lacZ reporter system. We then determine the roles of the membrane domain of the ComD in CSP recognition and quorum sensing activation. The results show that the membrane domain of the ComD receptor protein forms six transmembrane segments with three extracellular loops, loopA, loopB and loopC. Mutational analysis of these extracellular loops combined with luciferase report assays reveals that both loopC and loopB are required for CSP recognition and quorum sensing activation, while loopA plays little role in CSP detection. A deletion or substitution mutation of four residues NVIP in loopC abolishes CSP recognition for quorum sensing activation. Western blotting confirms that all the mutant proteins exist in the membrane fractions of the mutant strains, suggesting that a deletion or mutation of these extracellular loops does not affect the insertion of the mutant proteins into the membrane. Consistent with these findings, the loopC and loopB mutants are completely or partially defective in bacteriocin production. We conclude that both loopC and loopB are involved in CSP recognition and residues NVIP of loopC are essential for CSP perception and quorum sensing activation. The findings may have important implications for ligand‐receptor interactions in bacteria. POS037 Structural Modeling and Rational Design of Small Molecule Allosteric Agonists of GLP‐1 Receptor Tejashree Redij 1, Zhijun Li1 1University of the Sciences in Philadelphia, USA The Glucagon‐Like Peptide 1 Receptor (GLP‐1R) belongs to the pharmaceutically important Class B family of G‐protein coupled receptors and its incretin peptide ligand GLP‐1 analogs are used for the treatment of type‐2 diabetes. Despite remarkable anti‐diabetic effects, GLP‐1 peptide‐based agonists have several shortcomings. On the other hand, although considerable progress has been made in developing nonpeptidic small molecules targeting GLP‐1R, the success of such strategy remains elusive. A likely reason is because its orthosteric binding site is large and relatively flat, thus it is challenging to target by small molecules. In recent years, a novel venue has been reported to exploit the allosteric sites on GPCRs for the development of small molecule drugs to treat various diseases. For GLP‐1R, lack of high‐quality structure of the TM domain of GLP‐1R hinders the progress of this promising approach. In the present work, we carried out computer‐based molecule design studies by first constructing a 3D structural model of GLP‐1R in its active conformation using the modeling techniques developed in our lab and others. In silico screenings of drug‐like compounds against the predicted allosteric site on this structural model have identified a few compounds as potential GLP‐1R agonists. Their agonistic and modulating activities were subsequently confirmed using a cAMP response element (CRE)‐based reporting system. These results demonstrated that allosteric regulation exists in GLP‐1R and can be exploited for developing small molecule agonists to augment the activity of endogenous GLP‐1 or GLP‐1 analogs. The success of this work will help pave the way for small molecule drug discovery targeting other Class B GPCRs. POS038 Generating Ca2+ signaling via chimeras in mammalian cells Anam Qudrat 1, Anam Qudrat1 1University of Toronto, Canda The versatility of Ca2+ signals allows it to regulate diverse cellular processes such as migration, apoptosis, motility and exocytosis. In some receptors (e.g., VEGFR2), Ca2+ signals are generated upon binding their ligand(s) (e.g., VEGF‐A). Here, we employed a design strategy to engineer proteins that generate a Ca2+ signal upon binding various extracellular stimuli by creating fusions of protein domains that oligomerize to the transmembrane domain and the cytoplasmic tail of the VEGFR2. To test the strategy, we created chimeric proteins that generate Ca2+ signals upon stimulation with various extracellular stimuli (e.g., rapamycin, EDTA or extracellular free Ca2+). By coupling these chimeric proteins that generate Ca2+ signals with proteins that respond to Ca2+signals, we rewired, for example, dynamic cellular blebbing to increases in extracellular free Ca2+. Thus, using this design strategy, it is possible to engineer proteins to generate a Ca2+ signal to rewire a wide range of extracellular stimuli to a wide range of Ca2+‐activated processes. POS039 Synthetic MCSF sources attract protein chimeras Anam Qudrat 1 1University of Toronto, Canada Inflammatory lesions, often seen in diseases such as rheumatoid arthritis, atherosclerosis and cancer, feature an acidic (i.e. low pH) microenvironment rampant with pro‐inflammatory cytokines, such as CSF1. For targeted therapeutic intervention at these sites, engineered cells must be able to seek CSF1 sources. Here, we have assembled a system of four proteins in a cell to accomplish this feat; we introduced a CSF1 chimera receptor (named CSF1Rrec), the previously engineered CaRQ (a RhoA‐based Ca2+ sensor), VSVG and thymidine kinase (TK) into cells with no natural ability to seek CSF1 sources. Binding of CSF1 to the CSF1Rrec initiates a Ca2+ signal. This Ca2+ signal is then used by CaRQ to form non‐apoptotic blebs to migrate towards the CSF1 source. Next, the VSVG protein allows these engineered cells to fuse with the CSF1 source cells, upon low pH induction. Finally, these cells undergo death post‐ganciclovir treatment, via the TK suicide mechanism. Hence, with the assembly of a group of proteins, we have established the basis of engineering a cell to target inflammatory lesions in diseases featuring a microenvironment with high levels of CSF1 and low pH. POS069 Development of Novel Surfactants for Membrane Proteins’ Researches Toshihisa Mizuno 1 1Nagoya Institute of Technology, Japan The development of additional extraction surfactants for membrane proteins is necessary for membrane protein research, since optimal combinations for the successful extraction of target membrane proteins from biological membranes that minimize protein denaturation are hard to predict. In particular, those that have a unique basal molecular framework are quite attractive and highly desired in this research field. In this study, we successfully constructed a new extraction surfactant for membrane proteins, NPDGC12KK, from the peptide‐gemini‐surfactant (PG‐surfactant) molecular framework. The PG‐surfactant is a U‐shaped lipopeptide scaffold, consisting of a short linker peptide (‐X‐) between two long alkyl‐chain‐modified Cys residues and a peripheral peptide (Y‐) at the N‐terminal side of long alkyl‐chain‐modified Cys residues (Figure 1). Using Photosystem I (PSI) and photosystem II (PSII) derived from Thermosynecoccus (T.) vulcanus as representative membrane proteins, we evaluated whether NPDGC12KK could solubilize membrane proteins, while maintaining structure and functions. Neither the membrane integral domain nor the cytoplasmic domain of PSI and PSII suffered any damage upon the use of NPDGC12KK based on detailed photophysical measurements. Using thylakoid membranes of T. vulcanus as a representative biological membrane sample, we performed experiments to extract membrane proteins, such as PSI and PSII. Based on the extraction efficiency and maintenance of protein supramolecular structure established using clear native‐PAGE analyses, we proved that NPDGC12KK functions as a novel class of peptide‐containing extraction surfactants for membrane proteins. POS076 Plug and play: inserting a single amino acid into a stretch of leucines yields a surprising diversity of activities Ross Federman 1, Erin Heim2, Sophia Chen2 1Yale School of Medicine, Immunobiology Department, USA, 2Yale School of Medicine, Genetics Department, USA Artificial, single‐pass transmembrane (TM) proteins modeled on the bovine papillomavirus E5 protein can act as aptamers that modulate the expression or activity of naturally occurring transmembrane proteins. Previously, we isolated unique, chemically simple TM proteins consisting solely of leucines and isoleucines that specifically activated the platelet derived growth factor receptor beta (PDGFßR) or the human erythropoietin receptor (hEpoR). Further analysis revealed that, surprisingly, a single isoleucine at position 13 of a 26‐amino acid TM domain with leucines at all the remaining positions (polyLeu) was able to activate the PDGFßR. To determine what other amino acids at this 13th position within a polyLeu context displayed biological activity, we constructed the full set of 26‐amino acid long polyLeu TM proteins with all 20 standard amino acids present at this position. We found that six of these mutants activate the PDGFßR, and that a different subset activate the hEpoR. Yet another larger subset activate the mouse EpoR, which differs from the hEpoR at only three positions within the TM domain. Mapping experiments show the crucial difference between hEpoR and mouse EpoR (mEpoR) maps to a single TM amino acid. Additionally, a simple activator of both the mEpoR and hEpor can be tuned to specifically activate only the mEpoR through the substitution of a leucine to an isoleucine at certain positions flanking position 13. These results challenge our understanding of TM protein‐protein interactions, and suggest these ultrasimple proteins may serve as powerful new tools to better understand how proteins functionally interact within hydrophobic environments. POS081 Molecular level analysis of disease‐causing mutations in the human sulfonylurea receptor Claudia Alvarez 1, Marijana Stagljar2, Voula Kanelis2 1Chemistry Department, University of Toronto, Canada, 2Department of Chemical and Physical sciences, University of Toronto at Mississauga, Canada The sulfonylurea receptor (SUR) proteins form the regulatory subunit ATP sensitive potassium (KATP) channels found in the pancreas and other tissues. The SUR protein is a member of the ATP binding cassette family of transporters and thus it has the characteristic membrane spanning domain (MSD1 and MSD2) and two nucleotide binding domains (NBD1 and NBD2). The NBDs and MSDs are in direct contact by means of helices extending from the MSDs into the cytoplasm, called coupling helices. Binding and hydrolysis of MgATP at the SUR NBDs results in opening of the KATP channel pore. Disease‐causing mutations located in the NBDs or coupling helices impair the ability of the SUR protein to regulate the gating of the KATP channel. Mutations that reduce KATP channel opening cause hypersinsulinism, whereas mutations that increase channel opening cause diabetes. Here, we have used NMR, fluorescence spectroscopy and bio‐layer interferometry to determine the structural and functional consequences of hyperinsulinism‐ and diabetes‐causing mutations in the SUR protein. Our NMR assignment of NBD1 provides residue level information that enables residue‐level resolution of structural changes in NBD1with various mutations. Our results show that hyperinsulinism‐causing mutations alter the structure of NBD1 and impair its ATP binding activity, thermal stability and inter‐domain interactions. Diabetes‐causing mutations have fewer effects on the structure and stability but affect the ATP‐binding affinity and interaction of NBD1 with the coupling helices. Our results combined provide a structural and functional background for elucidating the mechanism by which mutations cause disease. Such studies are essential for improving design of drugs to target diabetes and hyperinsulinsm. POS105 Purification and functional reconstitution of TAAR13c into nanodiscs for the development of cadaverine‐detection biosensor Heehong Yang 1, Daesan Kim2, Seunghun Hong3, Tai Hyun Park1 1School of Chemical and Biological Engineering, Seoul National University, South Korea, 2Department of Biophysics and Chemical Biology, Seoul National University, South Korea, 3Department of Physics and Astronomy and Institute of Applied Physics, Seoul National University, South Korea Cadaverine (CV), the most death‐associated odor compound, generated by bacterial decarboxylation has been reported that the selective detection of CV can be applied to various fields such as scientific investigation and various industries. Trace amine‐associated receptors 13c (TAAR13c), belonging to G protein‐coupled receptors (GPCRs), could be a general diamine sensing element with selective binding to cadaverine, which can be applied to bioelectronic sensors. Moreover, the platform containing stable GPCRs can be powerful tool for a development of a practical biosensor, however; the development of stable device with high‐quality receptors remains challenging. In this study, we firstly purified and reconstituted the TAAR13c into nanodiscs, which leads to develop the TAAR13c‐conjugated bioelectronic sensor for selective‐detection of cadaverine. The TAAR13c and its biological characteristics were confirmed by tryptophan fluorescence assay and dual‐glo luciferase assay. Moreover, the nanodisc of TAAR13c was successfully produced and applied to biosensor for selective‐detection of cadaverine. The TAAR13c‐conjugated biosensor showed the high‐performance in selectivity, sensitivity and real‐sample detection. These results can be utilized as GPCR characterization and TAAR‐ligand interaction. Furthermore, TAAR13c‐based biosensor represents a novel method for the receptor‐based detection of death‐associated odor cadaverine. POS125 Membrane binding of S100A10 and annexin A2 proteins involved in cell membrane repair Xiaolin Yan 1, Élodie Boisselier1 1Laval University and CUO‐Research, Canada Objective: The protein complex S100A10/annexin A2 allows the recruitment of the protein AHNAK to the membrane in presence of calcium, before forming a platform which can initiate membrane repair. However, no molecular data are currently available on membrane binding of the different proteins involved in this complex. We aim to study the membrane binding of S100A10, annexin A2 and their complex to better understand their roles in cell membrane repair process. Methods: Firstly, S100A10 and annexin A2 will be overexpressed and purified. Langmuir monolayers membrane model will then be used to characterize the interactions between these proteins and different phospholipids found in membranes. The secondary structure, orientation and membrane organization of these proteins will be studied by Polarization Modulation Infrared Reflection‐Absorption Spectrometry. Their lateral localization will be determined through the influence of these proteins on the physical state of lipids by fluorescence microscopy. Results: S100A10‐GST was overexpressed and purified by affinity chromatography. The cleavage of GST tag was complete. The optimization of the purification procedure to obtain pure S100A10 is currently ongoing. Once pure S100A10 will be available, Langmuir monolayers model will be set up to investigate its membrane binding in different conditions. Conclusions: Our research will complete current knowledge on membrane binding of S100A10 and annexin A2. We could also identify the conditions leading to modifications of these membrane bindings, and possibly to the loss of function of proteins. Thus, this project helps to better determine their roles in membrane repair, as well as in other physiological mechanisms in which these proteins contribute. POS154 The Role of the C‐terminal Heptad Repeat (CHR) of HIV‐1 gp41 in Formation and Enlargement of Fusion Pores Zene Matsuda 1, Dehua Liu2, Hongyun Wang2 1Research Center for Asian Infectious Diseases, Institute of Medical Science, The University of Tokyo, Japan, 2Laboratory of Structural Virology and Immunology, Institute of Biophysics, CAS, China The gp41subunit, a class I fusion protein, of HIV‐1 envelope protein (Env) drives membrane fusion by forming a six‐helix bundle (6HB) structure. The 6HB formation is closely linked with the generation and enlargement of the fusion pores. In this study, we performed an alanine insertion mutagenesis of the N‐terminal and C‐terminal heptad repeats (NHR and CHR, respectively) including the connecting loop of gp41 in the HXB2 strain. The kinetics and extent of membrane fusion of each mutant were evaluated by the split luciferase‐based dual split protein (DSP) assay and the syncytia formation assay. The DSP assay measured the rate of fusion pore formation, and the syncytia assay provided the estimation of pore enlargement and membrane merge. We found that insertions in the NHR, loop, and proximal region of CHR (up to amino acid position 643: numbering is based on HXB2 Env) made gp41fusion‐incompetent by negatively affecting the processing of Env. An insertion at position from 644, 645, 647, 648, or 649 of CHR did not affect the processing of gp160. An insertion at position 644 or 645 reduced fusion pore formation and resulted in corresponding reduced syncytia formation. Interestingly, the mutant with an insertion at position 647, 648, or 649 showed similar or sometimes augmented fusion pore formation, yet a decreased rate of syncytia formation. Our data suggest that while the initial fusion pore can be generated by a partial zipping of CHR, the complete zipping beyond position 649 may be required for successful pore enlargement. POS181 Structural and Functional Analysis of the Bacterial Cell Division Proteins FtsB and FtsL Samuel Craven 1, Deena Mahbuba1, Samson Condon1, Claire Armstrong1, Loren LaPointe1, Ambalika Khadria1, Alessandro Senes1 1University of Wisconsin‐Madison, USA Cell division in prokaryotes is mediated by a protein complex called the divisome, which is responsible both for directing constrictive force at the division site and for restructuring the cell wall. In E. coli, the formation of the divisome from its various components occurs in an ordered fashion, and an essential event in this assembly pathway is the interaction between FtsB and FtsL to form a subcomplex. These bitopic membrane proteins are known to interact with other divisome proteins as well, and such interactions are required to complete assembly of the divisome at the division site. Largely due to the difficulty of obtaining structural information for integral membrane proteins, relatively little is known about the overall organization of the FtsB/L subcomplex or its specific interactions with other divisome proteins. To obtain a better understanding of the role that FtsB/L plays in cell division, we are employing cross‐disciplinary techniques to develop and validate structural models of the subcomplex. These models then direct experiments designed to probe the function of FtsB/L and, in turn, provide feedback to further refine the models. Specifically, FRET and single molecule photobleaching experiments support a 2:2 tetrameric model for FtsB:FtsL, co‐evolutionary analyses and computational modeling provide low‐energy structural models, and in vivo and in vitro assays of mutant proteins help validate these models. In conclusion, we have combined a variety of diverse techniques to develop rational models for the essential divisome FtsB/L subcomplex in lieu of “hard” structural data like X‐ray crystallography or NMR. POS219 Predicting deleteriousness of genetic variations in membrane proteins Julia Koehler Leman 1, Evan Baugh1, Richard Bonneau1 1Simons Foundation/NYU, New York, USA Through recent efforts in high‐throughput sequencing, the number of sequenced genomes has increased dramatically. Based on this wealth of data, genome‐wide association studies aim to identify which genetic variations or protein mutants are causing which disease, if any. This needle‐in‐a‐haystack problem has been approached by several methods, which currently mainly rely on sequence‐based information. The disadvantage with these tools is the lack of interpreting the effect of mutations onto protein structure and function. Recently, we trained the logistic regression classifier (VIPUR ‐ Variant Prediction and Interpretation Using Rosetta) to predict deleteriousness of mutations in soluble proteins, by combining sequence and structure‐based information, with the ability to identify the underlying cause of deleteriousness of the mutation in question. We used sequence features from multiple‐sequence alignments and structural features from existing protein structures or homology models, which were refined and scored with the Rosetta energy function. In parallel, we implemented a general framework for membrane protein modeling into Rosetta and created a new implementation of a high‐resolution refinement protocol that works for large to very large proteins, such as membrane proteins. We are now using Rosetta protocols that are adapted for membrane proteins, to train a classifier for the prediction and interpretation of sequence variation in membrane proteins. This new classifier is trained on membrane protein variations using a transfer learning approach. Its setup allows high‐throughput predictions and automated interpretations of what causes the deleteriousness from a structural point of view, which will be very useful to adapt therapies in the clinic. POS233 Signaling‐related Changes in Bacterial Chemoreceptor Protein Interaction Interfaces Localized by Hydrogen Deuterium Exchange Mass Spectrometry Xuni Li 1, Seena Koshy1, Stephen Eyles2, Lynmarie Thompson1 1Department of Chemistry in University of Massachusetts Amherst, USA, 2Department of Biochemistry and Molecular Biology in University of Massachusetts Amherst, USA Chemotaxis receptors are a great system for the study of the mechanism of transmembrane signaling. These receptors form remarkable 200 nm hexagonal arrays in the membrane with two additional proteins, an adaptor CheW and a kinase CheA. An outstanding question is how the signal propagates from the membrane to the cytoplasmic tip of the receptor, 200Å away, to control the kinase activity of CheA. Current proposals suggest that signal propagates through the receptor cytoplasmic domain via inverse changes in dynamics in different receptor subdomains. One challenge of testing these hypotheses is the need to form active complexes. To address this challenge, we use vesicle template assembly to prepare complexes of the receptor cytoplasmic fragment, CheA and CheW with native structural and functional properties. Previously, we used hydrogen deuterium exchange mass spectrometry of these complexes to measure differences in hydrogen exchange of the Asp receptor between the kinase‐on and kinase‐off signaling states. Results showed greater protection from exchange in the kinase‐on state in the protein interaction domain near the cytoplasmic tip of the receptor. With greater peptide coverage, we observe similar changes between two other constructs with kinase‐on and kinase‐off properties. These results are compared with structural models for the receptor/receptor, receptor/CheA and receptor/CheW interfaces. This research supported by NIH grant GM085288. POS271 Structural Identification of Phosphorylation Codes for Arrestin Recruitment by G protein‐Coupled Receptors Parker de Waal 1, X. Edward Zhou1, Yuanzheng He1, Xiang Gao1, Yanyong Kang Kang1, Ned Van Eps2, Yanting Yin1, Kuntal Pal1, Devrishi Goswami Goswami3 1Van Andel Research Institute, Michigan, USA, 2University of Toronto, Canada, 3The Scripps Research Institute, Florida, USA G protein‐coupled receptors (GPCRs) mediate diverse signaling in part through interaction with arrestins, whose binding promotes receptor internalization and signaling through G protein‐ independent pathways. High‐affinity binding of arrestins to GPCRs requires receptor phosphorylation, often at the receptor's C‐terminal tail. Here we report an X‐ray free electron laser (XFEL) crystal structure of the rhodopsin‐arrestin complex, in which the phosphorylated C‐terminal tail of rhodopsin forms an extended intermolecular ß‐sheet with the N‐terminal ß‐strands of arrestin. Phosphorylation was detected at rhodopsin C‐terminal tail residues T336 and S338. These two phospho‐residues, together with E341, form an extensive network of electrostatic interactions with three positively charged pockets in arrestin in a mode that resembles binding of the phosphorylated vasopressin‐2 receptor tail to ß‐arrestin‐1. Based on these observations, we derived and validated a set of phosphorylation codes that serve as a common mechanism for phosphorylation‐dependent recruitment of arrestin by GPCRs. POS276 Synthetic Antibody Reagents for Structure Determination of Membrane Proteins by Cryo‐EM Satchal Erramilli 1, Anthony Kossiakoff1, Pawel Dominik2, Przemyslaw Dutka1, Blazej Skrobek1, Tomasz Slezak1, Katarzyna Radziwon1, Somnath Mukherjee1 1University of Chicago, USA, 2University of California at San Francisco, USA The technological breakthroughs of cryo‐electron microscopy (cryoEM) have facilitated the rapid structure determination at atomic resolution of many difficult protein targets. However, there remain a number of challenges to its broader use; for instance, the size and conformational heterogeneity issues of many proteins, which limit the obtainable resolution of these images. Synthetic antibodies (sAb) are based on a Fab framework (50 kDa) and are generated using phage display mutagenesis to bind to target proteins to increase their size, act as fiducials for orientation, and reduce the sample heterogeneity. The selection conditions can be designed to produce region‐ and conformation‐specific binders to different functional states of membrane proteins. In this work, we demonstrate that sAbs can effectively trap the active state of CorA, a pentameric archaeal magnesium channel. Preliminary negative‐stain EM images and functional studies showed that CorA was trapped using a conformationally specific sAb in its open state. To further stabilize the open conformation, sAbs which recognize adjacent epitopes available on all subunits of CorA in this state were obtained. A series of 2nd generation Fab fiducials have been developed that further increase the size and symmetry of the particles. In combination, these CorA‐specific sAbs and Fab‐specific reagents provide the tools necessary for the structure determination of the active conducting state of CorA at high resolution by cryoEM. Because of the Plug‐and‐Play nature of the Fab‐specific reagent, the diversity of the phage display libraries, and the nanodisc platform for biopanning, this technology is applicable to virtually any membrane protein and greatly extends the capability of single‐particle cryoEM. POS350 Optical Dissection of the Assembly and Cooperativity of a Class C G Protein‐Coupled Receptor Joshua Levitz 1, Deo Singh1, Victor Vivcharuk1 1Weill Cornell Medicine, New York, USA G protein‐coupled receptors (GPCRs) provide a bridge between the extracellular and intracellular spaces by sensing stimuli, such as light, hormones, or neurotransmitters, and converting them into intracellular signals. The extraordinarily diverse GPCR family plays roles in nearly every disease and accounts for >60% of all current drug targets. Class C GPCRs, including the metabotropic glutamate receptors (mGluRs) and GABAB receptors, are particularly interesting allosteric signaling machines because of their propensity for dimerization and their large, extracellular ligand binding domains (LBDs) that couple, via a poorly understood mechanism, to a transmembrane domain (TMD). My work aims to use optical methods to develop a complete biophysical mechanism of how class C GPCRs assembly, activate and signal, and to use this insight to ultimately determine how these receptors modulate neural activity in physiology and disease. Using a variety of single molecule photobleaching assays we show that mGluRs strictly and specifically homo‐ and hetero‐dimerize via a complex combination of extracellular and transmembrane interface. We next used a combination of ensemble and single molecule FRET assays to decipher the characterize the intersubunit conformational dynamics of LBDs in response to ligand of various efficacy. Finally, as a complement to fluorescence imaging assays, we have also developed a family of optopharmacological tools for the manipulation of specific mGluR subunits. Using these tools in conjunction with time‐resolved functional assays and computational methods, we show that mGluRs display large ligand‐occupancy dependent cooperativity that is mediated in part by tune‐able inter‐LBD interactions. POS401 Essential Phenylalanine‐Anion Coordination in A Fluoride‐Specific Ion Channel Senmiao Sun 1, Nicholas Last1, Christopher Miller1 1Brandeis University, Massachusetts, USA F‐ ion channels of the Fluc family, built as dual‐topology antiparallel dimers, provide an efflux pathway for microorganisms to counteract the toxicity of environmental F‐. Recent crystal structures of a bacterial homologue reveal two conserved phenylalanines, F80 and F83, that contribute to the binding sites of pore‐resident F‐ ions through an electropositive, edge‐on aromatic‐halide coordination motif never before seen in proteins. This unprecedented aromatic‐anion coordination geometry, we conjecture, may underlie the channel's unusually high selectivity for F‐ over Cl‐ (>10^5‐fold). To test this idea, mutations of each phenylalanine residue were examined. Almost all substitutions, though well‐folded and homodimeric, cause complete loss of channel function, even tyrosine. Surprisingly however, methionine at position 80 retains wildtype channel activity, whereas F83M is inactive. A crystal structure of F80M reveals that the electropositive α‐carbon of methionine adopts a F‐ coordination position similar to the location of the F80 aromatic edge in wildtype. These results provide initial suggestions as to the delicate chemical requirements for highly selective F‐ binding sites in a largely anhydrous protein pore. POS414 The transmembrane protein otoferlin is a calcium sensitive scaffold linking SNAREs and calcium channels Colin Johnson 1, Nicole Hams1 1Oregon State University, USA Prelingual hearing loss is a common hereditary disorder, with approximately 1 of every 500 children suffering from profound deafness, and mutations in the 240kDa tail‐anchored transmembrane protein otoferlin have been identified as responsible for deafness in many of these cases. While it is hypothesized that otoferlin functionally replaces synaptotagmin as the synaptic calcium sensor for neurotransmitter release from sensory hair cells in the inner ear, the reason for this replacement is unknown, and current approaches to study otoferlin have proven inadequate in elucidating the exact function of otoferlin. We report these use of a two‐pronged approach that couples biophysical assays and zebrafish knockdown and rescue studies which allow us to probe function on both the molecular and organismal levels. Using zebrafish as a model, we find that otoferlin mediates exocytosis of neurotransmitter from sensory hair cell synapses involved in hearing and balance, and report the first rescue studies using full‐length and truncated forms of otoferlin to restore hearing in deaf zebrafish. Using total internal reflection fluorescence microscopy, we find that while both otoferlin and synaptotagmin bind membrane fusion SNARE proteins, only otoferlin interacts with the L‐type calcium channel Cav1.3, demonstrating a significant difference between otoferlin and synaptotagmin. Otoferlin was also found capable of interacting with multiple SNARE and Cav1.3 proteins simultaneously, forming a hetero‐oligomer complex. Our results support a model in which otoferlin acts as a calcium sensitive scaffolding protein, localizing SNARE proteins proximal to the calcium channel so as to synchronize calcium influx with membrane fusion during the encoding of sound. POS436 Trade‐off between GPCR folding and functional versatility Daniel Estevez Prado 1, Tilman Flock2, Alexander S Hauser3, Ramanujan S Hegde1, M. Madan Babu1 1MRC Laboratory of Molecular Biology, UK, 2Paul Scherrer Institute, Switzerland, 3University of Copenhagen, Department of Drug Design and Pharmacology, Denmark Membrane insertion of polytopic membrane proteins such as G‐protein coupled receptors (GPCRs) involves the thermodynamically driven partitioning of the emerging polypeptide into the lipid bilayer and the formation of native contacts between transmembrane helices (TMHs) as part of higher order folding and oligomerization. A sequential insertion model of TMHs faces several challenges: (a) How are hydrophilic domains processed, (b) which contacts are required to facilitate transition and assembly, and (c) why are energetically costly residues abundantly tolerated across all GPCRs. We have applied an interdisciplinary computational approach investigating the structural, biophysical and genomic signatures associated with GPCR folding, function and misfolding. The systematic analysis of TMH insertion probabilities and single‐residue energy contributions reveals the existence of thermodynamic insertion profiles (TIPs). These profiles largely reflect universal folding restraints and, surprisingly, show high variability across all receptors, presumably due to the need for evolving highly specific intra‐ and intermolecular interactions. This family/receptor specific optimization of folding versus function gives rise to distinct TIPs, but also demonstrates patterns that reflect conserved pan‐receptor constraints and mechanisms. Through functional annotation of high‐cost segments and the integration of genomic information, our analysis establishes a global roadmap to investigate GPCR stability and misfolding. The results shed further light on the experimentally proven context‐dependency of TMH insertion and conformational preferences of some receptor fragments. Our conclusions suggest receptors and regions that are vulnerable to mutations that can cause disease by affecting protein folding and trafficking. Such insights can guide the development of pharmacological chaperones that can rescue mutant receptors by assisting their folding and stabilizing energetically favorable and functional conformations. POS444 X‐ray Crystal Structures of the Influenza A M2 Proton Channel Bound to Amantadine, Rimantadine, and Inhibitors Jessica Thomaston 1, William DeGrado1 1University of California at San Francisco, USA Inhibition of the M2 proton channel in the influenza A virus prevents viral replication from occurring. Two of the four FDA‐approved drugs for the treatment of influenza infections, amantadine and rimantadine, target the M2 channel. However, because M2 is a membrane protein, structural studies of drug binding to the channel have been limited due to the challenging nature of the target. Here, we have obtained multiple crystal structures of M2 in the presence of drugs and inhibiting compounds using lipidic cubic phase (LCP) crystallization techniques. We present the first crystal structures of rimantadine bound to M2 in both the Cclosed and Copen conformations of the channel (2.0 Å, 2.5 Å), as well as amantadine bound to the Cclosed conformation (2.0 Å). At this resolution range the orientation of the bound drug is unambiguous, and the ammonium group of the adamantane drugs can be seen interacting with ordered water molecules present within the channel. Additionally, we report the binding of a dual‐inhibiting compound to both the wild type channel (2.6 Å) and the drug‐resistant V27A mutant (2.5 Å). The position of the bound inhibitor within the channel shifts in the presence of the V27A mutation. These structures further our understanding of drug binding and inhibition within the M2 proton channel and will help guide the design of compounds for the inhibition of drug‐resistant mutants of M2. POS469 Towards the Structural Characterization of Peripheral Myelin Protein 22 in Model Membrane by NMR Spectroscopy Geoffrey Li 1, Charles Sanders1, Melanie Ohi2, Charles Sanders1 1Department of Biochemistry, Vanderbilt University, Tennessee, USA, 2Department of Cell and Developmental Biology, Vanderbilt University, Tennessee, USA Peripheral myelin protein 22 (PMP22), a 160‐residue tetraspan membrane protein, is a vital component of the compact myelin, believed to regulate Schwann cell proliferation and myelin production. Gene duplication or mutation in PMP22 has been causally linked to a hereditary neuropathy of the peripheral nervous system known as Charcot‐Marie‐Tooth Disease (CMTD), for which no treatment is available yet. A detailed structural characterization of PMP22 will aid in the development of therapeutic agents of CMTD. To date, there is no experimentally determined structure of PMP22 except for a homology‐based model based on the crystal structure of claudin‐15. Here, we aim to determine the structure of PMP22 in model membrane using nuclear magnetic resonance (NMR) spectroscopy. We obtained the NMR spectra of PMP22 in different detergent micelles and bicelles to screen for the best conditions for structure determination. Previous NMR data have shown that the first transmembrane helix has higher propensity to dissociate from the rest of the transmembrane helix bundle (Sakakura‐M and Sanders‐CR, Structure 2011). The result of this finding will serve as the groundwork for the structural characterization of this important integral membrane protein and its CMTD mutant forms. POS503 A simulation‐guided fluorescence correlation spectroscopy tool to investigate the protonation dynamics of cytochrome c oxidase Ulrike Alexiev 1, Alexander Wolf1, Constantin Schneider1, T‐Y Kim1, Kristina Kirchberg1, Pierre Volz1 1Freie Universität Berlin, Germany Fluorescence correlation spectroscopy (FCS) is a single molecule based technique to temporally resolve rate‐dependent processes by correlating the fluorescence fluctuations of individual molecules traversing through a confocal volume. In addition, chemical processes like protonation or intersystem crossing can be monitored in the sub‐microsecond range. FCS thereby provides an excellent tool for investigations of protonation dynamics in proton pumps like cytochrome c oxidase (CcO). To achieve this, the pH‐dependent fluorescent dye fluorescein was attached as a protonation probe to the CcO surface via site‐specific labeling of single reactive cysteines that are located close to the entry point of a proton input channel (K‐pathway). The analysis of protonation dynamics is complicated by overlapping triplet and protonation rates of the fluorophore. A Monte Carlo simulation based algorithm was developed [1] to facilitate discrimination of these temporally overlapping processes thus allowing for improved protonation reaction rate determination. Using this simulation‐guided approach we determined precise local proton association and dissociation rates and provide information about protein surface effects, such as proton collecting antennae, on the transport properties of proton transfer channels [2]. [1] Wolf, A., Schneider, C., Kim, T.‐Y., Kirchberg, K., Volz, P., and U. Alexiev. A simulation‐guided fluorescence correlation spectroscopy tool to investigate the protonation dynamics of cytochrome c oxidase. Phys. Chem. Chem. Phys., 18, 1463–9076, (2016) [2] Kirchberg, K., Michel, H., and U. Alexiev. Net Proton Uptake Is Preceded by Multiple Proton Transfer Steps upon Electron Injection into Cytochrome c Oxidase. J. Biol. Chem. 287, 8187‐93 (2012) POS504 A Novel Affinity Chromatography Method for the Purification of Recombinant Binder of SPerm Proteins Samin Sabouhi 1, Bruno Prud'homme2, Puttaswamy Manjunath1 1University of Montreal, Canada, 2Research Centre Maisonneuve‐Rosemont Hospital, Canada Binder of SPerm (BSP) protein represent a super‐family exclusively expressed in the male reproductive tract and they play a role in fertilization. Recently, a human BSP homolog 1 (BSPH1) and two mouse BSP homologs (BSPH1/2) have been identified. Since these proteins are produced in very minute quantities in these organisms, we expressed His‐tagged recombinant proteins in E.coli and purified using His‐bind columns. However, recombinant proteins purified on His‐bind column were still impure and are not suitable for function analysis. In previous study, we have shown that BSP proteins interact with pseudo‐choline groups such as Diethylaminoethyl (DEAE) by affinity interaction rather than ionic interactions. The aim of the current study was to develop a method to purify recombinant BSP proteins using DEAE‐Sephadex. Mouse and the human recombinant BSP proteins were expressed in Origami B (DE3) pLysS. The recombinant proteins were then extracted from cells with B‐Per bacterial protein extraction reagent containing 3 M urea and passed through DEAE‐Sephadex column. The column was washed with 1 M NaCl in Tris‐buffer and the bound BSP proteins were eluted with 8 M urea. The fractions eluted with urea were pooled, desalted and lyophilized. In conclusion this simple and efficient novel method would be useful for further characterization and to clarify the role of BSP proteins in fertilization. (Funded by CIHR) POS506 Using Native Ion Mobility Mass Spectrometry to Investigate Membrane Protein – Lipid Interactions John Patrick 1, Arthur Laganowsky1, Xiao Cong1, Yang Liu1, Wen Liu2 1Texas A&M University, USA, 2Texas A&M Institute for Biosciences & Technology Integral membrane proteins are embedded in complex lipid environments of the biological membrane. Understanding how lipids influence the structure and function is of great biological importance. Native Ion Mobility coupled to Mass Spectrometry (IM‐MS) has been introduced in recent years as a vital biophysical tool for studying intact membrane protein complexes. Herein, we report the utility of IM‐MS to reveal unprecedented insight into membrane protein lipid interactions at the resolution of individual lipid binding events. Complex mixtures of lipids and protein were analyzed using IM‐MS to obtain their binding affinities. Analysis of the data uncovers structural rearrangements that occur as a result of lipid association that in turn enhance binding of other lipid types. Several lipids were investigated in association with a membrane protein complex in order to elucidate the cooperative nature of lipid bilayer interactions. Identifying cooperative effects is a vital step towards understanding the basis of membrane protein function and structure within the biological membrane and how the chemical complexity of lipids modulate protein function. POS511 Post‐Translational Control of Synthetic Notch Receptors Using Biotin Ligase Jeffrey McMahan1, John Ngo1 1Boston University, Massachusetts, USA Notch receptors regulate a variety of cell fate decisions and are activated by the binding and endocytosis of ligands presented on the cell surface of neighboring cells. Synthetic Notch (SynNotch) receptors are engineered version of the protein that can be programmed to yield user‐specified input and output relationships, and such receptors have been used to reprogram T cells to detect cancer antigens and induce cell‐killing transcriptional programs. In natural Notch signaling, various mechanisms are used to fine‐tune the activity of the receptor in order to achieve diverse context‐dependent signaling outcomes. For example, Notch is glycosylated in the endoplasmic reticulum (ER) to adjust its ability to recognize specific ligands, thus controlling its perception of certain cells. In contrast, the ligand sensing control mechanisms of SynNotch receptors have mostly been through substitution of the receptor's ligand‐binding region and intracellular transcriptional effector domain. Here, we describe a method for controlling SynNotch receptors through an analogous post‐translational mechanism using a chimera containing biotin accepting peptide, which can be enzymatically biotinylated in the ER by an E. coli–derived biotin ligase. We show that regulated biotinylation of SynNotch receptors can be used to enable or disable their signaling capacity in response to cells bearing biotin‐binding proteins (such as streptavidin and anti‐biotin antibodies). This method offers a new level of control in mammalian cells, enabling precise control over the synthetic functions of engineered therapeutic cells. 13. METABOLIC ENGINEERING/ENERGY APPLICATIONS POS323 Development of Scalable Toluate Induction System for small molecules Production by E. coli Cherry Lin 1, Michael Miller1, Geetha Veeramuthu1, Jeff Pucci1, Shauna Bowden1 1Dupont, USA Escherichia coli is a versatile bacterium and is the host of choice for producing small molecules. Isopropyl‐ ß‐D‐thiogalactoside (IPTG) is currently the most commonly used molecular inducer for heterologous genes expression. We achieved high level production of Isoprene in Escherichia coli using an IPTG‐inducible Ptrc promoter to overexpress six heterologous mevalonate pathway enzymes and isoprene synthase. However, IPTG is not a feasible inducer for large‐scale manufacturing due to its cost and toxicity. This poster describes the development of Pseudomonas putida‐derived positively regulated XylS/Pm expression system to control mevalonate pathway genes and isoprene synthase enzyme in Escherichia coli. With the benzoic acid‐derived inducer m‐toluate, we achieved robust, time‐ and dose‐dependent Isoprene production. However, compared to a Ptrc‐expressed pathway, growth was inhibited and productivity reduced using the wild type Pm promoter. Increased pathway intermediates in strains with the Pm promoter suggested growth inhibition was due to metabolite imbalance. The Pm promoter is tightly regulated compared to the Ptrc promoter; therefore, we hypothesized that isoprene synthase expressed from the Pm promoter did not accumulate fast enough to prevent accumulation of toxic intermediates. Pm1–16 promoter is a stronger and less tightly regulated than wild type Pm promoter. Combining the Pm1–16 promoter controlling isoprene synthase and a wildtype Pm promoter driving pathway enzyme expression relieved growth inhibition and maximized the productivity. 14. MOTORS AND MACHINES POS229 Oligomerization and peptide binding by the SecA subunit of preprotein translocase Brian Shilton 1, Aliakbar Khalili‐Yazdi1 1The University of Western Ontario, Canada Preprotein translocase is an essential bacterial protein secretion system with central components SecA and SecYEG. SecA is an ATPase that catalyzes translocation of unfolded pre‐proteins through the membrane‐integral SecYEG pore. The preprotein substrates must be translocated in an unfolded state, and therefore the functioning of this secretion system involves both anti‐folding activity and ATPase‐dependent movement of the preprotein through the SecYEG pore, both of which are mediated by SecA. To gain insight into the molecular mechanics of this process, we have used SecA‐N68 which is a truncation construct lacking the C‐terminal helical domains but retaining the ATPase and peptide binding properties of SecA. Size exclusion chromatography and analytical ultracentrifugation were used to demonstrate that SecA‐N68 forms monomers, dimers, and tetramers in solution. In contrast, SecA‐N68ΔNC, in which the unstructured termini of SecA‐N68 have been trimmed, is completely monomeric. Therefore, the unstructured termini of SecA‐N68 are solely responsible for oligomer formation. Diffracting crystals of SecA‐N68ΔNC were obtained by introducing surface entropy reduction mutations at two locations. Combining this crystal structure with small‐angle X‐ray scattering (SAXS) data, a molecular model of the SecA‐N68 tetramer was constructed. The unstructured N‐terminal residues are also important for dimerization of full‐length SecA and a new model for the SecA solution dimer is proposed based on SAXS data and the SecA‐N68 tetramer. A surface plasmon resonance (SPR) based peptide binding assay was developed. The SPR‐based assay was used to demonstrate that the unstructured N‐terminal residues of SecA are essential for peptide binding. Therefore, the extreme N‐terminus of SecA is important for both oligomerization and preprotein binding. POS296 Biophysical Characterization of the Chemomechanical Coupling of F1 ATPase of Paracoccus denitrificans Mariel Zarco ‐ Zavala 1, Duncan G.G. McMillan1, Toshiharu Suzuki1, Hiroshi Ueno1, Rikiya Watanabe1, Francisco Mendoza‐Hoffmann2, José J. García‐Trejo2, Hiroyuki Noji1 1Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan, 2Department of Biology, Chemistry Faculty, Autonomous University of Mexico, Ciudad Universitaria, Mexico city, México Over the last decade a multitude of biophysical experiments have been conducted to characterize the rotatory catalysis of the F1‐ATPase. Nevertheless, they have been carried out almost entirely on bacterial enzymes. Recent studies of the mitochondrial F1 have shown differences in chemomechanical coupling between enzymes derived from bacterial and eukaryotic sources. P. denitrificans (Pd) is a free living bacterium phylogenetically related to the protomitochondria that harbors a respiratory chain extremely similar of the mitochondria. Thus, the characterization of the PdF1‐ATPase rotatory catalysis will provide insight into the adaptations of ATPase machinery upon endosymbiosis. The PdF1‐ATPase has tightly regulated hydrolytic activity controlled by a unique alpha‐proteobacteria inhibitor, the ‘ζ subunit’. The ζ subunit is structurally different to the bacterial (e) and mitochondrial (IF1) regulators, but appears to functionally resemble both. Structural and biochemistry studies have proposed that the subunit blocks the rotation in PdF1; however, there is no direct evidence of this inhibitory mechanism. Here, we have established an E. coli expression system for PdF1 using molecular chaperones and have studied the PdF1 rotatory dynamics. We observed counter‐clockwise rotation; typical of ATP hydrolysis that fits a Michealis‐Menten kinetics. PdF1 has a Vmax of 363.4 revolutions/s using a 40nm gold nanoparticle. In addition we have observed PdF1 three‐step rotation and obtained preliminary results of the effect of the subunit on the rotatory behaviour of PdF1. We found similarities in the assembly machinery and the regulatory mechanism of the bacterial PdF1 and the mitochondrial F1. POS468 The Structure and Mechanism of a Viral Genome Packaging Motor Janelle A. Hayes1, Brendan J. Hilbert1, Nicholas P. Stone, 1 Caroline M. Duffy1, Banumathi Sankaran2, Brian A. Kelch1 1Biochemistry & Molecular Pharmacology, University of Massachusetts Medical School, USA, 2Berkeley Center for Structural Biology, Lawrence Berkeley National Library, USA Many dsDNA viruses use a multi‐component ATPase motor to package their genomes into procapsids during viral maturation. These motors are among the most powerful known molecular machines, and how they couple ATP hydrolysis to DNA translocation is still poorly understood. Using a multi‐faceted approach combining x‐ray crystallography, small angle x‐ray scattering, protein docking, and biochemical assays, we elucidate the mechanism of ATP hydrolysis‐mediated DNA translocation. We present a novel genome packaging system from a thermophilic virus that provides new insight into motor structure and mechanism. We show that interfacial contacts mediate ATP hydrolysis, identify essential DNA binding and cleavage motifs, and reveal ATP‐dependent conformational changes of the packaging motor. Additionally, we showed that the motor's ATPase domain is both necessary and sufficient for DNA binding, an attribute previously ascribed to the nuclease domain. Our findings lead to a mechanism of translocation with a long lever‐arm to generate high force, and a steric block mechanism for nuclease regulation. 15. PEPTIDES POS101 The antifungal peptide periplanetasin 2 from American cockroach Periplaneta americana activates apoptotic signaling via oxidative stress against Candida albicans Dong Gun Lee 1, Heejeong Lee1 1Kyungpook National University, South Korea The cockroach, which is a household insect, is an established model organism in research. Periplanetasin 2, derived from the American cockroach Periplaneta americana, exerted potent antifungal effect against pathogenic fungi without causing hemolysis. Periplanetasin 2 induced oxidative stress by generation of reactive oxygen species (ROS) and lipid peroxidation. Periplanetasin 2 also caused apoptosis by exposure of phosphatidylserine and fragmentation of DNA, exerted in a concentration‐dependent manner. Hence, we investigated the mitochondrial apoptotic mechanism of periplanetasin 2 in Candida albicans. After treatment with periplanetasin 2, we observed mitochondrial depolarization and calcium accumulation. Moreover, we observed a decrease in cytosolic glutathione, and an increase in mitochondrial glutathione, indicating that periplanetasin 2 induced oxidative stress and high ROS production in the mitochondria. Because of this mitochondrial dysfunction, cytochrome c was released from the mitochondria into the cytosol, and caspase was activated in a time‐dependent manner. In summary, the antifungal peptide periplanetasin 2 activates apoptotic signals in the mitochondria by induction of oxidative stress. POS193 Topologically Constrained Peptidic Systems for Cell Penetration and Targeted Drug Delivery Gaurav Jerath 1, Vibin Ramakrishnan1, Prakash Hazam1, Ruchika Goyal1, Vishal Trivedi1, T R Santhosh Kumar1, Vibin Ramakrishnan1 1Indian Institute of Technology Guwahati, Department of Biosciences and Bioengineering, India Cell Penetrating peptides (CPPs) or Protein Transduction Domains (PTDs) are short oligomeric peptides with the capability of translocating across the cell membrane while simultaneously employing multiple mechanisms of entry. Many CPPs with disordered structures in solution adopt an alpha‐helical conformation at the cell surface, indicating that structural re‐ordering to a helix is vital to penetrative capability. Herein, we describe a series of helical peptides (CHAPs) which are structurally optimized with an electrostatic potential distribution at their surfaces to impart cell penetration. The peptides were tested against both cancerous and non‐cancerous cell lines for cell penetration and exhibited preferential uptake in cancer cells, while uptake in non‐cancerous cells is minimal. CHAPs could also deliver a small molecule (methotrexate) inside the cell with its therapeutic function intact. Moreover, delivery of drug through peptide‐drug conjugates also reduced its inhibitory concentration for cells as compared to the native drug. This suggests the dual capability of CHAPs in selectively delivering the drug to cancer cells and also increasing its therapeutic value for development of novel anti‐cancer therapies. POS211 Identification of a Conformational Heparin‐Recognition Motif on the Peptide Hormone Secretin: Key Role for Cell Surface Binding Noé Quittot 1, Armelle Tchoumi Nerée1, Phuong Trang Nguyen1, Steve Bourgault1 1Université du Québec à Montréal, Canada Secretin is a peptide hormone that exerts pleiotropic physiological functions by specifically binding to its cognate membrane‐bound receptor. The membrane catalysis model of peptide‐receptor interactions states that soluble peptidic ligands initially interact with the plasma membrane. This interaction increases the local concentration and structures the peptide, enhancing the rate of receptor binding. However, this model does not consider the dense network of glycosaminoglycans (GAGs) at the surface of eukaryotic cells. These sulfated polysaccharide chains are known to sequester numerous proteic signaling molécules. In this study, we evaluated the interaction between the peptide hormone secretin and sulfated GAGs and its contribution to cell surface binding. Using GAG‐deficient cells and competition experiment with soluble GAGs, we observed by confocal microscopy and flow cytometry that GAGs mediate the sequestration of secretin at the cell surface. Isothermal titration calorimetry and surface plasmon resonance revealed that secretin binds to heparin with dissociation constants ranging between 0.9 and 4 µM. By designing secretin derivatives with a restricted conformational ensemble, we observed that this interaction is mediated by the presence of a specific conformational GAG‐recognition motif that decorates the surface of the peptide upon helical folding. This study identifies secretin as a novel GAG‐binding polypeptide and opens new research direction on the functional role of GAGs in the biology of secretin. POS380 Importance Of Cysteines In Surfactant Protein B Analogues For Treatment Of Premature Newborn Rabbits Oihana Basabe Burgos 1, Marie Hägerstrand‐Björkman2, Bim Linderholm2, Anna Rising1,3, Jan Johansson1,3, Tore Curstedt2 1Division for Neurogeriatrics, Center for Alzheimer Research, Department of NVS, Karolinska Intitutet, 141 57 Huddinge, Sweden, 2Department of Molecular Medicine and Surgery, Karolinska Institutet at Karolinska University Hospital, 171 76 Stockholm, Sweden, 3: Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Box 7011, 750 07 Uppsala, Sweden BACKGROUND: Pulmonary surfactant extracted from animal lungs is used for treatment of premature infants with respiratory distress syndrome (RDS). These natural derived surfactants, such as poractant alfa (Curosurf®), consist of phospholipids and two hydrophobic surfactant proteins, SP‐B and SP‐C. The production costs of natural surfactants are high and the supply is limited, therefore development of new synthetic surfactants is needed. However, SP‐B and SP‐C are difficult to produce and not stable without lipids and for this reason it is important to find functional analogues. AIM: To determine the effect of a set of SP‐B analogues with different cysteine patterns combined with an SP‐C analogue and phospholipids, on lung function in a model of neonatal RDS. METHODS: Premature newborn rabbits were treated with different surfactant preparations and ventilated with constant pressures for 30 min without positive end‐expiratory pressure. The tidal volumes were registered during the experiments and lung gas volumes, as a measure of functional residual capacity, were measured at the end of the experiments. RESULTS: Synthetic surfactant containing an SP‐B analogue with four cysteines prevents the alveolar collapse at end‐expiration similar to poractant alfa. Similar SP‐B analogues with two or zero cysteines give surfactants with a much lower ability to prevent the alveolar collapse. CONCLUSIONS: Both disulphide bridges in the SP‐B analogue are important for preventing alveolar collapse at end‐expiration in an animal model of neonatal RDS. POS410 Artificial Crown Ether Ion Channel as Promising Therapeutic Agents Jean‐Daniel Savoie 1, François Otis1, Jochen Bürck2, Anne Ulrich2, Christophe Moreau3, Michel Vivaudou3, Normand Voyer1 1Université Laval, Canada, 2Karlsruhe Institute of Technology, Germany, 3Institut de Biologie Structurale, France Ion channels are transmembrane proteins that regulate the flow of ions through cell membranes and are required for the proper functioning of the cell. However, some ion channels such as non‐gated nanopores may act as toxins by enabling the uncontrolled passage of ions, destroying the usual electrochemical gradients of a cell and leading to its death. Targeting non‐gated nanopores towards cancer cells would be very promising for the development of new nanochemotherapeutic agents to treat resistant cancer cells. In this intent, a new family of synthetic ion channels was developed in our laboratory using a transmembrane helical peptide as framework bearing six crown ethers to create a transmembrane channel for ions. Even though biophysical studies have shed light on several aspects of these channels, the mechanism of action by which they incorporate into membranes remains unclear. Therefore, in an attempt to assess what drives the incorporation of crown ether‐modified peptides into bilayer membranes, we have used oriented circular dichroism (OCD) spectroscopy and the two‐electrode voltage clamp method (TEVC). Studies in OCD showed a transmembrane orientation at very low peptide/lipid ratios in lipid bilayers and aggregation at higher ratios, while TEVC showed ionic current in genuine Xenopus laevis oocytes cells. Furthermore, oocytes tend to die, probably from depletion of energy, after a short period of incubation with the peptide, thus revealing its potential as a cytotoxic agent. POS417 Verification of candidate peptide markers in urine of pregnant women with pre‐eclampsia by Western Blot Baibakova Viktoriia 1, Bugrova Anna2, Sergeeva Viktoriia1, Zakharova Natalia3, Muminova Kamilla3, Starodubtseva Natalia31, Kononikhin Alexey3, Indeykina Maria2, Popov Igor1 1Moscow Institute Of Physics And Technology, Russia, 2Emanuel Institute for Biochemical Physics, Russian Academy of Sciences, Russia, 3V. I. Kulakov Research Center for Obstetrics, Gynecology and Perinatology, Ministry of Healthcare of the Russian Federation, Russia Preeclampsia (PE) biomarker search is performed by many modern techniques including proteomics, which represent a comprehensive analysis of thousands of proteins and peptides. Previously, qualitative results showed that peptides specific for PE were mainly fragments of alpha‐1‐antitrypsin (SERPINA1), a‐chains of collagen types I and III, uromodulin and serum albumin (ALBU) [1]. Here we tried to verify the presence of the most specific of these peptides by Western Blot. 100 urine samples from three groups of patients (with no, mild, and severe PE) were obtained at the V. I. Kulakov Research Center for Obstetrics, Gynecology and Perinatology. All patients included in the study provided written informed consent approved by the Commission of biomedical ethics. The peptides were extracted by ultrafiltration and size‐exclusion chromatography (SEC). HPLC‐MS/MS analysis was performed for all urinary peptides samples. Several techniques of PAGE were compared and 4–20% gradient Tris‐Tricine‐PAGE was selected. Peptides were transferred onto nitrocellulose membranes and probed with antibodies (Oligomer A11, ALBU and two Polyclonal Antibodies for epitopes of SERPINA1). Chemiluminescence of the observed bands indicated the presence of SERPINA1, ALBU and misfolded structures in some PE samples. Because of low peptides concentrations not every PE sample showed detectable fragments of proteins of interest. Nevertheless, we managed to verify some of the previous MS results. Acknowledgements: The work was supported by RFBR grants no. 17‐08‐01537 A, no. 16–54‐21011_SNF_a (PE samples preparation) and Russian Science Foundation grant no. 14–24‐00114 (FTICR MS measurements). Reference: [1] A.S.Kononikhin et al. J.Proteomics. 2016; 149: 38–43. DOI: 10.1016/j.jprot.2016.04.024 POS421 Characterizing a Peptide Therapeutic Derived from the Cholesterol Recognition Amino Acid Consensus (CRAC) Motif of a Bacterial Toxin Evan Koufos 1, Angela Brown1, Anxela Sinani1, Joanne Huang1 1Lehigh University, Pennsylvania, USA Treatment of bacterial illnesses has become increasingly difficult as the development of new antibiotics is being outpaced by the increasing number of antibiotic‐resistant organisms. This has led us to search for alternative therapeutic approaches to combat these illnesses. To accomplish this, we focus on inhibiting protein toxins, which are one of the many virulence factors that pathogenic bacteria produce. Many toxins, including the repeats‐in‐toxin (RTX) protein, leukotoxin (LtxA), secreted by Aggregatibacter actinomycetemcomitans, recognize and bind to cholesterol (Chol) on the host cell membrane as an initial step in their mechanism; however, a viable method of inhibiting this interaction has yet to be uncovered. LtxA recognizes Chol on the target cell membrane via a Chol recognition amino acid consensus (CRAC) motif within its primary structure, which has been previously identified and characterized. We designed a peptide based on the CRAC motif of LtxA and used biophysical methods to demonstrate that this peptide retains the affinity for Chol of the full‐length toxin. As a result, the peptide can prevent LtxA binding to Chol, thereby inhibiting LtxA cytotoxicity. We are currently characterizing the binding to Chol of a panel of related CRAC peptides to better understand this mechanism for the design of an improved peptide therapeutic. We anticipate that this approach to inhibiting Chol binding by bacterial toxins could have broad applications in treating bacterial diseases. POS495 Self assembly of collagen mimetic peptides Deepti Mahapatra 1 1University of Canterbury, New Zealand This research is part of a wider research programme that contributes to and focuses on the utilization of lower value red meat components to create new meat‐derived foods and ingredients. The more abundant protein in an animal meat muscle, collagen has been the focus; specifically collagen model peptides. Apart from bridging gaps in the study of oxidative modifications in red meat processing in the food industry, this study on the self assembly of collagen model peptides will also prove beneficial in medical applications like drug delivery, tissue engineering and biomedical applications related to collagen etc. Effects of chain lengths of (Glycine‐Proline‐Hydroxyproline)n peptide sequences on the triple helical first order self‐assembly formations and supramolecular assembly formations were studied (where n= number of residues). In‐depth investigations of the impact of temperature cycles on the assembly properties of collagen peptides were performed. Analytical characterization techniques were used to study protein chemistry (CD, DSC, DLS, TEM, AFM, Infrared Spectroscopy, Mass Spectrometry (ESI and MALDI) and SAXS were used). Structure determination of (Glycine‐Proline‐Hydroxyproline)10 collagen triple helical peptide was achieved at 0.89 Å by X‐ray crystallography. Hydrothermal insult (cooking) targeted at the primary structural level of the peptide sequence. This was found instrumental in impacting on the supramolecular assembly properties. From redox proteomic profiling approach, it was observed that middle portion of the peptide strand acted as target site for specific residue modifications, instead of either sides of the peptide chain. This trend will further impact on the texture of meat derived food products. POS500 Identification of hydroxyproline‐containing hairpin‐like peptide EcAMP1 from barnyard grass (Echinochloa crusgalli L.) seeds: structure determination and comparative functional analysis Eugene Rogozhin 1 1Shemyakin‐Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Russia Plants are known to contain a rich variety of defense proteins and peptides that provide “the first defensive position” against various phytopathogenic microorganisms. Wild plants, and especially weeds, are usually widespread worldwide, and some cereal species that are phylogenetically close to cultivated plants demonstrated a higher resistance level to environmental biotic stress factors, whereas crops mainly lost this key role while the breeding process was in progress. Barnyard grass (Echinochloa crusgalli L. Beauv.) is a weed plant that has a large pool of different defense polypeptides with antimicrobial and insect protease inhibition properties. Previously, a number of novel antimicrobial peptides from the hairpin‐like (alpha‐hairpinins) family EcAMP were isolated from seeds, characterized in detail, and displayed novel biological effects against plant pathogenic and animal pathogenic fungi, bacteria, and yeasts at micromolar active concentrations in vitro. Peptidomic analysis of seed peptide extract by a combination of MALDI‐TOF mass spectrometry and automated Edman degradation disclosed two similar isoforms of EcAMP1 peptide with the single difference: a proline to hydroxyproline substitution at the 19th position of the amino acid chain. The following comparative antimicrobial assays detected a contribution of the modified proline residue in the peptide's functional realization. This is the first report to display a presence of hydroxyproline in the composition of natural biologically active peptides from cereals. This work was supported by Russian Science Foundation (project no. 14–50‐00131). 16. PROTEIN INTERACTIONS AND ASSEMBLIES POS036 Regulated Proteolysis of the Alternative Sigma Factor SigX During Bacterial Competence Development Yung‐Hua Li 1 1Dalhousie University, Canada In the Genus Streptococcus, the alternative sigma factor SigX (sX) is the key regulator for transcriptional activation of competence genes essential for taking up exogenous DNA. However, it was not until recently that regulated proteolysis of SigX is involved in escape from competence in these bacteria. Here, we provide evidence that adaptor protein MecA and proteases ClpC/ClpP are required for escape from competence by a mechanism involving MecA‐mediated proteolysis of SigX in Streptococcus mutans. By analyzing cellular levels of SigX, we demonstrate that the synthesis of SigX is transiently induced by competence‐stimulating peptide (CSP), but the SigX is rapidly degraded following competence induction. A deletion of either MecA, ClpC or ClpP results in the cellular accumulation of SigX and prolonged competence state, while an overexpression of MecA enhances proteolysis of SigX and accelerates escape from competence. In vitro protein‐protein interaction assays confirm that MecA interacts with SigX via its N‐terminal domain (NTD1–82) and with ClpC via its C‐terminal domain (CTD123–240). Such an interaction mediates formation of a ternary SigX‐MecA‐ClpC complex, triggering the ATP‐dependent degradation of SigX in the presence of ClpP. A deletion of N‐terminal or C‐terminal domain of MecA abolishes its binding to SigX or ClpC. We have also found that MecA‐mediated proteolysis of SigX is ineffective when S. mutans is grown in a chemically defined medium, suggesting the possibility that an unknown mechanism may be involved in negative regulation of MecA‐mediated proteolysis of SigX under this condition. We conclude that adaptor protein MecA plays a crucial role in recognizing and targeting SigX for degradation by the proteases ClpC/ClpP. POS050 Nucleophosmin Interacts with PIN2/TERF1‐interacting Telomerase Inhibitor 1 (PinX1) and Attenuates the PinX1 Inhibition on Telomerase Activity Sai Tim Ho 1 1The Chinese University of Hong Kong, China Telomerase activation and telomere maintenance are critical in cancer progression and transformation. PinX1 is a telomerase regulator and aberrant expression of PinX1 can cause telomere shortening. Identifying PinX1 interacting proteins is important for understanding telomere maintenance. In particular, we have identified direct interaction between C‐terminal tail of PinX1 and nucleophosmin (NPM), a positive telomerase regulator. The interaction interface on NPM was mapped, and we further showed PinX1 acts as the linker to bridge the association of NPM and hTERT, the catalytic subunit of telomerase. Also, the recruitment of NPM by PinX1 to PinX1/hTERT complex could partially attenuate the PinX1 inhibition on telomerase activity. As a result we have reported a novel mechanism that regulates telomerase activation through the interaction between NPM, PinX1 and the telomerase complex. Furthermore, we investigated the binding and localization pattern of PinX1/NPM interaction and hTERT complex formation in different stages of cell cycle. POS059 A Complex of Arabidopsis DRB Proteins Can Impair dsRNA Processing Marie‐Aude Tschopp 1, Nathan Pumplin2, Taichiro Iki3, Christopher Brosnan1, Pauline Jullien4 1Swiss Federal Institute of Technology, Switzerland, 2University of California, Davis, USA, 3Graduate School of Frontier Biosciences, Osaka University, Japan, 4IRD, Montpellier, France Small RNAs play an important role in regulating gene expression through transcriptional and post‐transcriptional gene silencing. Biogenesis of small RNAs from longer double‐stranded (ds)RNA requires the activity of DICER‐LIKE ribonucleases (DCLs), which in plants are aided by dsRNA binding proteins (DRBs). To gain insight into this pathway in the model plant Arabidopsis, we searched for interactors of DRB4 by immunoprecipitation followed by mass spectrometry‐based finger printing and discovered DRB7.1. This interaction, verified by reciprocal co‐immunoprecipitation and bimolecular fluorescence complementation, colocalizes with markers of cytoplasmic siRNA bodies and nuclear dicing bodies. In vitro experiments using tobacco BY‐2 cell lysate (BYL) revealed that the complex of DRB7.1/DRB4 impairs cleavage of diverse dsRNA substrates into 24nt small interfering (si)RNAs, an action performed by DCL3. DRB7.1 also negates the action of DRB4 in enhancing accumulation of 21nt siRNAs produced by DCL4. Overexpression of DRB7.1 in Arabidopsis altered accumulation of siRNAs in a manner reminiscent of drb4 mutant plants, suggesting that DRB7.1 can antagonize the function of DRB4 in siRNA accumulation in vivo as well as in vitro. Specifically, enhanced accumulation of siRNAs from an endogenous inverted repeat correlated with enhanced DNA methylation, suggesting a biological impact for DRB7.1 in regulating epigenetic marks. We further demonstrate that RNASE THREE‐LIKE (RTL) proteins RTL1 and RTL2 cleave dsRNA when expressed in BYL, and that this activity is impaired by DRB7.1/DRB4. Investigating the DRB7.1‐DRB4 interaction thus revealed that a complex of DRB proteins can antagonize, rather than promote, RNase III activity and production of siRNAs in plants. POS072 Role of Proline in Three‐Dimensional Domain Swapping Yongqi Huang 1, Zhengding Su1 1Hubei University of Technology, China Many proteins function in the context of protein‐protein complexes. Three‐dimensional (3D) domain swapping is a mechanism to form protein homooligomers. It has been proposed that several factors, including proline residues in the hinge region, may affect the occurrence of 3D domain swapping. Although introducing prolines into the hinge region has been found to promote domain swapping for some proteins, the opposite effect has also been observed in several studies. So far, how proline affects 3D domain swapping remains elusive. In this work, based on a large set of 3D domain‐swapped structures, we performed a systematic analysis to explore the correlation between the presence of proline in the hinge region and the occurrence of 3D domain swapping. We further analyzed the conformations of proline and pre‐proline residues to investigate the roles of proline in 3D domain swapping. We found that more than 40% of the domain‐swapped structures contained proline residues in the hinge region. Unexpectedly, conformational transitions between the cis and trans isomers of proline residues were rarely observed during domain swapping. An analysis on the conformations of proline and pre‐proline residues showed that, while strain was relieved for some proteins upon swapping, strain was also introduced for several other proteins. Our analyses suggest that the critical roles of conformational constraints, backbone strain, and the cis‐trans isomerization in domain swapping proposed for proline is questionable. We propose that a role of proline in domain swapping may be to slow down the folding process and promote intermolecular interactions between unfolded monomers. POS084 Structure and function of the Toc159 M‐domain in membrane association and chloroplast protein import Matthew Smith 1, Emily Tran1, Nicholas Grimberg1, Simon Chuong2 1Department of Biology, Wilfrid Laurier University, Canada, 2Department of Biology, University of Waterloo, Canada Chloroplast biogenesis relies on the import of thousands of nuclear‐encoded preproteins from the cytosol. Preprotein import is supported by large protein complexes called the Toc and Tic (Translocon at the outer and inner envelope membranes of chloroplasts) complexes, which work cooperatively to translocate the preproteins across the double‐membrane envelope that surrounds chloroplasts. Toc159, one of the preprotein receptors of the Toc complex, is comprised of 3 distinct domains: 1. the N‐terminal Acidic (A‐) domain, which is intrinsically disordered; 2. the central GTPase (G‐) domain; and 3. the C‐terminal Membrane (M‐) domain that anchors the protein to the outer membrane using an unknown mechanism. The M‐domain has no known homologues and does not contain a predicted trans‐membrane domain, but does contain intrinsic chloroplast targeting information at the extreme C‐terminus. Three sub‐domains of the M‐domain have been identified, one of which contains a predicted beta‐helix motif, which may be important for anchoring the protein to the chloroplast outer membrane. We are interested in characterizing the structure of the M‐domain and determining the mechanism of Toc159 targeting, and the nature of the membrane association, as part of our larger goal of understanding the role Toc159 plays in protein import into chloroplasts. We will present our most recent data on the structure and function of the predicted beta‐helix sub‐domain of the Toc159 M‐domain. POS117 The Mechanisms for counting and handoff by human DNA primase: a role for the 4Fe‐4S cluster? Walter Chazin 1, Jacqueline Barton2, Matthew Thompson1, Elizabeth O'Brien2, Marilyn Holt1, Lauren Salay1, Aaron Ehlinger1 1Vanderbilt University, Tennessee, USA, 2California Institute of Technology, USA Generation of the complementary leading and lagging strands during DNA replication requires the action of a series of polymerases. The priming of template requires the action of the DNA‐dependent RNA polymerase, DNA primase, which synthesizes the first 8–10 nts de novo on the ssDNA template. Despite intense study over many years, the mystery of how primase counts to 10 and hands off the primed substrate to polymerase a remains unsolved. We are integrating crystallography, SAXS, and EM with specifically designed substrates to characterize the active configurations as primase initiates and elongates the primer and to test our model for the mechanism of primase counting. However, handoff of the primed template cannot be fully explained by structure alone. This question led us wonder if there is a role for the 4Fe‐4S cluster in the unique C‐terminal domain of the p58 regulatory subunit of human primase (p58C)? Although discovered ∼10 years ago, the function of this cluster in priming has remained enigmatic. Intriguingly, it has long been recognized that charge can be transported over long ranges through fully base‐paired duplex DNA. Using a nanoscale electrochemical device, we have shown that p58C is able to transport charge through DNA. Remarkably, DNA charge transport (CT) is dependent on the redox state of the cluster and a specific tyrosine‐mediated path that links the cluster to the DNA binding surface. Our results suggest that a redox switch in the primase 4Fe‐4S cluster, mediated by DNA CT, provides the missing factor that drives the handoff of the initial RNA primed template to DNA polymerase a. POS140 Negative Phosphoregulation of NCK1/2 Adaptor Proteins by the Tyrosine Kinase Receptor EphA4 Ugo Dionne 1, François Chartier1, David Bernard2, Michel Tremblay2, Gerald Gish4, Patrick Laprise1, Nicolas Doucet3, Christian Landry4, Nicolas Bisson1 1CRC, CHU of Quebec Oncology division, Molecular Biology, Medical Biochemistry and Pathology Laval University and PROTEO, Canada, 2CHU of Quebec, Canada, 3INRS and PROTEO, Canada, 4IBIS, Biology Laval University, PROTEO, Canada Cells respond to extracellular stimuli via membrane‐bound proteins, such as tyrosine kinase receptors (RTKs), in order to survive and to adapt to their environment. Activated RTKs bear phosphotyrosine (pTyr) docking sites for adaptor proteins such as NCK1 and 2 (NCK1/2). Their function is to couple pTyr on activated receptors via their single SH2 domain to cytoplasmic effectors containing Pro/Arg‐rich motifs via their three SH3 domains. The regulation mechanisms of NCK1/2 are poorly understood. We sought to determine whether NCK1/2 proteins are regulated by tyrosine phosphorylation. We used mass spectrometry to map pTyr residues on NCK1/2 and to analyse the effect of those modifications on NCK2 signaling networks in vivo. We identified 15 distinct pTyr on NCK1/2, including one that lays in the binding pocket of every SH3 domain of NCK1/2 and that is conserved in 57% of the 250 murine SH3 domains. We identified the RTK EphA4, a direct binder of NCK1/2, as a kinase that phosphorylates these residues both in vitro and in vivo. We demonstrated that phosphorylation of these Tyr abrogated NCK1/2 SH3 domains interactions with their substrates. We further showed that a phosphomimic triple mutant (Y/E) of the conserved Tyr of Dock, the Drosophila ortholog of NCK1/2, inhibited its SH3‐dependant functions in the development of the fly eye. Our data suggests that RTKs are able to terminate signaling directly by phosphorylating their substrates, including adaptor proteins such as NCK1/2. POS153 A comparative overview between in vitro and in vivo glucosylation of human serum albumin: Protein modification in diabetes mellitus Km Neelofar 1, Jamal Ahmad1 1Aligarh Muslim University, India Proteins modifcations in diabetes mellitus may lead to early glycation products and advanced glycation end products (AGEs). Whereas no extensive studies have been carried out to assess the role of early glycation products in chronic kidney disease (CKD), numerous research articles have demonstrated the role of AGEs. Bioethical committee, J.N. Medical College, Aligarh Muslim University, Aligarh did not find any objection for human samples collection and awarded ethical clearance for this study. Objective: This study has been design to compare the structural and functional changes in HSA glycated by glucose with HSA purified from diabetic patients with and without CKD. Methods: Structural changes in native and glycated‐HSA were observed by UV, fluorescence, circular dichroism spectroscopy, Fourier transform infrared spectroscopy, tryptophan fluorescence and free thiol group along with carbonyls estimation. Results: Enhanced changes in structural confirmations were observed in glycated‐HSA (75mM). Significant impairment in structure were observed in CKD‐HSA as compare to normal HSA. Consequently, these changes associated with glycation provoked a reduction in free thiol group and strong increment of protein carbonyl contents in Amadori‐HSA and diabetic‐HSA as compared to normal HSA. Conclusion: These findings reveal that structural comformation of glycated HSA, isolated from diabetic patients with and without CKD were significantly different from the native HSA. Additionally, HSA may not be available under extensive glycation, leading to the impairment of its important functions. It also suggests that glycated HSA may be involved in the pathogenesis of diabetes and its complications such as CKD and might be an important biomarker for monitoring diabetic pathophysiology especially diabetic kidney disease patients. POS161 Solving a Mystery of Coagulation Factor XIII: Dissociation of a Homodimer as Part of the Activation Process Boris Anokhin 1, Muriel Maurer1, Vilius Stribinskis2, William Dean2 1Department of Chemistry, University of Louisville, Kentucky USA, 2Brown Cancer Center, University of Louisville, Kentucky USA Factor XIIIA (FXIIIA) is a transglutaminase that cross‐links intra‐ and extracellular protein substrates in a calcium‐dependent manner. FXIIIA is the only member of the transglutaminase family found as a homodimer (A2) in zymogen form. It can be activated by thrombin‐mediated cleavage of the activation peptides (AP) or non‐proteolytically in the presence of high mM Ca2+. Activated FXIIIA has long been considered a homodimer, just like its zymogen. Accumulating but inconclusive evidence, however, suggests a monomeric state for active FXIIIA. In the current project, size exclusion chromatography and analytical ultracentrifugation were employed to assess the oligomeric state and hydrodynamic properties of FXIIIA. While intersubunit interactions in the dimeric zymogen form were tight (Kd 130 nM), both non‐proteolytic and thrombin‐mediated FXIIIA activation resulted in monomeric species (Kd 300 and 600 mM, respectively). Thrombin cleavage of a single AP on the FXIII A2‐homodimer initiated its transition to monomers. Remarkably, the catalytic activity of 100 mM Ca‐activated FXIIIA was lower than that of proteolytically activated enzyme. Thus, for the first time, a quantitative assessment of FXIIIA intersubunit interactions was performed and direct experimental evidence was obtained for the monomeric state of activated FXIIIA in solution. A dimeric state of the zymogen is proposed to stabilize FXIIIA in a physiological setting and to prevent premature protein cross‐linking. By contrast, cleavage of the AP and dissociation of the homodimer are crucial for full expression of FXIIIA function. POS223 Structural, Functional and Evolutionary Analysis of Domain‐Swapped Dimerization within the Interferon Induced Proteins with Tetratricopeptide Repeats Yazan Abbas 1, Bhushan Nagar1, Irene Xie1, Zixian Li1 1McGill University, Canada The interferon induced proteins with tetratricopeptide repeats (IFITs) are a family of innate‐immune, antiviral effectors which in humans comprises 4 well‐characterized members: IFIT1, IFIT2, IFIT3, and IFIT5. IFITs have been shown to interact with RNA to limit viral replication. Additionally, they can form a complex made up of IFIT1, IFIT2, IFIT3, and other host factors (referred to as the IFIT interactome), which may have a role in antiviral immunity. A recent crystal structure of RNA‐free IFIT2 revealed N‐terminal domain‐swapping, whereby 3 central helices are exchanged between two protomers to form an intertwined dimer, thus forming a positively‐charged RNA‐binding channel. The origins and mechanisms of domain‐swapping, its role in IFIT complex formation, and its impact on RNA binding are unknown. Using X‐Ray crystallography, we show that the N‐terminal domain of IFIT3 also forms domain‐swapped dimers, mediated by two hinge‐loops, similar to IFIT2. Sequence and structure comparison of these hinge‐loops shows that IFIT2 and IFIT3 harbor deletions in these sites in comparison to the non‐domain‐swapped IFIT1 and IFIT5. Insertion mutagenesis into the first hinge‐loop disrupts IFIT2 and IFIT3 homo‐dimerization. Bacterial co‐expression assays suggest that IFIT2 and IFIT3 form hetero‐domain swapped dimers. Our work therefore suggests that domain‐swapping in IFIT proteins arose from an IFIT precursor following gene duplication and deletions in structured hinge‐loops, allowing IFIT2 and IFIT3 to homo‐ and hetero domain‐swap, which may be important for IFIT interactome assembly. Current work is focused on the biophysical and structural characterization of IFIT2 and IFIT3 monomeric mutants, the IFIT2‐IFIT3 hetero‐dimer, and investigating the RNA binding properties of IFIT2 and IFIT3 monomers and dimers. POS278 Understanding the structure and function of the capsid protein of Zika, West‐Nile and Dengue viruses, namely their ability to interact with host lipid systems Ana Martins 1, Ivo Martins1, André Faustino1, André Nascimento1, Filomena Carvalho1, Nuno Santos1 1Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Portugal Flaviviruses, such as Dengue (DENV), West‐Nile (WNV) and Zika (ZIKV) viruses are transmitted to humans mainly by the bite of mosquitos, and constitute a serious public health treat. Despite the global spread and disease severity, there is no specific and effective treatment for these Flaviviruses infection, in part due to a poor understanding of the viral life cycle. The capsid protein (C) is a major drug target, since it mediates key viral life cycle steps, including viral assembly and encapsidation. Here, we investigated WNV and ZIKVC ability to bind host lipid systems. Zeta potential show that WNVC interacts with LD surface proteins, in a potassium dependent manner, as previously shown for DENVC by us. ZIKVC also interacts with LD, in a similar manner, although, in contrast, potassium ions can be replaced by sodium. Dynamic light scattering measurements show that WNVC interacts with very low‐density lipoproteins (VLDL) but not with low‐density lipoproteins (LDL). ZIKV C also interacts with VLDL, as observed for DENV C. Moreover, WNVC (un)binding forces upon interaction with LD and VLDL were quantitatively determined by atomic force microscopy (AFM)‐based force spectroscopy. AFM confirmed that WNVC specifically binds to LD and VLDL (but not LDL), in a process requiring K+ ions. Furthermore, ZIKV, WNV and DENV C protein sequences reveal similar predicted hydrophobicity, a‐helical propensity and tertiary structure, that can thus be targeted via similar approaches. Combining all the above with our background on DENVC protein and pep14–23 (an inhibitor of DENVC interaction with lipid systems, designed by us) will thus pave the way for Flavivirus drug development strategies. POS326 Biophysical Characterization of the Interaction between Caspase‐2 and 14‐3‐3 Protein Veronika Obsilova 1, Tomas Obsil2, Dana Kalabova1, Miroslava Alblova1, Aneta Smidova1, Olivia Petrvalska2 1Institute of Physiology, The Czech Academy of Sciences, Prague, Czech Republic, 2Faculty of Science, Charles University, Prague Czech Republic Caspase‐2 (C2), a cysteine‐dependent and aspartate‐specific intracellular protease, has multiple roles in the DNA damage response, cell cycle regulation and tumor suppression. C2 functions as a central coordinator between the cell metabolism and apoptosis and its function is regulated by phosphorylation at several Ser residues. Phosphorylated procaspase‐2 (proC2) binds to the 14‐3‐3 protein and this interaction blocks proC2 activation through an unknown mechanism. To elucidate this regulatory mechanism we have identified sites responsible for the 14‐3‐3 protein binding to proC2, performed the biophysical characterization of the 14‐3‐3:proC2 complex using analytical ultracentrifugation and mapped the binding interface of the 14‐3‐3:proC2 complex using hydrogen‐deuterium exchange kinetics coupled to mass spectrometry. Our results provide the first structural insight into the 14‐3‐3‐dependent regulation of C2. This work was supported by the Czech Science Foundation (Project 17–00726S). POS328 Structural Characterization of Complexes between 14‐3‐3 Protein and Protein Kinases CaMKK2 and ASK1 Tomas Obsil 1, Veronika Obsilova2, Olivia Petrvalska1, Katarina Psenakova1, Salome Kylarova2, Dana Kalabova2 1Faculty of Science, Charles University, Prague, Czech Republic, 2Institute of Physiology, The Czech Academy of Sciences, Prague, Czech Republic Many protein kinases have been shown to be regulated in the 14‐3‐3 protein‐dependent manner; however, the underlying molecular mechanisms are only partially identified, mainly due to the lack of structural data. In this work, the role of 14‐3‐3 in the regulation of two protein kinases ASK1 and CaMKK2 has been investigated. ASK1 (apoptosis signal‐regulating kinase 1) is a member of the mitogen‐activated protein kinase kinase kinase (MAP3K) family that plays a crucial role in immune and stress responses. CaMKK2 (Ca2+/calmodulin‐dependent protein kinase kinase 2) is a member of CaMK signaling cascades that detect changes in intracellular concentration of calcium ions and transduce this signal by activation of corresponding transcription factors. To investigate the role of 14‐3‐3 in the regulation of these kinases, we have performed biophysical and structural characterization of their complexes with 14‐3‐3 using kinase activity measurements, analytical ultracentrifugation, small angle X‐ray scattering, NMR and fluorescence spectroscopy. Our results indicate that these complexes are dynamic and conformationally heterogeneous. Structural analysis also indicated that 14‐3‐3 interacts with the kinase domain of ASK1 in close proximity to its active site, thus indicating this interaction might block its accessibility and/or affect its conformation. This work was supported by the Czech Science Foundation (Project 16–02739S). POS329 The AAA+ chaperone‐proteases ClpXP and Lon target MinD for proteolysis in E. coli Chris LaBreck 1, Jodi Camberg1 1University of Rhode Island, USA The Min system of E. coli, including MinC, MinD, and MinE, promotes the placement of the FtsZ‐ring at midcell by preventing FtsZ assembly at the cell poles. MinD is an ATPase that associates with the membrane in the ATP‐bound conformation, and dissociates from the membrane following ATP hydrolysis stimulated by MinE binding. The Min system exhibits pole‐to‐pole oscillations that are driven by the binding and release of MinD from the membrane. MinC oscillates with MinD through a direct interaction and inhibits FtsZ polymerization. ClpXP and Lon are major prokaryotic AAA+ chaperone‐proteases. ClpX is a AAA+ ATPase that associates with the ClpP protease to unfold and degrade substrates. Lon contains both AAA+ and protease modules in a single polypeptide. ClpXP degrades several cell division proteins in vivo, including FtsZ and ZapC. Here, we report that MinD is degraded by ClpXP and Lon in vitro, and further characterize the mechanisms governing MinD recognition and proteolysis. We monitored degradation of purified MinD and observed slower degradation in the presence of small unilamellar phospholipid vesicles derived from E. coli. The MinD N‐terminal region contains a putative ClpX recognition motif. We purified the MinD mutant protein MinD(R3E), containing a substitution in this region, and observed impaired degradation. Finally, in vivo we observed altered MinC oscillation rates in cells overexpressing ClpXP, suggesting that ClpXP degradation modifies the Min oscillation cycle. Together, our results show that MinD is a ClpXP substrate and further suggest that recognition depends on the MinD N‐terminal region. Furthermore, membrane association protects MinD from degradation, suggesting that ClpXP may target cytoplasmic MinD. POS343 Restricted HIV‐1 Env Glycan Engagement by Lectin‐Engineered DAVEI Protein Chimera is sufficient for Lytic Inactivation of the Virus Bibek Parajuli 1, Kriti Acharya1, Harry Bach2, Cameron Abrams3, Irwin Chaiken3 1Drexel University College of Medicine, Pennsylvania, USA, 2Drexel University, Pennsylvania, USA, 3Drexel University College of Engineering, Pennsylvania, USA, 4Drexel University College of Medicine/A.J. Drexel Institute of Basic and Applied Protein Science, Pennsylvania, USA The first generation recombinant CVN‐DAVEI constructs composed of cyanovirin‐N (CVN) fused to a membrane proximal external region (MPER) exhibited potent and irreversible inactivation of both pseudotyped and fully infectious HIV‐1 viruses. Additionally, we engineered the chimeric protein to identify molecular determinants important for virolytic activity. In the CVN‐DAVEIs made, the CVN domain binds to gp120 and provides sufficient binding affinity to steer the MPER for gp41 engagement eliciting virolysis. However, the promiscuity of CVN to associate with multiple glycosylation sites in gp120 and its multivalency limits understanding of the molecular arrangement of the DAVEI molecules on trimeric spike needed for virolysis. In this study, we constructed and investigated the virolytic function of second generation DAVEI molecules using a simpler lectin domain derived from microvirin (MVN). Unlike cyanovirin, microvirin has a single glycan binding site, exhibits no toxicity or mitogenic activity, and binds to a small repertoire of glycans in gp120, on the outer domain. We found that, like CVN‐DAVEI‐L2–3Trp, MVN‐DAVEI2–3Trp exploits similar mechanism of action for inducing virolysis, but by more selective gp120 glycan engagement. By sequence redesign, the potency of MVN‐DAVEI2–3Trp protein was significantly increased. Re‐engineered MVN‐DAVEI2–3Trp(Q81K/M83R) protein binding was competed by gp120 specific mAb, 2G12, both in binding and virolytic assays. That the lectin domain in DAVEIs can utilize MVN without loss of virolytic function argues that simple monovalent and restricted HIV‐1 Env glycan engagement is sufficient for inducing virolysis. Since the improvised MVN‐DAVEI2–3Trp(Q81K/M83R) construct has a defined binding site on gp120, it provides an improved tool to elucidate productive molecular arrangements of Env‐DAVEI enabling virolysis. POS345 Study of the Interaction Between Parkin and Endophilin A1 Marjan Seirafi 1, Zlata Plotnikova1, Guennadi Kozlov1, Jean‐Francois Trempe1, Kalle Gehring1 1McGill University, Canada Mutations in the parkin and PINK1 genes are responsible for a common inherited form of Parkinson's disease (PD) with an early onset. The gene products E3 ubiquitin ligase parkin and kinase PINK1 are involved in autophagy of damaged mitochondria termed mitophagy. In this pathway, PINK1 phosphorylates parkin and ubiquitin, thus activating parkin ligase activity. Parkin contains a ubiquitin‐like (Ubl) domain at the N‐terminus that inhibits its activity. Autoinhibited parkin is activated by phosphorylation at the Ubl domain by PINK1 and binding to phospho‐ubiquitin, both releasing Ubl domain from the E2 binding site on parkin. Parkin Ubl domain also binds SH3 domain of Endophilin A1, a brain specific protein, with an affinity comparable to proline‐rich domains (PRDs) from well‐established SH3 partners. Parkin structure reveals that Ubl uses similar surfaces for binding to the RING1 domain of parkin and SH3 domain of Endophilin A1. This could explain why SH3 binds to full‐length parkin with low affinity, and that conditions that promote phosphorylation enhance the interaction between full‐length proteins at nerve terminals. Here, we report that phosphorylated Ubl also binds to SH3 domain with similar affinity in vitro. Moreover, phosphorylation of parkin, its binding to phospho‐ubiquitin, and parkin mutants that release Ubl domain increase the binding of full‐length parkin to the SH3 domain of Endophilin A1. Current work is directed towards studying the effect of this interaction on parkin activity in vitro and in cells. The findings may identify the link between synaptic vesicles endocytosis and mitophagy. POS355 A Phosphorylation and Ubiquitylation Circuitry Drives Homologous Recombination on RPA‐ssDNA Jean‐Christophe Dubois1, Alexandre Maréchal1, Maïlyn Yates1, Geneviève Clément1, Laurent Cappadocia2, Luc Gaudreau1, Lee Zou4 1Université de Sherbrooke, Quebec, Canada, 2Memorial Sloan Kettering Cancer Center, New York, USA, 3Massachusetts General Hospital Cancer Center, Massachusetts, USA Impediments to replication fork progression induce RPA‐coated single‐stranded DNA (RPA‐ssDNA) accumulation. RPA‐ssDNA orchestrates the recruitment and activation of many genome maintenance factors to signal and repair DNA damage. The RPA complex is heavily phosphorylated by ATR, ATM and DNA‐PK kinases during replication stress which redirect its function from DNA replication to damage signaling and repair. RPA is also ubiquitylated by the PRP19 and RFWD3 ubiquitin ligases in response to damage. Both E3 ligases also promote replication fork repair and homologous recombination (HR). However, how DNA damage stimulates RPA ubiquitylation is still unclear and whether the ubiquitin ligase activity of PRP19 on RPA‐ssDNA is important for HR remains unexplored. Here, we find that whereas RFWD3 constitutively interacts with the RPA complex, PRP19 assembles on RPA‐ssDNA only upon fork damage. Ubiquitylation of RPA and its interaction with PRP19 correlate with RPA32 phosphorylation. Interestingly, a non‐phosphorylatable RPA32 mutant still interacts with RFWD3 but cannot bind PRP19 and is poorly ubiquitylated upon damage. A positively charged pocket on the PRP19 WD40 domain interacts with RPA suggesting that PRP19 recognizes phosphorylated RPA through this surface. Finally, the ubiquitin ligase activity of PRP19 and its RPA‐interacting surface are required for optimal HR and ATR activation. We propose that RPA phosphorylation promotes the accumulation of PRP19 on RPA‐ssDNA which together with RFWD3 stimulates RPA ubiquitylation. Similarly to the phosphorylation‐ubiquitylation cascade underlying the g‐H2AX chromatin‐based DNA damage response, this would allow the spreading of RPA phosphorylation and ubiquitylation over large segments of RPA‐ssDNA found at impaired forks and promote robust ATR activation and HR‐mediated DNA repair. POS357 Characterization of Monobody Interactions with a Fluoride Ion Channel by Fluorescence Anisotropy Abraham Cheloff 1, Daniel Turman1, Christopher Miller1 1Department of Biochemistry, HHMI, Brandeis University, Massachusetts, USA The Fluc ion channel family is comprised of dimeric membrane proteins whose function is to expel excess F‐ from the cytoplasm of microorganisms to resist inhibitory effects of this environmental xenobiotic anion. Recent structures of an E. coli Fluc homolog bound to engineered “monobody” proteins selected from phage display libraries reveal multiple side‐chain contacts at the channel‐monobody interface. Two such monobodies, S9 and S12, share a similar interface structure, but their nanomolar‐range binding affinities differ by ∼20 fold. We focus on the per‐residue energetic contributions to the binding affinity of S12 to Fluc by introducing point mutations at polar contacts on either size of the interface, assessing the change in binding affinity using fluorescence anisotropy. We have found that residues Y88, Y86, Y79, S81, and T30 on the diversified loops of S12 all contribute significantly to the binding energy. Future studies will mutagenize the monobody‐channel interface to derive information on how these different monobodies interact with Fluc channels. POS363 Investigating Trim5a RING and B‐Box Self‐Association And Its Role In Antiretroviral Signalling Joy Yang 1, David C. Goldstone1, Jeremy R. Keown1 1School of Biological Sciences, University of Auckland, New Zealand The anti‐retroviral restriction factor Trim5a prevents infection by diverse retroviruses, including HIV‐1, disrupting early post‐entry stages of the retroviral lifecycle. As a member of the TRIM protein family Trim5a has a conserved N‐terminal domain architecture consisting of an N‐terminal RING domain with E3 ubiquitin ligase activity, and a B‐box domain and antiparallel coiled‐coil involved in self‐assembly. To restrict a particular retrovirus, Trim5a must recognise the capsid of the incoming retrovirus. This recognition results in RING‐mediated ubiquitylation, leading to the activation of downstream signalling events that induce a cellular antiviral state. To better understand how capsid recognition translates into antiretroviral activity, we have studied the self‐association and ubiquitylation activity of the RING and B‐box domains. Oligomeric states were determined by size‐exclusion chromatography with multi‐angle light scattering and sedimentation velocity analytical ultracentrifugation. Ubiquitylation activity was tested with in vitro ubiquitylation assays involving E2 enzyme partners established in the literature. Previous work has established that a monomeric RING domain has no activity. The RING‐B‐box construct exhibits higher order self‐assembly and ubiquitylation activity. Mutations were made to disrupt self‐assembly, which also resulted in a decrease in ubiquitylation activity. These results demonstrate that ubiquitylation activity is closely dependent upon Trim5a higher‐order assembly, linking recognition of the retroviral capsid to ubiquitylation and the activation of restriction. POS364 Diverging from eukaryotic to prokaryotic expression system for PP2A phosphatase catalytic subunit Priyanka Sandal 1, Shweta Shah1, Gururaj Rao1 1Iowa State University, Iowa, USA Protein Phosphatase 2A (PP2A) is a serine/threonine phosphatase that exists as a hetero‐trimeric complex composed of Scaffold ‘A’ subunit, catalytic ‘C’ subunit and substrate specific variable ‘B’ subunit. PP2A‐3 and PP2A‐4 are the two isoforms of the PP2A ‘C’ subunit in Arabidopsis. We have previously demonstrated that one of the key steps in the regulation of formative cell division in root is the interaction between the 'C' subunit of PP2A‐3 (PP2A‐3c) and the kinase domain of receptor like kinase Arabidopsis CRINKLY4 (ACR4). Importantly, PP2A‐3c is phosphorylated by ACR4 and, became the first described novel substrate for the receptor kinase {(Yue & Sandal et al, PNAS (2016)}. A more comprehensive biochemical/biophysical characterization of the interaction between the two proteins invitro can significantly inform and drive in planta investigations. However, such studies require substantial quantities of protein. Expression in eukaryotic systems such as insect cell or mammalian cell cultures has been the preferred method for obtaining catalytically active human PP2Ac owing to the intrinsic availability of all necessary regulatory proteins necessary for differential regulation and activation. However, the paucity of pure protein is a rate‐limiting step. To overcome this limitation, we have expressed Arabidopsis PP2A‐3c in the more traditional E. coli system. Although the protein initially is expressed in an inactive form, distinct but measurable activity can be detected over time at room temperature suggesting a slow conformational change to active state. Herein we characterize the biochemical properties of the E.coli expressed PP2A‐3c and demonstrate the ability to recover phosphatase activity in vitro both in presence & absence of PP2A phosphatase activator‐PTPA. POS370 Extension protein engineering (EPE), a technique for the engineering of novel proteins Matthew Dominguez 1, Elliott Stollar1 1Eastern New Mexico University, New Mexico, USA Protein engineering increases stability and improves function with the potential to develop new binding partners with increased affinity and specificity. To our knowledge, libraries of extended protein variants have not been explored. As such, we have developed extension protein engineering (EPE), a method that explores the extension of the natural protein sequence to improve stability. In this study, an SH3 domain and an SH3 domain‐peptide hybrid were subjected to EPE to discover a stabilized domain and a highly specific peptide. The screening process involves a recently developed high throughput protein folding equilibrium and kinetic assays and a custom data analysis program. Screening results show variants with a range of equilibrium and kinetic stability compared to wild type. Tyrosine appears to be a key residue in increasing binding affinity, while multiple prolines decrease binding affinity. Our top variants can be subjected to a second round of EPE to further enhance its properties. This study is the beginning of what could become a highly selective way to develop high affinity peptides which could assist in the development of novel therapeutics for protein related diseases. POS407 Structural Studies of the SH3 domain family of yeast. Rebecca Rhode 1, Matthew Dominguez1 1Eastern New Mexico University, New Mexico, USA The SH3 domain family is a group of proteins, typically 60 amino acid residues long. SH3 domains play key parts in protein‐protein interactions. There are 28 Domains in the Yeast SH3 domain family, which is a good model for the approximately 300 human SH3 domains. By probing the structure and stability of each domain, insights into their binding properties can be obtained. Using CD, we can measure secondary structure for all SH3 domains to compare between the various members. This experimental data can be compared to known or predicted structural models. Simultaneously, melting temperatures can be measured to determine thermal stability, using different salt concentrations. Overall, we have purified 20 out of 28 domains. Preliminary results show a variety of melting temperatures and secondary structure content. The range of melting temperatures is expected as previous studies indicate varying stabilities across the domain family. However, we did not expect the secondary structure to change as drastically because the family has a common fold. Combining structure and stability data will help in determining the ideal conditions to study each domain member. POS412 Phosphorylation of Anabaena Sensory Rhodopsin Transducer: a Putative Signaling State in Sensory Rhodopsin Mediated Protein‐Protein Cross Talk Vishwa Trivedi 1, Tashmay Jones1, Renee Walker1, Ravi Kumar Gundampati2, Thallapuranam Suresh Kumar2 1Bethune Cookman University, Florida, USA, 2University of Arkansas‐Fayetteville, Arkansas, USA Anabaena sensory rhodopsin transducer, ASRT a tetrameric soluble protein indicated to function as downstream signaling partner to sensory rhodopsin. Both solution and crystal structures has reveled that this primarily beta stranded protein exhibit a helical face at carboxyl terminus. Besides receptor binding, ASRT is been shown to serve as a novel eukaryotic‐like interaction with DNA. We have demonstrated that carboxyl helical face of tetramer is involved in unusual tetrameric stability. Our bioinformatics analysis has revealed a phosphor transfer along with unique structural fold that may transform it as a unique carbohydrate binding module. However, the signaling state/mechanism of ASRT is obscure. Our initial data with efficient donor, acetyl phosphate, acP using fluorescence quenching of lone Trp‐9 suggests that ASRT is involved in phosphorylation. We noticed two notable consensus phosphor transfer residue motif in ASRT, SDKE [53–56] and TRLD [105–108]. Acetyl phosphate has been used as phosphoryl donor in vitro to numerous response regulators [CheY/PhoB/OmpR]. The extent of quenching in presence of Mg2+ by increasing concentration of acP yielded lower KM [∼21mM] compared to PhoB [∼8mM]. It is likely that proximity of phosphor accepting residue (s) to the lone Trp is not comparable to others. Interestingly the ASRT mutant E56Q and D108N indicate the loss of phosphor transfer. We hypothesize that putative position shown as red in figure is involved in ASRT phosphorylation. This motif is in close proximity to receptor binding deduced by CABS docking. Phosphor transfer data along with plausible impact on tetrameric stability will be discussed in this study. [Supported by NIH‐NIGMS SCORE SC3GM113803 award to VT]. POS413 LC‐MS/MS Analysis of Cytochrome c Peroxidase Interactors in Yeast Mitochondria Alan de Aguiar Lopes 1, Ann English1, Heng Jiang1 1PROTEO and the Centre for Biological Applications of Mass Spectrometry, Department of Chemistry and Biochemistry, Concordia University, Canada Heme is an essential protein prosthetic group for biological processes such as antioxidant defense and respiration. Heme synthesis finalizes with the insertion of ferrous iron into the protoporhyrin ring, which is catalyzed by ferrochelatase (FECH) on the matrix side of the inner mitochondrial membrane. However, no acceptors of heme from FECH have been identified. Cytochrome c peroxidase (Ccp1), which is a heme protein produced under fermentative conditions, might be an early acceptor of heme in yeast cells. Since Ccp1 is localized in the intermembrane space, we hypothesize that the ADP/ATP and putative heme transporter, Pet9, is an intermediate in heme trafficking between Ccp1 and FECH. As a starting point, we used high‐performance LC‐MS/MS to profile the proteins in the mitochondrial‐enriched (P10) fraction of 1‐day fermenting BY4741 yeast cells ± 20 mM N‐octylglucoside detergent. Ccp1, Pet9 and FECH were among the 776 proteins we detected so we pulled down interactors of GST‐apoCcp1 and GST (negative control) in the P10 fraction. Twelve mitochondrial proteins were identified including Pet9 and the matrix proteins, aconitase and succinate and malate dehydrogenases, but FECH was not detected. Further in vitro and in vivo screening is underway to probe FECH, Ccp1 and Pet9 interactors in yeast mitochondria using GST‐pulldown and proximity‐dependent BioID assays. POS426 The Role of Electrostatic Interactions in the Abp1 SH3 Domain Benjamin Lantz 1, Matthew Dominguez1 1Eastern New Mexico University, New Mexico, USA The SH3 domain from the yeast protein, Abp1p (AbpSH3) is essential for its role in actin cytoskeleton rearrangement. This highly acidic 60 residue domain (net −12 charge) binds to an extended proline‐rich peptide, involving electrostatic interactions. We study the electrostatic interactions in the domain and a domain‐peptide hybrid in a variety of salt concentrations using a high‐throughput stability assay with the denaturants urea (neutral) and guanidine (charged). The optimum conditions found were used in crystallization trials for select proteins, which led to determining the structure of a hyper‐stabilized AbpSH3 triple mutant in high salt conditions. We also found stability in different salt concentrations was affected by different denaturants. Domains were more stable in higher salt concentrations regardless of the denaturant, while the domain‐peptide hybrid was found to be most stable in high salt with guanidine, but no salt gave the highest stability with urea. We believe the high ionic strength of guanidine already disrupts the favorable peptide‐domain electrostatic interactions in the hybrid and allows salt to show a net positive effect due to stabilization of the domain. In the neutral denaturant urea, high salt decreases domain‐peptide hybrid stability due to the screening of the strong electrostatics still present between the peptide and domain. Our high‐throughput methods have determined optimal conditions for our domain and domain‐peptide hybrid for future structural and functional studies that will characterize novel binding peptides. POS438 An Improved Method to Purify and Activate Wild‐Type and Chimeric Botulinum Neurotoxins (BoNTs) Sulyman Barkho1, Min Dong1 1Boston Children's Hospital, Massachusetts, USA Botulinum Neurotoxins (BoNTs) are highly potent protein toxins produced by spore‐bearing Clostridium botulinum. In the last few decades, these deadly agents were found useful in treating numerous neuromuscular disorders and in aesthetic applications by blocking neurotransmitter release in the injected muscles. Now established therapeutic agents, BoNTs are widely produced in large scales by several manufacturers around the world. Available data suggest manufacturing procedures rely on decades‐old methodologies that utilize sporulating strains, and toxin isolation is achieved by many laborious and inefficient bulk purification steps. Here, we utilize recent structural and biochemical knowledge of specific interactions between BoNTs and their non‐toxic partners to formulate an improved method for the direct purification and activation of therapeutic BoNTs. POS463 Structures Of Urease Accessory Proteins And Their Insights Into Urease Maturation In Helicobacter Pylori Man Hon Yuen 1, Kam Bo Wong1, Yu Hang Fong1, Yap Shing Nim1 1The Chinese University of Hong Kong, School of Life Science, China Urease is a nickel‐¬containing metalloenzyme that catalyzes the hydrolysis of urea, which produces the acid ¬neutralizing ammonia that is essential for the survival of Helicobacter pylori (a peptic ulcer causing pathogen) in the human stomach. Maturation of urease involves carbamylation of an active lysine residue and insertion of two nickel ions at its active site. This process is facilitated by four urease accessory proteins, UreE, UreF, UreG and UreH. To understand how urease accessory proteins interact with each other to facilitate urease maturation, we have determined the crystal structures of UreF2H2 and UreG2F2H2 complexes. UreF2 contains a flexible C‐terminal tail that forms an extra helix and F‐tail loop stabilized by Arg‐250. These newly formed structures facilitates the recruitment of UreG to form UreG2F2H2 complex. From the structure of UreG2F2H2 complex, it illustrates the dimerization of UreG assemble its nickel binding site by bringing the two conserved CPH motif at the dimerization interface. Addition of nickel and GTP to the UreG2F2H2 complex releases a GTP/nickel‐¬bound UreG dimer that can activate urease in‐vitro in the presence of UreF2H2 complex. Recently, we have determined the crystal structure of the GTP/nickel‐¬bound UreG dimer, which shows the conformational difference of UreG at different nucleotide‐bound states. This reveals how GTP hydrolysis induces conformational changes that dissociate the UreG dimer and release its bound nickel. Using mutagenesis study along with activity assay and interaction study by static light scattering, it provides insights into the role of UreG in the delivery of nickel during urease maturation. POS483 Clustering of Ig‐like domains during CRTAM‐Necl2 Interaction Juan Carlos Barragan‐Galvez 1, Vianney Ortiz‐Navarrete1 1National Autonomous University of Mexico, Center for Research and Advanced Studies (CINVESTAV), Mexico CRTAM is a transmembrane protein belonging to the immunoglobulin superfamily that is expressed in activated T lymphocytes and constitutively in epithelial cells, composed of a variable (V) and constant (C) immunoglobulin‐like domain on the extracellular region, and interacts through the V‐domain with Nectin‐Like 2 (Necl2), ligand that is expressed constitutively on the dendritic cell surface (DC) and epithelial cells. However, the role of the constant Ig‐Like domains during the interaction of CRTAM‐Necl2 have not been studied. The extracellular region of CRTAM and Necl2 were cloned and expressed in bacterial system. The recombinant proteins were purified for affinity chromatography and subjected to gel filtration, and the kinetic parameters of the interactions were measured by Surface Plasmon Resonance. The results show that the C‐domain of CRTAM is present in oligomeric forms in solution, and that the affinity in CRTAM‐Necl2 interaction is KD = 3.72x10‐8 M. This suggests that the C‐domains in both molecules could be improving the affinity during the interaction, compared to the affinity of only the variable domains (KD = 1.25 x 10‐6 M) already reported. POS486 A Dynamic KaiA‐KaiC Interaction Maintains the Oscillation of the KaiABC Circadian Clock Sen Liu 1, Qiang Chen1 1China Three Gorges University, China The design of biological oscillators is an intriguing topic in Synthetic Biology. Although there have been many artificially designed biological oscillators, none of them has a period of around 24 hours like a circadian clock oscillator, especially solely based on proteins. The core circadian oscillator of cyanobacteria consists of three proteins, KaiA, KaiB, and KaiC. This circadian oscillator could be functionally reconstituted in vitro with these three proteins, and therefore has been a very important model in circadian rhythm research. KaiA can bind to KaiC and then stimulate its phosphorylation, whereas KaiB antagonizes KaiA's function leading to the de‐phosphorylation of KaiC. Using this protein‐based circadian clock oscillator as a model, we aim to find its controlling point and re‐design this oscillator. To this end, we combined the tools in bioinformatics, evolutionary biology, protein design, molecular biology, and mathematical modeling to study the interaction between KaiA and KaiC. We found that there exist complicated but critical structural movements during the binding of KaiA and KaiC, and these movements are determinant to the oscillation of the KaiABC system. We further revealed that the KaiA has an asymmetric structural flexibility, which regulates its auto‐inhibition and the interaction with KaiC. Based on our findings, it could be possible to redesign this oscillator with more interesting functionalities, which would provide useful insights to the design of protein‐based oscillators. POS493 Two mAbs Binding to Different Sites on Clostridium difficile Toxin B Induce Similar Conformational Changes Jianxin Zhang 1, Fuqin Ma1, Sophia Mundle1, Stephen Anderson 1Sanofi Pasteur, Massachusetts, USA C. difficile infection (CDI) is identified as the leading cause of hospital‐acquired diarrhea, and disease is primarily mediated by two exotoxins, A and B, that damage the lining of the intestine. Both toxins are multi‐functional proteins with similar domain architecture. Monoclonal antibodies neutralizing these two toxins were isolated and identified, and a variety of methods were employed to analyze the antibodies‐toxin interactions. Using the FortéBio Octet Bio‐Layer Interferometry (BLI) system, we were able to map to which domain each mAb binds. Using SEC‐HPLC, we found some antibodies have one binding site while others have multiple sites. The interactions of two mAbs with the N‐terminal domain of toxin B were further analyzed with Hydrogen‐Deuterium eXchange Mass Spectrometry (HDX‐MS). Although these two antibodies bind to different sites of the N‐terminal domain, binding resulted in a similar conformational change in both instances. One neutralizing antibody was found to bind to a region distant from the substrate binding subdomain but the binding led to a flexibility change in the substrate binding subdomain, suggesting a possible mechanism for neutralization involving an induced or allosteric conformational change. POS507 Antibody‐Antigen Interactions Studies Using Nuclear Magnetic Resonance Spectroscopy Gaohua Liu 1, Alan Gibbs2, Ruth Steele2, Gaetano Montelione3, Rong Xiao1 1Nexomics Biosciences, Inc., USA 2Janssen Research and Development LLC, USA, 3Center for Advanced Biotechnology and Medicine, Rutgers, the State University of New Jersey, USA The specificity of the antibody molecule to its cognate antigen has been exploited for the development of a variety of immunoassays, vaccinations, and therapeutics. Almost all of the high‐resolution structures currently available for antibodies have been determined by X‐ray crystallography. Although very informative, these structures are limited in their insight into functional conformational dynamics. 19F‐NMR has been applied to study protein‐protein interactions dynamics, as well as fragment‐based drug discovery. Nexomics Bioscience is leveraging advanced NMR technologies, including 19F‐NMR, to study antibody‐antigen interactions to enable drug discovery. Three humanized Fab's were selected, and successfully expressed in E. coli with uniform 15N‐enrichment and with 19F‐Trp labeling. 2D 15N TROSY‐HSQC and 1D 19F NMR spectra demonstrate that these isotope‐specific labeled Fab samples are very well suited for antibody‐antigen interaction studies by NMR. Significant chemical shift perturbations due to antigen binding were observed in both 2D 15N TROSY‐HSQC and 1D 19F NMR spectra for three different Fab's. This study demonstrates that our strategy for specific isotope labeling of Fab's is suitable for preparing samples of Fab's for NMR studies, and for characterizing the interactions between Fab‘s and antigens. This platform can become an extremely useful tool for the pharmaceutical and biotech communities for drug discovery, especially in the field of antibody engineering. POS521 Identification of Biologically Relevant Binding Partners of SH3 Domains Using Compensatory Mutation Approach Yun Zhu 1, Alan Davidson1 1Department of Molecular Genetics, University of Toronto, USA SH3 domains are among the most common peptide recognition modules in eukaryotes. It has been difficult to understand their interaction networks in vivo. In this study, we developed the compensatory mutation approach to identify biologically relevant binding targets of SH3 domains in S. cerevisiae. Compensatory mutations are a pair of mutations of SH3 domain and its binding motif that do not interact with its wildtype binding partner(s) but can recognize each other. Through engineering these mutations into the wildtype SH3 domain and its binding motif, we were able to isolate each interaction of interest and determine its biological importance in vivo. Using this approach, we examined the interaction network of Nbp2p in S. cerevisiae. Nbp2p is an adaptor protein containing an SH3 domain. This SH3 domain has been shown to interact with the proline‐rich motif in Bck1p, Ste20p, Cla4p, Skm1p and Pbs2p in vitro. We confirmed that Bck1p is a biologically relevant binding target of Nbp2p SH3 domain under high temperature condition. We also identified Ste20p as a biologically relevant target in mating pathway regulation. The Nbp2p‐Ste20p interaction is important for the adaptation of yeast cells to mating pheromone. Interestingly, Nbp2p is also important for the activation of mating pathway, but this function is not related to the Nbp2p‐Ste20p interaction. In summary, we established the compensatory mutation approach and demonstrated its effectiveness to identify biologically relevant binding targets. This concept has the potential to be applied to the understanding of other protein‐protein interaction networks. POS527 Dissecting the Mechanism of Bacterial Amidase Activation by EnvC Paola Di Lello 1, Yiming Xu1, Kevin Clark1, Rina Fong1, Bob Lazarus1, Patrick Lupardus1, Man Wah Tan1 1Genentech, USA In bacteria, remodeling of the peptidoglycan network is crucial for cell growth and division. AmiA and AmiB are two of the three amidases that cleave the peptidoglycan network during cell division in E. coli. The activity of these two enzymes is tightly regulated by a complex network of proteins including EnvC, FtsX and FtsE. Both AmiA and AmiB adopt an inactive conformation where the catalytic site is occluded by an auto‐inhibitory helix. However, upon binding to the LytM domain of EnvC, both AmiA and AmiB have been postulated to undergo a conformational change that releases the auto‐inhibitory helix from the catalytic site, thus leading to enzyme activation. Here we sought to gain structural information on how the LytM domain of EnvC modulates AmiA and AmiB activity. This type of information is essential for the design of small molecules that would activate these two amidases, interfere with bacterial proliferation and could potentially be developed into potent antibiotics. Using Nuclear Magnetic Resonance (NMR) we characterized the interaction between AmiB and its activator, the LytM domain of EnvC. Mapping studies by NMR revealed the binding interface of the AmiB/LytM complex and also showed that, contrary to the current hypothesis, the interaction between AmiB and the LytM domain of EnvC does not appear to be mediated by the auto‐inhibitory helix of AmiB, but by a different region within the AmiB surface. POS537 Mechanism of RNA Recognition by the M2‐1 Transcription Antiterminator from Respiratory Syncytial Virus Gonzalo de Prat Gay 1, Ivana G. Molina1, Sebastian A. Esperante1, Lucia B. Chemes1 1Fundación Instituto Leloir, Argentina RNA transcription of mononegavirales decreases gradually from the 3’ leader single promoter towards the 5’ end of the genome, due to a decay in transcription processivity. In the syncytial respiratory (RSV) and metapneumoviruses (MPNV) the M2‐1 protein, unique to these viruses, is responsible for transcription anti‐termination. Despite being a homotetramer, RSV M2‐1 binds two molecules of RNA of 13 nt or longer per tetramer, while four molecules of shorter RNAs bind per tetramer. RNA oligomers of 20nt show temperature sensitive secondary structure which is unfolded by stoichiometric interaction with M2‐1. Fine quantitative spectroscopic analysis of the binding data show positive cooperativity, indicative of conformational asymmetry in the tetramer. Binding to shorter RNAs respond to a model of four independent identical binding sites of lower affinity. RNA binds to M2‐1 through a fast bimolecular association followed by two slow rearrangements corresponding to an induced‐fit mechanism on the protein side. The data provide a sequential description of time events of the cooperativity, where a first binding event of half of the RNA molecule to one of the sites increases the affinity of the second binding event, product of increased effective concentration by the entropic link. This mechanism allows high affinity binding with an otherwise loose sequence specificity, and suggests a structural recognition signature in the RNA for modulating gene transcription. This work provides a basis for the understanding of the mechanism of transcription anti‐termination, an essential event for ensuring transcription polarity in pneumoviruses and constitutes a one of a kind example among RNA viruses. POS539 Raf Isoform Dependent Binding Of Oncogenic Kras Mutants Ryan Thurman 1, Natalie Hewitt1, Tikvah Hayes2, Samuel George3, Channing Der3, Sharon Campbell1 1University of North Carolina, Department of Biochemistry and Biophysics, USA, 2UNC Department of Genetics, USA, 3UNC Lineberger Comprehensive Cancer Center, Department of Pharmacology, USA RAS genes encode small GTPases that function as molecular switches to regulate cellular growth. Approximately one‐third of all human cancers contain activating mutations in RAS genes, with codon hotspots at positions 12, 13 and 61. These point mutations render RAS proteins insensitive to down regulation, resulting in chronic Ras activation and constitutive, oncogenic signaling. As such, they have historically been considered oncogenic equivalents. However, recent observations suggest that codon‐specific RAS mutations show differences in their relative ability to function as molecular switches and engage regulators and effectors. Signaling and tumorigenic properties also differ. Differences have also been observed in the response and resistance to specific anti‐cancer therapies. Thus understanding these differences will likely have important clinical and biological implications. To better understand cancer‐specific RAS mutation differences, we conducted structural and biochemical characterization studies on oncogenic KRAS mutations. We find that distinct oncogenic KRAS mutants differentially alter binding to RAF kinases. We employed NMR to elucidate differences in binding determinants between RAS and A‐, B‐ and C‐RAF RAS binding domains (RBD), and identified a point mutation in the c‐Raf RBD that reverts isoform dependent binding. BRET and signaling assays are in process to interrogate how isoform dependent binding alters signaling properties in cells. 17. PROTEINS IN CELLS POS027 Mammalian Cell Programming Targets Tnfa Sources Anam Qudrat 1, Anam Qudrat1 1University of Toronto, Canada In certain diseases (e.g. atherosclerosis and cancer), there is a local formation of cell masses (e.g. plaques and tumours), a low pH extracellular microenvironment and the secretion of various pro‐inflammatory cytokines such as TNFa. The ability to engineer a cell to seek these TNFa sources allows for targeted and local delivery of therapeutic intervention to disease sites. To impart this ability into cells that do not naturally target TNFa sources, here we introduced a system of proteins: an engineered TNFa chimeric receptor (named TNFR1chi), a previously engineered Ca2+‐activated RhoA (named CaRQ), VSVG and thymidine kinase. Upon binding of TNFa, TNFR1chi generates a Ca2+ signal. This Ca2+ signal in turn activates CaRQmediated non‐apoptotic blebs that allow migration towards the TNFa source. Next, with the addition of VSVG, upon low pH induction, these engineered cells fuse with the TNFa source cells. Finally, postganciclovir treatment, the cells undergo death via a suicide mechanism assisted by thymidine kinase. Hence, we assembled a system of proteins that forms the basis of engineering a cell to target inflammatory disease sites characterized by the secretion of TNFa and a microenvironment with low pH. POS066 Simultaneous Visualization of a Gene and its Nascent Transcripts in Live Cells João Pessoa 1, João Pessoa1, Célia Carvalho1, Maria Carmo‐Fonseca1 Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Portugal Live‐cell visualization of fluorescently labeled DNA and RNA is a powerful approach to study genome organization and gene expression. Here, we targeted the human ß‐globin gene using an inactive mutant of the DNA endonuclease CRISPR‐associated protein 9 (dCas9) fused to the green fluorescent protein (GFP). Nascent ß‐globin transcripts were tagged with MS2 stem loops and labeled with the MS2 phage coat protein (MCP) fused to the fluorescent protein mCherry. We generated a human U2OS cell line stably expressing dCas9‐GFP and a tandem array of ∼100 human ß‐globin gene copies each modified with 24 MS2 stem loops. MCP‐mCherry and a gRNA were transiently expressed. By fluorescence microscopy, we visualized the ß‐globin DNA integration locus surrounded by nascent transcripts. 3D reconstructions of z‐stack images provided insights into the DNA‐mRNA interaction and their structural variability. DNA was usually compacted into a single moderately elongated globular shape, with one or two protruding bundles of mRNA. The DNA‐mRNA overlap was highly variable. Usual particle dimensions were 0.6‐1.2 µm for DNA and 0.3‐0.6 µm for mRNA. Our approach opens new perspectives for addressing the organization of nascent transcription in the nucleus of living human cells. This work was supported by Fundação para a Ciência e a Tecnologia (grant PTDC/BEX‐BCM/5899/2014 and fellowship SFRH/BPD/102323/2014 to J.P.). POS075 Screening of compounds rescuing non‐sense p53 gene using p53‐GFP fusion protein as indicator Jingjing Zhou 1, Jingjing Zhou1, Sicong Li2, Yuhui Sun2, Zhengding Su1 1Institute of Biomedical and Pharmaceutical Sciences, Hubei University of Technology, China, 2School of Pharmaceutical Sciences, Wuhan University, China Premature termination codons (PTCs) cause many diseases including the cancers which contain premature terminated p53 gene. Aminoglycoside antibiotics have been proved to enable to induce the read‐through of PTCs and restore the production of functional full‐length p53 protein. In this work, we demonstrated a quick system for evaluating the biological activity of compounds for rescuing the read‐through of p53‐PTCs. The system was built up on an E. coli protein expression strain for the expression of the full‐length of p53‐GFP protein. Our data revealed that the full‐length p53 protein could be reproduced upon the treatment of the strain with aminoglycoside compounds, by monitoring the fluorescence of GFP protein. The expression of the full‐length p53 protein was also confirmed by Western Blotting assays. We believe that the strategy reported in our system should be extendable to screen new compounds beyond aminoglycoside antibiotics as anti‐cancer agents. POS184 Characterization of Sigma Anti‐sigma Complexes Central to the Iron Homeostasis in Pseudomonas aeruginosa G. Patricia Casas G 1, Iain A Lamont2, Matthew A Perugini1, Megan J Maher1 1Department of Biochemistry and Genetics, Institute of Molecular Bioscience, La Trobe University, Australia, 2Department of Biochemistry, University of Otago, New Zealand Cystic fibrosis (CF) is the most common genetic lethal disease among the Caucasian population. The environment in the lung of CF patients is prone to bacterial infection, being Pseudomonas aeruginosa, the most common isolate. Iron is an essential metal required for in vivo growth and pathogenesis. Thus, in iron depleted environments this bacterium synthesises siderophores, such as pyoverdine, which enable iron acquisition. The expression of genes for iron uptake in P. aeruginosa is controlled by two sigma factor: sPvdS and sFpvI, and their anti‐sigma factor, FpvR. Of particular interest is the molecular mechanism by which a single anti‐sigma factor, like FpvR, can complex two different sigma factors. A protocol was developed for the co‐expression and purification of sPvdS and sFpvI in complex with a truncated form of FpvR (FpvR1–89), confirming the interaction between those proteins. Analytical Ultracentrifugation (AUC), showed that the sPvdS/FpvR1–89 and sFpvI/FpvR1–89 are expressed as 1:1 stoichiometric complexes. Furthermore, the characterisation of the biophysical properties of the complex by AUC indicated similar binding affinities between FpvR1–89 and the sigma factor sPvdS and sFpvI. These findings revealed a model by which iron acquisition is controlled by P. aeruginosa. POS212 Effects of Macromolecular Crowding are Modulated by Shape of Protein Complex Alex Guseman 1, Gerardo Perez Goncalves1, Gary Pielak1 1Department of Chemistry, UNC Chapel HIll, USA Proteins in the crowded cellular interior are influenced by interactions not found in simple buffer solutions, hardcore repulsions and transient chemical interactions between the crowding molecules and the protein. Scaled‐particle theory predicts that hardcore repulsions have little effect on the stability of dumbbell‐shaped dimers and a stabilizing effect on ellipsoidal/spherical dimers. Here, we test this idea using the A34F variant of GB1 which forms a side‐by‐side dimer and the L5V;F30V;Y33F;A34F variant which forms a domain‐swapped dimer. These dimers are useful for testing ideas from scaled‐particle theory, because the side‐by‐side dimer is dumbbell shaped and domain‐swapped dimer is ellipsoidal. Importantly, these dimers have similar surfaces, meaning they have similar chemical interactions with crowders. We used 19F NMR to quantify the free energy of dissociation (ΔG°`DΔM) at 25°C for both dimers. As predicted by scaled‐particle theory, hardcore repulsions had a small effect on ΔG°`DΔM (=ΔG°`DΔM,cosolute‐ΔG°`DΔM,buffer) in 300 g/L Ficoll‐70 and 200 g/L 8 kDa PEG (0.150 ± 0.04 kcal/mol and −0.22 ± 0.03 kcal/mol, respectively). As predicted ΔG°`DΔM for the domain‐swapped dimer is larger (0.70 ± 0.03 kcal/mol and 0.550 ± 0.01 kcal/mol, respectively). Supporting the idea that dimer shape modulates hardcore repulsions. To further support this conclusion we tested the effects of the protein cosolutes bovine serum albumin (68 kDa, 100 g/L) and lysozyme (14 kDa, 50 g/L). These protein cosolutes are predicted by scaled particle theory to have minimal contributions from hardcore repulsions. Furthermore, ΔG°`DΔM values are within error of each other for both dimer systems, supporting our hypothesis that protein crowders interact by chemical interactions. We are now investigating these dimers in cells using 19F NMR. POS216 Cosolutes and the Stability of a Domain‐Swapped Dimer Gerardo Perez Goncalves 1, Alex Guseman1, Gary Pielak1 1Department of Chemistry, UNC Chapel Hill, USA The cellular interior is a complex environment where the concentration of macromolecules exceeds 300 g/L. Under such crowded conditions, proteins experience hard‐core repulsions and chemical interactions with cytoplasmic components that are absent in buffer alone. Hard‐core repulsions stabilize globular proteins. Chemical interactions can be stabilizing if repulsive, but destabilizing if attractive. Studies of these interactions have broadened our understanding of protein biophysics in cells, but for simplicity sake, have focused on folding. However, proteins rarely work alone, and protein‐protein interactions give rise to complex architectures. The L5V;F30V;Y33F;A34F variant of the B1 domain of protein G (GB1) forms a domain‐swapped homodimer. We labeled its sole tryptophan with fluorine and used 19F nuclear magnetic resonance spectroscopy to quantify the effect of small and large cosolutes on the equilibrium thermodynamics of dimerization at 298 K. The synthetic polymers Ficoll (70 kDa) and polyethylene glycol (8 kDa) stabilize the dimer by 0.70 ± 0.03 kcal/mol and 0.50 ± 0.01 kcal/mol, respectively. Additionally, we observe a macromolecular effect in that the monomer of Ficoll, sucrose, stabilizes the dimer by only 0.40 ± 0.01 kcal/mol, and ethylene glycol destabilizes the dimer by −0.30 ± 0.01 kcal/mol. We also investigated biologically‐relevant macromolecular cosolutes and find that they act primarily via chemical interactions. Preliminary data show that bovine serum albumin, which has the net charge of the same sign as GB1, stabilizes the dimer by ∼0.5 kcal/mol, while lysozyme, which has a charge of opposite sign, destabilizes the dimer by ∼0.3 kcal/mol. Our data provide new insight into the biophysics of protein‐protein interactions under physiologically relevant conditions. POS272 Expansion Pathology Enables Super‐Resolution Optical Investigation of Proteins for Diagnostic Pathology and Research Octavian Bucur 1, Yongxin Zhao2, Humayun Irshad1, Andreea Stancu1, Astrid Weins3, Stuart Schnitt2, Martin Pollak4, Ananth Karumanchi4, Edward Boyden2 1Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Massachusetts, USA, 2Media Lab, Massachusetts Institute of Technology, USA, 3Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Massachusetts, USA, 4Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Massachusetts, USA BACKGROUND: While in the past decades the research community focused on developing super resolution microscopes for tissue imaging, which are expensive and need long recording times, a new approach, Expansion Microscopy (ExM), was recently discovered, enabling physical magnification and high resolution imaging of fixed cell lines and mouse brain tissue with conventional, optical microscopes (Chen, Tillberg, Boyden, 2015, Science). In the present study, we aimed to improve the ExM method for imaging of proteins in human clinical tissue samples, for diagnostic pathology and research. METHODS: We developed a clinically optimized variant of ExM called Expansion Pathology (ExPath), by optimizing the ExM chemistry, labeling, and imaging methodologies. ExPath enables morphological and protein imaging and analysis of tissue microarrays, by providing ∼70nm resolution imaging of proteins in any type of human tissues, using optical microscopes (currently limited to a 250nm resolution). RESULTS and CONCLUSIONS: This ExPath protocol enabled expansion of human normal and cancer tissues ∼4.5x in linear dimension and ∼100x in volume, with a post‐expansion measurement error of <5%. We demonstrate that the nanoscale changes of kidney podocyte foot processes, now diagnosed with electron microscopy (EM), can be accurately diagnosed with ExPath, by physical tissue expansion followed by imaging of the ACTN4 protein expression pattern with conventional light microscopy. This process is fast, inexpensive, and reliable, facilitating morphological and multiplexed protein investigation of large tissue regions. Our findings indicate that ExPath can be applied to many clinical samples, enabling super‐resolution optical investigation of protein expression/localization and morphology in both whole tissue slide and tissue microarray formats, with fluorescent microscopy. POS315 Production of fungal biomass protein from Trichoderma harzianum Sibtain Ahmed 1, Muhammad Rajoka2 1University of California San Diego, USA, 2Government College University, Pakistan The aim of this study was to access the potential of Trichoderma harzianum to produce fungal biomass protein (FBP) from rice polishings. Maximum biomass yield was obtained at 5% (w/v) rice polishings after 72 h of incubation at 28°C at pH 4. Carbon and nitrogen ratio of 20 : 1 gave significantly higher production of FBP. The FBP in the 75 L fermenter contained 49.50% crude protein, 32.00% true protein, 19.45% crude fiber, 9.62% ash, 11.5% cellulose content, and 0.325% RNA content. The profile of amino acids of final FBP exhibited that all essential amino acids were present in great quantities. The FBP produced by this fungus has been shown to be of good nutritional value for supplementation to poultry. The results presented in this study have practical implications in that the fungus T. harzianum could be used successfully to produce fungal biomass protein using rice polishings. POS341 Peroxiredoxins are Key Players of the Enzymatic Antioxidant System in Human Spermatozoa Maria Fernandez 1, Cristian O´Flaherty2, Adel Moawad3, Cristian O´Flaherty1 1McGill University, Canada, 2Surgery Department, McGill University and RI‐MUHCm Canada, 3Research Institute ‐McGill University Health Center, Canada Introduction and aim: Our lab has been pioneer to show that a family of antioxidant called peroxiredoxins (PRDXs) has a major role in the protection against oxidative stress in spermatozoa. However, how spermatozoa maintain PRDXs active remains unknown. Here, we aimed to determine the impact of different reactive oxygen species (ROS) levels produced by inhibiting specific players of the PRDX system on sperm viability and DNA oxidation. Experimental Approach: Highly motile spermatozoa from healthy donors were incubated with different inhibitors of the PRDX system and viability, ROS levels or DNA oxidation were asses by flow cytometry. Results: We observed a significant decrease in viable cells in a dose dependence manner when incubated with inhibitors of the 2‐Cys PRDXs, calcium independent phospholipase A2 (PRDX6‐PLA2) activity or PRDX re‐activation system compared to controls (p<0.05). Inhibition of PRDX6‐PLA2 had the strongest detrimental effect on viability promoting a significant increase of –OCl, HO•, ONOO‐, O2•‐ and DNA oxidation labeling compared to controls (p<0.05). The inhibition of NADPH donors for the 2Cys‐PRDXs re‐activation system has no effect in sperm viability or the levels of O2•‐. However, we found a significant increase in the levels of others ROS (ROO• and ONOO‐). Conclusions: PRDXs, especially PRDX6‐PLA2 activity, are essential in maintaining sperm viability and DNA integrity. POS473 Contribution of the chromosomal ccdAB operon to bacterial drug tolerance Kritika Gupta 1, Raghavan Varadarajan1, Arti Tripathi2 1Indian Institute of Science, India, 2Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA A large number of free‐living and pathogenic bacteria are known to harbor multiple toxin‐antitoxin systems, on plasmids as well as on chromosomes. The F‐plasmid CcdAB system is extensively studied and known to be involved in plasmid maintenance. In contrast, little is known about the function of its chromosomal counterpart, found in several pathogenic E.coli strains. We show that both the native ccd operon of the E.coli O157 strain as well as the ccd operon from the F‐ plasmid, when inserted on the E.coli chromosome lead to protection from cell death under multiple antibiotic stress conditions, with the O157 operon showing higher protection. While the toxin encoded from the plasmidic ccd operon is a potent gyrase inhibitor and leads to bacterial cell death, the chromosomally encoded toxin leads to growth inhibition. In vitro gyrase binding of purified CcdBF and CcdBO157 using SPR shows that CcdBO157 have five fold lower affinity for its target GyrA than CcdBF. Analysis of the CcdBF‐Gyrase bound structure indicates that substitutions N95D and W99D observed in CcdBO157 are likely to disrupt Gyrase binding, confirmed by our study involving saturation mutagenesis on the CcdBF protein. This study demonstrates an important role for chromosomal ccd systems in bacterial persistence and has implications for generation of potential therapeutics that target the TA systems in these pathogens. POS491 Unraveling a new role for bacterioferritin (BfrB) in Pseudomonas aeruginosa: a step toward rational targeting of bacterial iron homeostasis. Huili Yao 1, Mario Rivera1, Achala Hewage1 1The University of Kansas, USA There is an urgent need to discover novel antibiotics due to severe multidrug resistance developed by many pathogens. Among them, Pseudomonas aeruginosa (PA) is the major pathogen responsible for the lethal infections among CF (cystic fibrosis) patients and immune‐compromised patients. Bacterial iron homeostasis plays a crucial role on cell growth and cell fitness. Among the proteins involved in regulating bacterial iron concentrations, the iron storage protein‐bacterioferritin (BfrB) is probably a unique target for new antibiotic development, due to the following reasons: 1) It only exists in bacteria. 2) To mobilize iron stored in BfrB into the bacterial cytosol a physiological partner (Bfd) is needed. 3) Our previous studies identified the key residues stabilizing the BfrB‐Bfd interaction. Our new results from inhibiting the BfrB‐Bfd interaction in PA cells demonstrate that targeting the BfrB‐Bfd interface exposes bacterial vulnerabilities. Perturbation of the BfrB‐Bfd interaction causes irreversible iron accumulation in BfrB and causes low free iron levels in the cytosol. Irreversible iron accumulation in BfrB also causes secretion of high levels of iron chelators, whose function is to help bacterial cells acquire iron from the environment, which is a signal of acute bacterial iron starvation. Finally, the study also showed that iron stored in BfrB is the main source of iron for incorporation into iron‐utilizing proteins. Hence, when iron is irreversibly “trapped” in BfrB due to inhibition of the BfrB‐Bfd interaction, bacterial cell growth is inhibited. These findings take us a step closer to our long‐term goal of developing novel antibacterial strategies that target bacterial iron homeostasis. POS519 Impact of perturbing the BfrB:Bfd interaction on Pseudomonas aeruginosa biofilms Anabel Soldano 1, Mario Rivera1 1The University of Kansas, USA Pseudomonas aeruginosa, a Gram‐negative opportunistic pathogen, forms diverse bacterial communities enclosed within an extracellular matrix (biofilm) that aid its survival in a variety of environments. While growing in static cultures form biofilms at the air‐surface interface, also known as pellicles, which appear to impart a growth advantage. Several studies have suggested that iron availability is important to support the various stages of biofilm development. In previous work, we demonstrated that the main iron storage protein in P. aeruginosa is a bacterioferritin (BfrB) and that iron mobilization from BfrB, which requires specific interactions with a ferredoxin (Bfd), establishes a dynamic equilibrium that buffers the concentration of free iron in the cytosol. In the present study we investigated the implications of perturbing the BfrB:Bfd interaction on the formation and integrity of P. aeruginosa biofilms. We compared the establishment and stability of pellicles formed by the wild type strain with mutant strains where the BfrB:Bfd interaction was blocked by in frame deletion of the bfrB or bfd genes and in a variant with the bfrB allele encoding BfrB (L68A/E81A). The effects of the mutations were evaluated by the time‐dependent microscopic inspection of the pellicles, and by quantitative determination of (i) total biofilm mass, (ii) concentration of iron, pyoverdine and pyocyanin in the spent culture media. The results reveal that perturbing iron homeostasis by disrupting the BfrB:Bfd interaction causes early cell detachment and concomitant biofilm dissolution. Consequently, our findings suggest that inhibiting the BfrB:Bfd protein‐protein interaction adversely affects the integrity of P. aeruginosa biofilms. 18. PROTEOMICS POS015 Application of gamma‐linked ATP‐Sepharose capture technology to the study of NLRP inflammasomes. Christina Sandall 1, Kuo‐Chieh Liao2, Annegret Ulke‐Lemee1, David Carlson3, Timothy Haystead3, Daniel Muruve1, Justin MacDonald1 1Department of Biochemistry & Molecular Biology, University of Calgary, Cumming School of Medicine, Canada, 2Departments of Biochemistry & Molecular Biology and Medicine, University of Calgary, Cumming School of Medicine, Canada, 3Department of Pharmacology & Chemical Biology, Duke University School of Medicine, USA The nucleotide‐binding domain and leucine‐rich repeat, pyrin domain‐containing (NLRP) proteins are innate immune sensors that regulate inflammatory signaling in response to a wide range of microbial and endogenous danger signals. The prototypical member, NLRP3, can initiate the assembly of a multiprotein inflammasome complex that results in the autoproteolytic cleavage and activation of pro‐caspase‐1, a cysteine protease that regulates multiple host defense pathways, including inflammatory cytokines. The 14 NLRP family members are characterized by a conserved ATP‐binding domain that controls inflammasome assembly. NLRP inflammasomes are under‐represented in biomarker and mechanistic studies of disease due to factors that preclude their facile examination: low expression, lack of specific antibodies, and peculiar biophysical characteristics. ‐linked ATP Sepharose with a 1,10‐diaminodecane linker provided effective for NLRP capture, with ∼90–95% efficiency observed for NLRP1–4, 6–12, and 14. NLRP3 binding was particularly sensitive to the ATP linkage, and N‐linked ATP Sepharose binding efficiency was low, at ∼20%. The gamma‐linked ATP Sepharose was then applied to targeted proteomic studies of NLRP3. The fluorescence‐linked enzyme chemoproteomic strategy (FLECS) was used to screen a 4,000‐member small molecule library. Several molecules could competitively elute NLRP3 from ATP‐Sepharose. A lead compound, a benzo[d]imidazol‐2‐one, inhibited inflammasome‐triggered pro‐caspase‐1 activation and interleukin(IL)‐1ß in ATP‐stimulated THP‐1 monocytes. Next, ATP‐Sepharose enrichment was successfully applied to the antibody‐independent identification of NLRP3 with selected‐reaction monitoring mass spectrometry (SRM‐MS) methods. In summary, ATP capture technologies offer opportunities for identifying disease‐associated NLRP protein biomarker profiles and drug discovery initiatives that specifically target the ATP‐binding properties of the NLRPs. POS041 Identification of APAP Covalent Binding Protein Targets In Rat and Mouse Liver by 2D‐LC‐HRMS/MS Ghazaleh Moghaddam 1 1Université du Québec à Montréal, Chemistry Department, Montréal (Qc), Canada Acetaminophen (APAP) is one of the most commonly over the counter medicines and is also the main cause of acute liver failure in North America. This hepatotoxicity has been related to covalent binding of APAP's reactive metabolite, N‐acetyl‐p‐benzoquinoneimine (NAPQI), to proteins. The aim of this study was to identify in vivo protein targets of APAP in both rat and mouse models and compare the results from both species. Using a bottom‐up proteomic approach, sample preparation involved protein extraction and digestion from rat and mouse liver samples. Liver extracts were digested with trypsin and pepsin then subjected to strong cation exchange (SCX) prior to reversed‐phase UHPLMS/MS. Data processing involved using ProteinPilot software to find potential modified peptides, followed by verification of peak integration and compare APAP‐treated samples to controls to remove any potential false positive. Currently, several proteins have been identified as modified by NAPQI including ubiquitin‐conjugating enzyme, 5‐hydroxyisourate hydrolase, carboxylesterase 1C, SH3 domain‐containing RING finger protein 3 in mouse and two modified protein found in both mouse and rat including carbonic anhydrase 3, triosephosphate isomerise. These proteins are known to be involved in several important biological pathways involved in cell survival during oxidative stress and could potentially be linked to hepatotoxicity. APAP modified proteins will be compared between rat and mouse to strengthen our understanding of acetaminophen toxicity and species differences. Future verification of modified peptides as potential biomarkers for hepatotoxicity using peptide standards, for MS/MS and retention time matching will follow. POS064 A Study to Establish the Impact of Glycoxidation on Structural and Immunological Characteristics of IgG Isolated from Rheumatoid Arthritis Patients Sidra Islam 1 1Department of Biochemistry, J.N Medical College, A.M.U., Aligarh Immunoglobulin G (IgG), a 150 kDa molecule and the most abundant serum protein has been described as sensitive to glycation, nitration, oxidation and other modifications. Amongst these post translational modifications, glycation and oxidation being most common, deserves special attention. Increasing evidences suggest the role of glycoxidation in the onset and progression of Rheumatoid Arthritis (RA). This study was designed to elaborate the cumulative effect of glycation (using Methylglyoxal) and oxidation (using Hydroxyl Radical) on IgG with reference to RA. We found appreciable binding of RA auto‐antibodies towards epitopes in hydroxyl radical modified methylglyoxal glycated IgG (OH•‐MG‐IgG). Furthermore, spectroscopic characterization of IgG isolated from RA patients (RA‐IgG) revealed structural changes in comparison to IgG from healthy human subjects (NH‐IgG); with hyperchromicity in UV absorbance spectroscopy, quenching in fluorescence spectroscopy, decreased ß sheet content in far‐UV CD spectroscopic analysis and shifting of amide I and II bands in FTIR spectroscopy. OH•‐MG induced damage in RA‐IgG was evaluated by anti‐OH•‐MG antibodies (generated in female rabbits) using competitive binding immunoassay. Compared to NH‐IgG, RA‐IgG was observed to be more specific towards the immunogen (OH•‐MG‐IgG). Our results confirm that IgG in RA patients is prone to glycoxidation induced structural damage leading to the generation of neo‐epitopes that renders the protein immunogenic. POS078 Comparative Proteomics on Mytilidae Species Revealing Potential Byssus‐Related Proteins using 2D‐LC‐MS/MS Maxime Sansoucy 1, Réjan Tremblay2, Isabelle Marcotte1 1UQAM, Canada, 2UQAR, Canada Byssus is an intriguing collagen‐based polymer secreted by mussels used to secure these animals under harsh marine conditions. Mature filaments have unique mechanical and adhesive properties and therefore have potential applications for bio‐inspired materials. Considering that the mechanism of byssogenesis is yet to be fully understood, a high‐resolution tandem mass spectrometry (HR‐MS/MS) study has been performed to identify potential byssus related proteins from the foot organ of three Mytilidae mussels. Mussel foot and mantle tissues were homogenized with mechanical and sonication probes prior to protein precipitation. Extracts were digested with complementary enzymes (trypsin and pepsin, separately). Fractionation was performed on the protein and peptide level using off‐line chromatography. LC‐MS/MS analysis was then performed on a hybrid quadrupole‐time‐of‐flight platform. Proteins exclusively found in the mussel foot were mined for biological and physicochemical information. Solubilisation issues adversely affected protein‐level fractionation, therefore did not give satisfactory results. Differential analysis between foot and mantle (control) datasets has yielded a list of proteins exclusively found in the mussel's foot. The total number of unique foot proteins from strong cation exchange (SCX) and reverse‐phase (RP) peptide fractionation were compared to no pre‐fractionation. SCX and RP fractionation gave similar results with 124 and 130 proteins, respectively, whereas less than the half was identified for the non‐fractionated sample. Protein coverage was also much better with the fractionation strategies. Further biological investigation on thirty candidates revealed crucial proteins for securing adhesion, toughness promotion, and transcription promoter. An efficient multi‐dimensional LC‐MS/MS approach was developed for the analysis of mussel tissues. Strong cation exchange and reverse phase fractionation have demonstrated similar protein identification performance. POS133 Intracellular/Surface Moonlighting Proteins Constance Jeffery 1, Wangfei Wang1 1University of Illinois at Chicago, USA Cell surface proteins of bacterial pathogens play key roles in invasion and virulence. Colonization requires adhesion of the bacterium to host tissues, so some surface proteins bind to the extracellular matrix or to host cells. Others bind to plasminogen, which, when converted to the active protease plasmin, aids in degradation and invasion of host tissues. Surprisingly, dozens of cell surface proteins that bind to host cells, extracellular matrix, or plasminogen were previously identified as intracellular enzymes or chaperones, and we refer to them as intracellular/surface moonlighting proteins. It is not known how most of these proteins are secreted. They do not possess signal peptides or other motifs for secretion by the Sec secretion system or the non‐canonical secretion systems. They do not contain sequence motifs known to be involved in attachment to the cell surface. Secretion and cell surface attachment may involve novel versions of known mechanisms or as yet unknown mechanisms. Our project involves identifying additional intracellular/surface moonlighting proteins and the mechanisms involved in their secretion and cell surface attachment. With the increasing problem of antibiotic resistance, new targets for inhibiting bacterial infection are needed. Understanding how intracellular/cell surface moonlighting proteins are targeted to the surface of a pathogen might lead to methods to decrease the ability of bacteria to bind to and degrade host tissues and could provide new targets for developing therapeutics to treat infections. POS143 Use of halogenated derivatives of the ferulic acid as matrices in proteomic analysis with Matrix‐Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry. Yosuke Kato1, Takeshi Sakamoto1, Narumi Hirosawa2, Jun Takayama1, Maiyan Xuan1, Mari Okazaki1, Yasushi Sakamoto2 1Josai University, Japan, 2Saitama Medical University, Japan In about 70 kind of synthesized ferulic acid derivatives we made, a few halogenated ferulic acid derivatives had a property like a matrix for matrix‐Assisted laser desorption/ionization time of flight mass spectrometry. Particularly, they seemed to be usable for the identification of proteins and the structural analysis of peptides. Then the properties of them compared with the general matrices such as alpha‐Cyano‐4‐hydroxycinnamic acid (CHCA) and 2,5‐Dihydroxybenzoic acid (DHBA). These 5‐bromo ferulic acid (5‐BFA) and 5‐chloro ferulic acid (5‐CFA) showed unexpected good quality mass spectrum in mass finger print (PMF) analysis of the transferrin. They gave higher intensity than CHCA about molecular weight region of 2,500 ‐ 4,000. Therefore, these halogenated matrices seemed to be available to detect the relatively large molecular peptides. Moreover, 5‐BFA and 5‐CFA were tested as matrix for MS/MS analysis to carry out the rapid amino acid sequencing by use of Matrix‐Assisted Laser Desorption/Ionization Quadrupole Ion Trap Time of Flight Mass Spectrometry. Both of them generated ion clusters reflecting the cleavage of peptide bonds and the scores in Mascot database search were relatively better than DHBA about tryptic peptides of transferrin and some synthesized peptides. Therefore the derivatives were applied to actual identification of proteins prepared from mouse brains sample and seemed to have some advantages for analyzing the bigger size peptides. Additionally, they suggested being effective matrices on the peptides including proline. POS162 Cloning, Expression, and Characterization of Putative Cyclooxygenases from Three Bacterial Species Barry Selinsky 1, Rebecca Skaf1, Alecia Cunniff1, Lauren Fahrenkrog1 1Villanova University, Chemistry Department, Pennsylvania, USA Cyclooxygenases are dual function enzymes that catalyze sequential cyclooxygenase/peroxidase reactions on lipid substrates. In mammals, cyclooxygenases convert arachidonic acid to prostaglandin H2, which is the precursor for the synthesis of all biologically active prostaglandins. Examples of cyclooxygenases have been reported for many lower eukaryotes, including coral and the red algae Gracilaria. Bioinformatics analyses have identified eleven structural homologs to eukaryotic cyclooxygenase with bacterial origins. In this study, we describe the cloning, expression and characterization of putative cyclooxygenases from three different bacterial species. The enzymes were expressed with N‐terminal his tags and purified to greater than 90% homogeneity. Two of the three enzymes exhibit heme‐dependent peroxidase activity, which is lacking in the third, a previously characterized linoleate 10S‐dioxygenase. Assays exploring lipid oxidation are in progress, and results on lipid substrate specificity and oxidation products will be presented. A comparison of predicted structures of the enzymes from the three species will be used to help understand the observed differences in activity. POS169 BioID Identification of Novel Effector Proteins in Eph Receptor Signaling Sara Banerjee 1, Nicolas Bisson1,2 1Centre de Recherche sur le Cancer, PROTEO, Division Oncologie, Centre de Recherche du Centre Hospitalier Universitaire (CHU) de Québec, Canada,2Biochimie Médicale et Pathologie, Université Laval, Division Oncologie, Centre de Recherche du Centre Hospitalier Universitaire (CHU) de Québec, Canada The Eph family of receptor tyrosine kinases (EphRs) regulates a wide range of cell‐cell communication events, such as cell adhesion, migration and tissue boundary formation. However, the molecular mechanisms by which EphRs mediate these processes is far from being understood. To address this question, we aim to identify new EphR downstream effector proteins and to determine their requirement for Eph function. First, to unravel EphR‐dependent signaling complexes we applied the mass spectrometry‐based BioID approach. We obtained a proximity network for four EphRs, namely EphA4, ‐B2, ‐B3 and ‐B4. We identified 188 proteins, the majority of which have not been previously linked to Eph signaling. Further bioinformatics analysis revealed a core signaling network of 34 proteins shared between the four EphRs. Next, we started to explore effect of a loss‐of‐function of BioID‐identified candidates in Eph‐dependent cell sorting and repulsion assays. Preliminary experiments showed that depletion of PARD3 blocked segregation of EphB2 cells from ephrinB2 cells, demonstrating that PARD3 could be a downstream mediator of EphRs. Overall, this research will lead to a better understanding of EphRs signaling pathways, providing invaluable insight on the mechanisms through which EphRs affect cell behavior. POS195 Illuminating the Specificity Landscape of the HCV NS3 Protease using Computation and Next Generation Sequencing Manasi Pethe1, Aliza Rubenstein2, Sagar Khare1, Dmitri Zoraine1 1Chemistry and Chemical Biology, Rutgers University, New Jersey, USA, 2CBMB, Rutgers University, New Jersey, USA Proteases are central to the processing and transfer of biological information. Viral systems encoding proteases need them for precise cleavage of the polyprotein during replication and viral assembly e.g. HCV NS3 protease. The HCV NS3 protease is a multifunctional protease, which is likely a result of both positive selection pressure to maintain cleavability of its five native substrates, i.e. known sites on the polyprotein, and negative selection pressure to avoid cleavage of other sites in the polyprotein. We mapped out the specificity landscape of the HCV NS3 protease to obtain a comprehensive understanding of the protease–substrate interaction network. We used an in vivo yeast surface display assay in order to isolate populations of cleaved, partially cleaved, and uncleaved substrates using Fluorescence Assisted Cell Sorting. These populations were analyzed using Next‐Gen Sequencing technology and computational modeling using Rosetta and Amber packages. Through generating force directed graphs of the library we discovered that the partially cleaved sequences act as “pit stops” in the evolutionary trajectory from functional substrates to non‐functional substrates. We were able to reconstruct the entire (3.2 million sequences) HCV NS3 substrate landscape learning from the sequences identified in our experiment, using a SVM based approach to predict and validate sequences that lie in the network. We further explored the influence of prevalent Drug Resistant protease mutants (R155K, A156T, D168A and the triple mutant) on the substrate specificity landscape. This study deepens our understanding of the robustness of the protease‐ substrate landscape and also paves the way to creating designer proteases. POS240 Survey of the Proteomic and Transcriptomic Profile of Sea Anemone Anthopleura dowii Verril (1869) from Mexico Claudia Rodríguez Almazán 1, Jorge Tonatiuh Ayala Sumuano1, Santos Ramírez Carreto1, Rosario Vera Estrella1 1Universidad Nacional Autónoma de México, Mexico Sea anemones produce a variety of peptides and proteins containing biological activities of pharmacological and biotechnological interest. These components have been isolated from crude extracts of the whole body, tentacles and mucous secretions that cover the entire body of these marine organisms. Here we describe the analysis of the proteomic and transcirptomic profile of mucus and tentacles samples, obtained from the sea anemone Anthopleura dowii Verril (1869) of the Mexican Pacific. We used a proteomic approach to profile the protein components of A.dowii Verril (1869). A total of 141 proteins were identified as proteins involved in various biological functions such as secretion, adhesion, neurotoxicity, regulating enzyme activity, antioxidant, structural, proliferation and cell death, metabolic processes, and others. The use of RNA‐seq has allowed the identification and quantification of bioactive compounds of biotechnological importance. In the transcriptome of A. dowii Verril (1869) there were 70,097,332 raw readings of Illumina technology for the sequencing of transcirptome. These readings were assembled by the Trinity pipeline resulting in 72,684 contigs with an N50 = 1179 bp, average length of 707 bp. This work provides the first transcriptome and proteome of the sea anemone A. dowii Verrill (1869). The information presented here may be useful to identify new molecules for biotechnology and pharmaceutical relevance. This work was supported by Consejo Nacional de Ciencia y Tecnología (CONACYT) [grant number 178151]. POS346 Identification of Substrates of the PRP19 E3 Ubiquitin Ligase in Response to DNA Damage Mailyn Yates 1, Alexandre Marechal1, Samuel Picard1, Jean‐Christophe Dubois1, Antoine Gaudreau‐Lapierre1, Pauline Kaczmarek1 1Universite de Sherbrooke, Quebec, Canada The DNA damage response (DDR) relies on specialized protein sensors to detect, signal and repair DNA lesions thereby maintaining genomic stability. The single‐stranded DNA (ssDNA)‐binding complex RPA is a critical DDR factor which detects persistent stretches of ssDNA induced by DNA breaks and replication impediments. RPA‐ssDNA acts as a platform to recruit a large number of genome maintenance factors including the ATR checkpoint kinase to signal replication problems and repair the genome. We recently identified the ubiquitin ligase PRP19 as a key player in this pathway. PRP19 is rapidly recruited onto RPA‐ssDNA and functions as a ubiquitin ligase to enhance ATR activity, DNA repair and replication fork restart. The specific PRP19‐mediated ubiquitylation substrates that regulate all of these processes are still unclear. Here, we use a label‐free proteomics approach to systematically identify constitutive and DNA damage‐induced PRP19 interactors. We show that PRP19 interacts with critical DNA helicases on the RPA‐ssDNA platform in response to DNA damage. Using immunofluorescence assays, we establish that PRP19 regulates the accumulation of a specific DNA helicase on RPA‐ssDNA. PRP19 also regulates the ubiquitylation of this helicase during replication stress to promote genome stability. Our data provides a comprehensive landscape of PRP19 interactors and provides much needed insight into its many roles in genome maintenance. POS352 Assessment of the effects of pollutants in the Great Lakes on the human proteome Emmalyn Dupree 1, Costel Darie1, Bernard Crimmins2, Thomas Holsen2, James Pagano3, Brooke Thompson4, Krista Christensen4, Michelle Raymond4 1Clarkson University, Biochemistry & Proteomics Group, Department of Chemistry & Biomolecular Science, USA, 2Clarkson University, Department of Civil and Environmental Engineering, Clarkson University, USA, 3SUNY Owsego, Department of Chemistry, USA 4Wisconsin Department of Health Services, USA The Great Lakes fishery provides a bountiful source of protein to indigenous peoples. However, there are concerns about fish consumption and contaminant exposure to these populations. Sera from fish consumers in the Great Lakes basin were analyzed for legacy chemicals such as polychlorinated biphenyls and organochlorine pesticides to assess the differential accumulation of these contaminants with respect to fish consumption and proteome related to contaminant enrichment. The current study is a double‐blinded study: legacy contaminant residues are being analyzed in random order with those sera exhibiting the lowest levels adopted as controls. In parallel, the sera are also analyzed blindly by proteomics methods. The control/exposed samples will then be segregated based on contaminant levels where samples withe the lowest levels of PCBs considered controls and those with elevated levels denoted exposed. Legacy chemical analysis was performed using CDC methods employing isotope dilution mass spectrometry. Proteome analysis was performed by albumin depletion and separation by SDS‐PAGE, followed by the in‐gel trypsin digestion and nanoLC‐MS/MS analysis on a NanoAcquity UPLC coupled to a Q‐TOF Ultima API mass spectrometer (both from Waters). In addition, the sera are also digested by trypsin in‐solution followed by nanoLC‐MS/MS analysis. The two methods (in‐gel and in‐solution digestion complement each other). The initial scout runs for the in‐solution digestion and nanoLC‐MS/MS analysis is completed. Additional more in‐depth analyses are currently underway. POS353 Mass Spectrometry based Proteomic Investigation of Induced Obstructive Sleep Apnea (OSA) in Rat Atria Devika Channaveerappa 1, Costel C. Darie1, Jacob Lux2, Kelly L. Wormwood1, Meredith McLerie2, Brian K. Panama2 1Clarkson University, New York, USA, 2Masonic Medical Research Laboratory, New York, USA Obstructive sleep apnea (OSA) affects up to 24% of the adult population and is associated with several atrial diseases. It is characterized by transient cessations in respiration lasting >10 seconds due to narrowing or occlusion of the upper airway during sleep. Although clinical evidence linking OSA to proarrhythmaic atrial changes is well known, the molecular mechanisms by which OSA causes atrial disease remain elusive. We have initiated a recently developed rat model which closely recapitulates the characteristics of OSA, to study OSA‐induced cardiac changes. Male Sprague Dawley rats, aged 50–70 days, received surgically implanted tracheal balloons which were inflated to cause transient airway obstructions. Apnea groups experienced 60 apneas per hour of either 13 seconds (moderate apnea) or 23 seconds (severe apnea) for 2 weeks and 8 hours per day. Control rats received surgeries but no inflations. Proteomics analysis was done on the rat atria homogenates to identify dysregulated proteins in moderate and severe apnea when compared to control. SDS‐PAGE was performed and the gel bands were trypsin digested to obtain the peptide mixtures. The peptides were analyzed by a Nano Acquity UPLC coupled with Xevo G2 Mass Spectrometer. The proteomics analysis revealed that 3 of the 9 enzymes in glycolysis and 2 proteins related to oxidative phosphorylation were down‐regulated in the severe apnea group. In contrast, several structural and pro‐hypertrophic proteins were up‐regulated with chronic OSA. The data suggests the chronic OSA causes protein changes which lead to cessation of glycolysis, a diminished capacity to generate reducing equivalents (i.e. NADH) as well as promotion of cardiac hypertrophy. POS356 Nicotine‐Induced Proteome of Arthrobacter nicotinovorans pAO1 Marius Mihasan 1, Costel Darie1, Cornelia Babii2, Roshanak Aslebagh1 1Biochemistry & Proteomics Group, Department of Chemistry & Biomolecular Science, Clarkson University, USA, 2Biochemistry and Molecular Biology Laboratory, Department of Biology, Alexandru Ioan Cuza University of Iasi, Iasi, Romania Several key points in the plasmid‐encoded nicotine catabolic pathway of the soil bacterium Arthrobacter nicotinovorans pAO1 are still missing. More data on the regulators of the nicotine catabolic genes and nicotine‐transport proteins is required before this pathway could be successfully used for the detoxification of nicotine‐containing waste and/or production of useful nicotine‐derivatives. The current work attempts to provide this data and to identify the nicotine‐catabolism related proteins from A. nicotinovorans pAO1 using nanoLC‐MS/MS. Cell free extracts of A. nicotinovorans grown with and without nicotine were separated on 9–16% SDS‐PAGE maxi gradient gels and stained by Commasie Brilliant Blue. The proteins are currently digested by trypsin and the resulting peptide mixtures analyzed by a NanoAcquity UPLC coupled to a QTOF Ultima API mass spectrometer. Data processing and analysis will be achieved by the in‐house Protein Lynx Global Server (v.2.4), Mascot Server (v.2.5) and Scaffold software (v.4.3). Simple examination of the Coomassie‐stained gels revealed some differences between the proteomes of the nicotine‐untreated and nicotine‐treated bacteria. Several extra bands in the range of 60 and 30 kDa could be identified in the nicotine‐grown bacteria extracts. One of these nicotine‐related bands was identified as a KatA, a chromosomal heme‐binding catalase. The KatA might help protecting the bacterial cells against the reactive oxygen species generated by the one‐electron reductions of nicotine end‐products. A comprehensive proteomic analysis is currently under way. These experiments will allow us to identify the nicotine‐catabolism related proteins and to better understand how the pathway is integrated into the general metabolism of the cell. The nicotine catabolic enzymes might also find applications in biotechnology. POS404 Analysis of differential protein expression in post‐mortem human bone of rib and skull Rubén Darío Díaz Martín 1, Javier R Ambrosio Hernández2, Lorena Valencia Caballero1, Javier R. Ambrosio Hernández2 1Departamento de Anfiteatro, Facultad de Medicina Universidad Nacional Autónoma de Mexico, Mexico, 2Departamento de Parasitología y Microbiología, Facultad de Medicina Universidad Nacional Autónoma de Mexico, Mexico Bone tissue holds information regarding the physiological status of individuals because the cellular components and mineralized extracellular matrix participate in homeostatic events. Despite this, the specific protein expression profile of the different skeletal components is unknown. Our aim is identify organ‐specific protein markers in bone tissue from the skull and rib to obtain information regarding pre‐mortem events such as pathologies or causes of death. To perform this analysis, bone pieces of skull and rib were obtained from post‐mortem individuals admitted in the Institute of Forensic Sciences (INCIFO) in Mexico City. Protein extraction was performed following a demineralized protocol to recover proteins associated with the bone matrix. The proteins were separated by SDS‐PAGE and the expression patterns were determined by densitometric analysis and mass spectrometry. Finally, Western Blot assays were performed to determine the presence of some protein markers. The extraction method allowed the recovery of proteins from two types of bone with different structural and physiological characteristics. The analysis of the electrophoretic pattern of proteins markers indicates that there are differences in protein expression between the two analyzed bone types. These results suggest that the expression of proteins in bone tissue is an event that depends of the physiological and mechanical requirements to bone is subjected. The identified proteins could be useful for establishing possible pre‐mortem conditions. Additionally, the strategy of identification of biomarkers could be applied for forensic and anthropological purposes. This project has been submitted to the approbation of the ethical and research institutional committees of FM‐UNAM. RDDM is supported by a scholarship from the DGAPA‐UNAM postdoctoral scholarship program. POS462 Targeted Proteomics Identification of Post‐Translationally Modified KRAS Forms Oleg Chertov 1, William Gillette1, Shelley Perkins1, Esposito Dominic1, Elena Chertova2, Joseth Knotts3, Simona Colantonio3, Anna Maciag1, Andrew Stephen1 1NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc, USA, 2AIDS and Cancer Virus Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc, USA, 3Antibody Characterization Laboratory, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc, USA Oncoprotein KRAS4b is frequently mutated in pancreatic, colon and lung cancers. It is localized on the cytoplasmic membrane, which requires farnesylation, proteolytic cleavage and carboxymethylation at the C‐terminal Cys residue. Inactivation of KRAS processing enzymes genetically or by small molecule inhibition may result in the generation of different forms of KRAS that lack the complete set of C‐terminal modifications (prenylation, proteolysis, and/or methylation). To develop a method which would allow us to identify these protein forms by proteolysis and mass spectrometry we expressed and purified GKEKMSKDGKKKKKKSKTKC(farn)‐Me peptide. The peptide was expressed as a fusion protein (His6‐MBP‐tev‐HVR) in a baculovirus/insect cell expression system that was engineered to co‐express human farnesyl transferase. The protein was affinity purified (IMAC) via the 6x‐His tag, followed by CEX, and then cleavage of the His6‐MBP‐fusion with His6‐TEV protease. The final step of the purification was removal of all His‐tagged proteins by a final IMAC step. This peptide was cleaved by Glu‐C protease to generate KMSKDGKKKKKKSKTKC(farn)‐Me and GKKKKKKSKTKC(farn)‐Me which were purified by HPLC and quantified by amino acid analysis to use as standards for targeted proteomics. Peptide corresponding to C‐terminus of unprocessed KRAS4b (KMSKDGKKKKKKSKTKCVIM) was purified from Glu‐C digest of KRAS4b (1–188). These peptides were used to develop a targeted proteomics method for detection and quantification of KRAS C‐terminal peptides on Shimadzu LCMS‐8050 system that consists of an HPLC pump and a triple quadrupole mass spectrometer. The method allows to study the state of KRAS4b C‐terminus in cell expressing wild type and mutant KRAS, as well as differently tagged KRAS. Supported by NCI contract HHSN261200800001E. POS489 Deciphering the role of ANGPTL8 and its Variants in Insulin Resistance and Lipid Metabolism by Proteomics Mohamed Abu‐farha 1, Preethi Cherian1, Irina Al‐khairi1, Jehad Abu‐baker1 1Dasman Diabetes Institute, Kuwait ANGPTL8 has been recently shown to induce beta‐cell proliferation and regulates triglyceride (TG) and fatty acid metabolism. We showed that ANGPTL8 was increased in T2D and a sequence variant in its gene R59W was associated with higher FBG level in Kuwaitis. Our objective is to identify proteins that are regulating the function of ANGPTL8 in vitro and identify new targets proteins that might be involved in its function in lipid regulation. A cross sectional study was designed to examine the level of ANGPTL8 in 283 non‐diabetic Arabs, and to identify its sequence variants using Sanger sequencing and their association with various clinical parameters. The study conformed to the principles outlined in the Declaration of Helsinki and was approved by the Ethical Review Committee at Dasman Diabetes Institute (DDI). An informed written consent was obtained from all the participants before their enrolment in the study. Our data shows for the first time that Arabs with the heterozygote form of (c.194C>T Rs.2278426) had higher level of Fasting Blood Glucose (FBG) than wild type. ANGPTL8 and its variant were flagged tagged and their expression was tested using FLAG antibody. Overexpression of ANGPTL8 in HepG2 cells resulted in changes in a number of proteins expression. A selected number of differentially expressed proteins were identified by LC/MS such as SEC24 family members that are involved in lipid metabolism. 19. PROTEOSTASIS AND QUALITY CONTROL POS031 Structural Basis for Inactivation of an HtrA2/Omi variant ‐ Implication in Neurodegeneration Ajay Wagh 1, Kakoli Bose 1ACTREC, India Background & Objective: Serine protease HtrA2/Omi, (High temperature requirement protease A2), is involved in apoptosis and protein quality control. However, one of its murine inactive mutants, (S276C aka mnd2) is associated with motor neuron degeneration 2. Similarly, this conserved mutation in human HtrA2 (hHtrA2) also renders the protease inactive, implicating pathogenicity. However, the structural determinants for its inactivation have not been yet elucidated. Therefore, our objective is to understand the structural correlates of inactivity and pathogenicity associated with this mutation in hHtrA2 using multidisciplinary approach. Methods: Secondary and tertiary structural characterization of wildtype and mutant was done using CD and fluorescence spectroscopy. Chromatography and Dynamic Light Scattering were employed to compare their oligomeric properties. The structural comparison at atomic resolution was performed using X‐ray crystallography. Results: While enzyme kinetics showed inactivity, spectroscopic probes did not identify any major structural changes in the mutant. Structural analyses at 2Å resolution showed subtle yet critical structural changes in the C‐terminal regulatory domain and residues critical for oxyanion hole formation. Crystallographic data also highlight the importance of water molecules, which might play critical role in mediating intermolecular interactions for maintaining the functional ensemble of the protease. Conclusion: Overall, the crystallographic data along with ongoing functional studies helped in deciphering not only the structural basis of (S276C) HtrA2 inactivity and its implication in neurodegeneration, but also shed light on the residues important for its complex allosteric mechanism of activation. POS052 Intrinsically Disordered Segments Regulate Cellular Protein Abundance by Encoding a Multilayer Degron Architecture Mainak Guharoy 1, Peter Tompa1, Pallab Bhowmick1, Tamas Lazar1 1VIB‐VUB Center for Structural Biology, Belgium Programmed protein degradation via the ubiquitin‐proteasome system is an important controller of signaling and regulatory pathways. The specific targeting of substrates is performed by about 1000 E3 ligases (in humans), with a complexity parallel to transcriptional and translational regulation. Our recent work has highlighted the role of protein structural disorder in regulating substrate turnover. From the experimental literature, we first collected datasets of E3 ligase‐substrate pairs in order to study degradation signals (‘degrons’) that trigger protein turnover. Based on systematic bioinformatics analysis, we proposed a multi‐level degron architecture that tightly controls decision‐making in protein degradation. The primary degron specifies substrate recognition by cognate E3s. Short peptide motifs constitute an important class of primary degrons that are almost exclusively present within disordered regions. Primary degron sequences are strongly conserved among orthologs and occur in disordered segments that undergo E3‐induced folding‐upon‐binding. Posttranslational modifications can switch primary degrons into E3‐binding‐competent states, thereby integrating degradation with signaling pathways. Secondary degron(s) comprise single or multiple neighboring ubiquitinated substrate lysine(s) that are often located proximal to the primary degron and therefore are part of the disordered segment. Finally, experiments and predictions show that initiation of degradation at the 26S proteasome requires a partially disordered region (tertiary degron) to facilitate substrate entry into the proteasome core. The distributed nature of degrons ensures specificity and regulatory control of degradation. Furthermore, since disordered segments may accommodate multiple binding partners, it enables degron masking via overlapping binding sites for partners not involved in triggering degradation. The balance of competing interactions determines the spatio‐temporal control of protein degradation. POS126 Ubiquitin Receptors Mediate Proteasomal Processivity Daniel Kraut 1, Mary Cundiff1, William Dewey1, Eden Reichard1, Nicholas Nassif1 1Villanova University, Pennsylvania, USA In eukaryotic cells, proteins are targeted to the proteasome for degradation by polyubiquitination. These proteins bind to ubiquitin receptors, are engaged and unfolded by proteasomal ATPases, and are processively degraded. The factors determining to what extent the proteasome can successfully unfold and degrade a substrate are still poorly understood. We previously found that the architecture of polyubiquitin chains attached to a substrate affects the proteasome's ability to unfold and degrade the substrate, with K48‐ or mixed‐linkage chains leading to greater processivity than K63‐linked chains. Ubiquitin‐independent targeting of substrates to the proteasome gave substantially lower processivity of degradation than ubiquitin‐dependent targeting. Thus, even though ubiquitin chains are removed early in degradation, during substrate engagement, remarkably they dramatically affect the later unfolding of a protein domain. How do ubiquitin chains differentially activate the proteasome? One attractive candidate was the ubiquitin receptor Rpn10. Mutation of Rpn10 to prevent ubiquitin binding led to drastic unfolding defects early in the degradation process. Using a destabilized substrate that allowed initial degradation to occur more easily, we found that processivity defects were dependent on ubiquitin chain linkage on the substrate. Similar results were found for the ubiquitin receptor Rpn1, while a double mutant essentially eliminated any ubiquitin‐based activation of the proteasome. We conclude that linkage‐based differences allow a substrate to interact with one or more ubiquitin receptors, activating the proteasome's unfolding ability. POS213 The Structure of Yeast tRNA Ligase Reveals a Competition between Non‐Conventional mRNA Splicing and RNA Decay Peter Jirka 1, Peter Walter1 1UCSF, California, USA Yeast tRNA ligase (Trl1) is an essential trifunctional enzyme consisting of a cyclic phosphodiesterase, a polynucleotide kinase and an ATP‐dependent RNA ligase. It catalyzes exon‐exon ligation of tRNA half‐molecules during tRNA splicing. Trl1 is also required for HAC1 mRNA splicing during the unfolded protein response (UPR). The UPR is an intracellular signaling network that monitors the protein folding capacity of the endoplasmic reticulum (ER). Upon sensing protein folding perturbations in the ER, the kinase/endonuclease Ire1 initiates the UPR signal by a unique mechanism: In a non‐conventional, cytoplasmic splicing reaction, Ire1 removes an intron from HAC1 mRNA followed by exon‐exon ligation by Trl1, allowing the production of the Hac1p transcription factor that drives the transcriptional response. How the splicing reaction is orchestrated with fidelity to ensure stress signaling remains an outstanding question. Here, we report the crystal structure of the RNA ligase domain of the thermophilic fungus Chaetomium thermophilum Trl1 at 1.9 Å resolution. The molecular architecture of the active site reveals the principles of RNA substrate recognition. A Trl1 mutant variant that uncouples both functional outputs in vivo allowed us to identify a competition between RNA ligation and degradation during HAC1 mRNA splicing. Our results show the functional importance of mRNA decay pathways and ribosome‐associated quality control in maintaining the fidelity of this non‐conventional splicing reaction. Furthermore, Trl1 enzymes are found in all human fungal pathogens and they are potential drug targets because of the difference to mammalian tRNA splicing enzymes. Our findings provide structural insights into ligand binding that can enable chemical inhibition of tRNA ligases from pathogenic fungi. POS222 Calpain‐ and proteasome‐dependent proteolysis in the control of salmonid fish growth Liudmila Lysenko 1, Nadezhda Kantserova1, Mikhail Ruchyev1, Nina Nemova1 1IB KarRC RAS, Russia Most Teleostei, including salmonid fish, are indeterminate growers with maximal growth rate observed at the first years. Young salmonids of northern, low‐production watercourses of the White Sea basin are characterized by decreased growth rate at the river period of their life and increased smoltification age comparing to their conspecifics from the lower‐latitude watercourses. Fish growth is mostly determined by the accumulation of skeletal muscle proteins relying on the balance between protein synthesis and degradation. Although proteolysis limits the rate of muscle growth in fish, the role of proteolytic systems responsible for degrading myofibrillar proteins is not well defined. Among protein‐degrading systems, calcium‐dependent proteolysis dominates in total muscle protein degradation in teleost fish while proteasomal digestion plays a minor role. Negative correlation of protease activities in fish muscles and growth increments was shown in Atlantic salmon (S. salar) and brown trout (S. trutta). Significant decrease in calpain and proteasome activities as well as continuous decrease in growth increments were show during the first years of salmonid life. Lowest levels of calpain and proteasome activities has been detected in the larger individuals of salmon (but not trout) entering smoltification stage. Some common and specific mechanisms controlling proteolysis in salmonid growth such as growth hormone and endogenous inhibition were evaluated. The research was supported by the Russian Scientific Foundation, project no. 14–24‐00102. POS393 Intracellular checks and balances: Not an infinite account Ipsita Roy 1, Ratnika Sethi1, Ankan Bhadra1 1S.A.S.Nagar, National Institute of Pharmaceutical Education and Research (NIPER), India Aggregation‐induced proteotoxicity and proteostasis collapse have been linked to progression of a number of protein misfolding disorders. These include Huntington's diseases (HD), which is caused by expansion of the trinucleotide repeat sequence at the 5'‐end of the huntingtin gene. This results in misfolding and aggregation of mutant huntingtin which is associated with various deleterious effects on the cellular phenotype. We have studied aggregation of mutant huntingtin using the yeast model of HD. Dietary restriction (DR) has been reported to have beneficial effects on survival of many lower organisms although its benefits in higher animals are not clear. DR induces mild stress which activates the cellular stress response machinery with consequent advantages for the cell. Our studies show that DR offers no benefits and is, in fact, detrimental for a cell which is proteotoxically challenged. We find that although the cell does activate stress response pathways when challenged individually by protein aggregation or DR, combination of the two factors (huntingtin aggregation in DR‐induced cells) dampens the cellular stress response. Our work also shows that the levels of the two components of the heat shock response, viz. the chemical chaperone trehalose and the heat shock protein Hsp104, are finely tuned. In a cell expressing aggregated huntingtin, the level of one component does not exceed a threshold level in response to aggregation‐induced stress even in the absence of the other component. We conclude that various arms of proteostasis are tightly regulated and uncontrolled stress does not lead to an unregulated response. POS415 Characterization of Recombinant Human Mitochondrial Processing Peptidase Andrew Bayne 1, Jean Francois Trempe1 1McGill University, Montreal, Canada Mitochondrial processing peptidase (MPP) is a metallopeptidase that cleaves mitochondrial targeting signals from the majority of nuclear‐encoded mitochondrial proteins. Mutations in both MPP and its substrates have been implicated in various neurodegenerative diseases, including Parkinson's disease (PD) and non‐progressive cerebellar ataxia. One of these implicated substrates is PINK1 ‐ a kinase whose mutations are known to cause early‐onset autosomal PD. In healthy mitochondria, PINK1 is constitutively imported, cleaved first by MPP, and then retrotranslocated to the cytosol for proteasomal degradation. In this regard, MPP acts as a key junction for PINK1 import, proteolysis, and overall mitochondrial quality control. However, both the MPP cleavage site on PINK1 and its binding conformation remain unknown. To gain insight into the proteolytic mechanisms concerning PINK1 and other disease‐implicated substrates, we have begun to characterize the human MPP heterodimer. We demonstrate that the human MPP dimer can be successfully purified from a co‐expression system in E. coli. We have also developed a proteomics‐based method to monitor MPP activity, using a synthetic presequence from malate dehydrogenase as a positive control. Research into the PINK1 cleavage site and mechanism of cleavage are currently ongoing in our laboratory. 20. SINGLE MOLECULE STUDIES POS132 Probing the Determinants of Collagen Flexibility using Atomic Force Microscopy Aaron Lyons 1, Nancy Forde1, Naghmeh Rezaei1, Nancy Forde1 1Department of Physics, Simon Fraser University, Vancouver, Canada As the primary load‐ and tension‐bearing protein in mammals, the mechanical properties of collagen are of significant biomedical interest. By virtue of its high aspect ratio, the flexibility of the collagen monomer – a 100 kDa triple helical structure, 300 nm in length and 1.5 nm in diameter – can be described with the tools of polymer physics. However, experimental estimates of collagen's rigidity span nearly an order of magnitude, leaving the mechanical properties of the protein unresolved. To address this disparity, we have developed methods for extracting quantitative metrics of collagen's flexibility from atomic force microscope (AFM) images. Our results demonstrate that collagen's flexibility is strongly influenced by environmental conditions, including pH and ionic strength. Surprisingly, we find minimal variations in rigidity between different forms of collagen, despite their different physiological contexts. With these new tools, our future goals include the extension of these methods to the sequence‐dependent analysis of collagen flexibility. 21. STRUCTURE (X‐RAY/NMR/EM) POS044 Pseudo‐pseudophosphatases: phosphocysteine as a regulator of protein phosphatase interactions Kalle Gehring1 1McGill University, Montreal, Canada Phosphatases of regenerating liver (PRLs) are highly oncogenic, yet their mechanism of action remains controversial. Recent studies have proposed that PRL oncogenicity is mediated through their regulation of a family of magnesium transporters, CNNMs. Here, we present the X‐ray crystal structure of three different PRL‐CNNM complexes to reveal the molecular basis complex formation (Zhang et al, Scientific Reports, 2017, 7(1):48). We show that PRLs function as pseudo‐pseudo‐phosphatases where the binding to CNNM proteins is controlled by PRL phosphorylation of its catalytic cysteine residue (Gulerez et al, EMBO Reports, 2016, 17(12):1890). In vivo, PRLs are endogenously phosphorylated on cysteine to high levels and this phosphorylation changes in response to magnesium levels. These studies suggest PRLs act as molecular switches combining properties of pseudo‐phosphatases and true phosphatases. POS055 Polycomb Repressive Complex 2 Structure with Inhibitor Reveals a Mechanism of Activation and Drug Resistance Alexei Brooun 1, Ketan Gajiwala1 1Pfizer, USA Polycomb repressive complex 2 (PRC2) mediates gene silencing through chromatin reorganization by methylation of histone H3 lysine 27 (H3K27). Overexpression of the complex and point mutations in the individual subunits of PRC2 have been shown to contribute to tumorigenesis. Several inhibitors of the PRC2 activity have shown efficacy in EZH2‐mutated lymphomas and are currently in clinical development. We have used hydrogen deuterium exchange mass spectroscopy (HDX‐MS) and small angle X‐ray scattering (SAXS) to engineer a minimal PRC2 complex consisting of EZH2, Suz12 and EED that is active and amenable to crystallization. The crystal structures of the inhibitor‐bound wild‐type and Y641N PRC2 reveal a surprising ligand binding mode. The structures illuminate an important role played by a stretch of 17 amino acid residues in the N‐terminal region of EZH2, we call the activation loop, in the stimulation of enzyme activity, inhibitor recognition and the potential development of the mutation mediated drug resistance. While crystal structures of inhibitor 1 with WT and Y641 PRC2 are nearly identical and consistent with sensitivity of Y641N PRC2 to inhibitor 1, HDX‐MS analysis of the oncogenic mutant PRC2 suggests that Y641N substitution has far reaching consequences on EZH2 protein dynamics rather than just creating a more spacious substrate binding site. The work presented here provides new avenues for the design and development of next generation PRC2 inhibitors through establishment of a structure‐based drug design platform and offers key insights into the interplay of PRC2 activation and pyridone based inhibitor recognition. POS057 Structural and Functional Study of Legionella pneumophila Effector lpp0008 Ivy Yeuk Wah Chung 1 1University of Saskatchewan, Canada Legionella pneumophila is an intracellular pathogen that causes Legionnaire's disease in human. L. pneumophila can be found in fresh water environment as a free‐living organism but it is also an intracellular parasite of protozoa. L. pneumophila becomes infectious to human when the aerosolized pathogen is inhaled and comes into contact with the alveolar mucosa. L. pneumophila primarily replicates in macrophages. Macrophages and amoeba defense themselves against bacterial pathogen by phagocytosis. The subsequent fusion of the phagosome to endosomal compartment kills the pathogen. Interestingly, L. pneumophila enters the host cell by phagocytosis but the L. pneumophila containing phagosomes are segregated from the phagocytic maturation pathway. Instead, they fuse with ER‐derived secretory vesicles and membranes resulting in the formation of Legionella containing vacuole where L. pneumophila replicates intracellularly. L. pneumophila achieves this and subverts many host cellular processes (e.g. apoptosis, transcription, secretory transport and ubiquitination) by secreting effector molecules through its Dot/Icm type IV secretion system (T4SS). Here we present the structural and functional study of L. pneumophila effector RavA (lpp0008). Not much is known about lpp0008 but its carboxyl terminal glutamate rich sequence has been reported to be important for its translocation. Expression of RavA‐GFP in HEK293 cells showed a juxtanuclear localization and this localization depends on the C‐terminal region. Structural analysis reveals that the N‐terminal region of lpp0008 consists of four structural repeats that are likely involved in interactions with host proteins. The potential RavA targets are presently investigated by the yeast two‐hybrid system and proteomics approaches. POS060 Visualizing the first steps of a megaenzyme making an antibiotic Janice Reimer 1 1McGill University, Montreal, Canada Nonribosomal peptide synthetases (NRPSs) are true macromolecular machines, using modular assembly‐line logic, a complex catalytic cycle, moving parts and multiple active sites to make their structurally diverse small molecules. We have determined a series of crystal structures of the initiation module of the antibiotic‐producing NRPS, linear gramicidin synthetase. This module includes the specialized tailoring formylation domain, and we captured states that represent every major step of the assembly‐line synthesis in the initiation module. The structures illustrate how the formylation domain is incorporated into the NRPS architecture and how it has evolved to act in concert with the other domains in the initiation process. Substantial conformational changes occur between sequential steps in the synthetic cycle, with both the peptidyl carrier protein and the adenylation subdomain undergoing immense movements to shuttle substrates over 50 Å between distal active sites. The structures highlight the great versatility of NRPSs, as small domains repurpose and recycle their limited interfaces to interact with their various binding partners. Together our published and unpublished work presents a holistic view of the function of this elegant NRPS initiation module. POS061 Kinetic and Structural Characterization of the Effects of Membrane on the Complex of Cytochrome b5 and Cytochrome c Katherine Gentry 1 1University of Michigan, USA Cytochrome b5 (cytb5) is a membrane bound protein vital for the regulation of cytochrome P450 (cytP450) metabolism and is capable of electron transfer to many redox partners. Here, using cyt c as a surrogate for cytP450, we report the effect of membrane on the interaction between full‐length cytb5 and cyt c for the first time. As shown through stopped‐flow kinetic experiments, electron transfer capable cytb5 ‐ cyt c complexes were formed and incorporated into both isotropic bicelles and lipid nanodiscs. Chemical shift perturbations and differential line broadening data, measured from NMR experiments, were used to map the binding interface between cytb5 and cyt c. Our experimental results identify differences in the binding epitope of cytb5 in the presence and absence of membranes. Notably, in the presence of membrane, cytb5 only engaged cyt c at the lower and upper clefts while the membrane‐free cytb5 also uses a distal region. Using restraints generated from both cytb5 and cyt c, a complex structure was generated and an electron transfer pathway was identified. These results demonstrate the importance of studying membrane protein‐protein complex formation in their native lipid environment. Our results also demonstrate the successful preparation of a novel peptide‐based lipid nanodisc system, which is detergent‐free and possesses size flexibility, and its use for NMR structural studies of membrane proteins. POS079 Structural Insights to the Functions of C‐terminal Domains of Topoisomerase I Kemin Tan 1, Nan Cao2, Qingxuan Zhou2, Bokun Cheng3, Andrzej Joachimiak1 1Structural Biology Center, Biosciences Division, Argonne National Laboratory, USA, 2Department of Chemistry and Biochemistry, Florida International University, USA, 3Department of Biochemistry and Molecular Biology, New York Medical College, USA Topoisomerase I has an essential function in preventing hypernegative supercoiling of DNA. The topoisomerase I of Mycobacterium tuberculosis (MtTOP1) is essential for the viability of the organism and survival in a murine model and is being pursued as a novel target for the discovery of new therapeutic agents for the treatment of drug‐resistant tuberculosis. The structures of the toroid‐shaped N‐terminal domains of E. coli topoisomerase I (EcTOP1) were reported more than ten years ago while the structures and even the domain arrangement of the C‐terminal region of topoisomerase I had remained elusive until recent studies. In the past two years we have determined a full‐length structure of E. coli topoisomerase I, which unveiled how C‐terminal domains (including three 4‐Cys zinc ribbon domains and two zinc ribbon‐like domains) bind ssDNA with primarily stacking interactions. We have also analyzed the domain arrangement of C‐terminal region of MtTOP1 and successfully obtained the first MtTOP1 crystal structure, which includes four N‐terminal domains and the first C‐terminal domain. The first C‐terminal domain, an expected representative of other four C‐terminal domains, reveals a novel a/ß fold and key potential DNA‐binding residues. POS103 Understanding the Bacillamide Nonribosomal Peptide Synthetase System Camille Marie Fortinez 1, Kristjan Bloudoff1, Martin Schmeing1 1McGill University, Montreal, Canada Nonribosomal peptide synthetases (NRPSs) are large multimodular enzymes that produce interesting bioactive products. Typically, a module contains the three domains required for peptide chain elongation – the adenylation (A), condensation (C) and peptidyl carrier protein (PCP) domains. Each module activates the amino acid substrate, attaches it to the PCP domain and adds it to the growing peptide through condensation in the C domain. This process occurs in every module until the product is released in the final module. In some NRPSs, the C domain is replaced with a heterocyclization (Cy) domain. These domains first perform condensation, adding that module's cysteine, threonine or serine substrate to the peptide chain, and then perform a second reaction, the intramolecular cyclodehydration of the side chain and the backbone carbonyl to form a thiazoline or (methyl)oxazoline ring. Bacillamide synthetase (BmdB), from Thermoactinomyces vulgaris, is a 3‐module, 6‐domain NRPS which includes a Cy domain that catalyzes peptide bond formation between alanine‐PCP and cysteine‐PCP, followed by thiazoline ring formation. Afterwards, the next C domain performs a condensation reaction with free tryptamine to form bacillamide E. Additional trans‐acting proteins have been identified within this biosynthetic cluster. BmdA decarboxylates tryptophan to produce tryptamine, and BmdC oxidizes the thiazoline ring of bacillamide E to make bacillamide D. Our goal is to elucidate the timing and details of bacillamide biosynthesis through crystallography and biochemical assays. We have reconstituted the entire bacillamide biosynthetic pathway in vitro and determined the structures of the Cy domain of BmdB and the BmdC oxidase protein, providing valuable insight into bacillamide synthesis. POS120 Dodecyl Melibiose: A Novel Detergent for Membrane Protein Work James Hutchison 1, Zhenwei Lu1, Justin Marinko1, Catherine Deatherage1, Ben Travis2, Ritesh Mittal2, Charles Sanders1 1Vanderbilt University, Tennessee, USA, 2Anatrace, Ohio, USA The choice of membrane mimetic when studying integral membrane proteins is critical to experimental success; detergents are often the easiest mimetic to implement, but an extensive detergent screen may be needed to find a suitable protein‐detergent pair. Non‐ionic detergents have become a key tool for studying challenging multi‐component or unstable membrane proteins. Dodecyl melibiose (DD‐MB) is a novel non‐ionic detergent whose aptness for membrane protein work had not been investigated. Dynamic light scattering of DD‐MB revealed a micelle stable over a wide range of common biochemical conditions. Membrane proteins in DD‐MB resulted in high quality TROSY‐HSQC spectra which were often equal to or slightly better than previously used ionic detergents. Unlike most detergents, DD‐MB possesses the ability to protect Diacylglycerol kinase (DAGK) from thermal inactivation. DD‐MB is capable of solubilizing multiple lipid raft component mixtures to form bilayered micelles. Given the stable micelle, ability to dissolve lipid raft components, high quality spectra and protective nature, DD‐MB should be included in all initial screens for membrane protein work. POS156 Selective Inhibition of Calcineurin Activity in Pathogenic Fungii Sophie Gobeil 1, Leonard Spicer1, Ben Bobay2, Ron Venters2 1Duke University Biochemistry Department, North Carolina, USA, 2Duke University NMR Center, North Carolina, USA Invasive fungal infections remain a leading cause of death in immunocompromised patients. Current antifungal agents have a multitude of issues including limited efficacy, host toxicity and an alarming increase in resistance. Current research in our laboratory is focused on targeting the calcineurin signaling pathway that has been shown to be required for fungal pathogenesis. Calcineurin is a key regulator of a signal transduction network required for survival of the most common pathogenic fungi in humans, making it an ideal target for fungal drug development. Calcineurin is also the target of the immunosuppressant FK506, which functions as an inhibitor by first complexing with the peptidyl‐prolyl cis‐trans isomerase immunophilin, FKBP12. The FKBP12‐FK506 complex subsequently binds to calcineurin and inhibits its activity. Although fungal calcineurins are targeted by FK506, this drug also targets mammalian calcineurin and is thus immunosuppressive in the host. In order to improve therapeutic efficacy, we have undertaken a unique effort that utilizes structural biology, molecular dynamics and molecular mycology aimed at identifying biophysical features of these complexes that might confer fungal specificity for inhibiting calcineurin activity with the objective of designing novel drugs to treat invasive fungal diseases with reduced human immunosuppression. NMR studies have focused on determining the resonance assignments, solution structures, inhibitor binding and dynamics for the FKBP12 proteins from Human and the pathogenic fungi C. albicans, A. fumigatus, C. glabrata and M. circinelloides with and without bound inhibitors. Importantly, these studies have already determined that specific residues are affected differentially between the human and the pathogenic fungal FKBP12 proteins. POS160 Solution scattering at the Life Science X‐ray Scattering Beamline of National Synchrotron Light Source II Lin Yang 1, Shirish Chodankar1, Vito Graziano1 1Brookhaven National Laboratory, New York, USA We present early results from the Life Science X‐ray Scattering (LiX) beamline at the National Synchrotron Light Source II. The LiX beamline supports several methods for investigating the structure of biological macromolecules in solution. Most of the experiments are carried out in the high throughput mode, in which data are collected from ∼50 microliter of sample in two minutes. For samples that contain multiple species of particles (e.g. coexisting complex and subunits) or may contain aggregates, the instrument can instantaneously switch to the in‐line size exclusion chromatography mode, essentially using the beamline as one of the detectors of the purification system. The instrument is equipped with an x‐ray fluorescence detector to facilitate anomalous scattering measurements. We are also exploring time‐resolved solution scattering measurements based on flow mixers. In all measurements, the scattering data are recorded on three detectors to cover a wide range of scattering vectors, typically from 0.005 to 2.4 Å‐1. The LiX beamline is part of the LSBR research resource jointly funded by the NIH and DOE‐BER, together with a pair of macromolecular crystallography beamlines, FMX and AMX. These beamlines are currently operational. Access is flexible through regular general user, rapid access, and block allocation group proposals. POS185 Insight into the origin of the formylation tailoring domain found in the linear gramicidin nonribosomal peptide synthetase Ingrid Harb 1, Janice M. Reimer1, Olga Ovchinnikov2, Jessie Jiang1, Chris Whitfield2, T. Martin Schmeing1 1Dept. of Biochemistry, McGill University, Montreal, Canada, 2Dept. of Molecular and Cellular Biology, University of Guelph, Ontario, Canada The presence of tailoring domains in nonribosomal peptide synthetases (NRPSs) dramatically increases the chemical space nonribosomal peptides can access. Linear gramicidin synthetase has evolved to include a formylation (F) domain at the start of its initiation module. The F domain is responsible for formylating the first residue, valine, and this formylation event is crucial for linear gramicidin's bioactivity. Our earlier bioinformatics studies suggest that the F domain originates from a sugar formyltransferase (FT) and was fused to the NRPS through horizontal gene transfer. We have now identified a gene for a sugar FT in Anoxybacillus kamchatkensis that is likely to be similar to the pre‐transfer gene transfer FT that is the ancestor of the F domain. This FT is located in a gene cluster like that for the CMP‐pseudaminic acid pathway, which is involved in glycosylation of flagellin. In CMP‐pseudaminic acid pathway, UDP‐GlcNAc is processed by a series of Pse enzymes, with the third enzyme (PseH) being an N‐acetyltransferase that acetylates the C4‐amino group. In the A. kamchatkensis gene cluster, PseH has been substituted with the putative FT (PseFT). Using biophysical techniques and X‐ray crystallography, we show that PseFT can replace PseH to bind and formylate its sugar‐nucleotide substrate, and that it exhibits remarkable structural resemblance to the linear gramicidin F domain. Together, these experiments display compelling evidence that PseFT is representative of the precursor sugar FT prior to incorporation into the NRPS. POS189 Understanding the structure and function of UDP‐glucose:glycoprotein glucosyltransferase (UGGT), a unique sensor of misfolded glycoproteins in ER Meng Yang 1, Daniel Calles1, Guennadi Kozlov1, Maire Menade1, Naoto Soya1, Justin Kollman2, Yukishige Ito3, Kalle Gehring1 1McGill University, Canada, 2University of Washington, USA, 3RIKEN Advanced Science Institute, Japan The enzyme UDP‐glucose:glycoprotein glucosyltransferase (UGGT) specifically recognizes misfolded glycoproteins and further catalyzes the attachment of a glucose to the N‐glycan in order to regenerate the glucosylated form for additional cycles of calnexin and calreticulin assisted refolding process. As a quality sensor, the function of UGGT ensures each ER targeted glycoprotein adopts its proper three‐dimensional fold, thus avoiding cell malfuntion or damage resulting from misfolded protein accumulation. However, the structure of UGGT and its mechanism for selection of misfolded proteins has been unknown since it was identified 25 years ago. Here, we expressed Drosophila melanogaster UGGT and characterized its physical properties. Besides, we characterized the interaction between UGGT and its well‐known binding partner, Sep15. Through hydrogen‐deuterium exchange‐mass spectrometry (HDX‐MS) analysis, the binding site of Sep15 on UGGT is revealed. Small‐angle X‐ray scattering (SAXS) provides us a model of full length UGGT. Furthermore, the full length structure of UGGT has been solved and refined to 16 Å through single particle construction by negative stain electron microscopy (EM). The structures of UGGT indicate the advantages of UGGT to screen and accommodate a variety of substrates. We propose a model in which there is one big central binding pocket in UGGT where UDP‐glucose and the misfolded portion of glyco‐substrate seat simultaneously. Upon well positioned in UGGT, the substrate will be precisely glucosylated. In the end, we identified the domain organizations of Penicillium chrysogenum UGGT through internal labeling strategy and single particle EM. Our model is a big step for uncovering mysteries of UGGT. POS210 Structural Basis for Substrate Sequence Specificity in Single Domain Human APOBEC3 Cytidine Deaminase Tania Silvas 1, Shurong Hou1, Nese KurtYilmaz1, Mohan Somasundaran1, Brian Kelch1, Celia Schiffer1 1University of Massachusetts Medical School, USA The APOBEC3 (A3) family of human cytidine deaminases is renowned for providing a first line of defense against many exogenous and endogenous retroviruses. Recently, however, it has been discovered that the ability of these proteins to deaminate cytidines in ssDNA has made A3s a double‐edged sword. When overexpressed, A3s can mutate endogenous genomic DNA resulting in a variety of cancers. Although the sequence context for mutating DNA varies among A3s has been known for some time, the mechanism for substrate sequence specificity is not well understood. To characterize A3's substrate specificity, a systematic approach was used to quantify affinity for substrate as a function of sequence context, pH, and substrate secondary structure. With our co‐crystal structure of A3 bound to ssDNA, we were able to develop a model explaining the molecular mechanism underlying A3 sequence specificity. Results of this work will not only provide key insights into the mechanism of A3's beneficial roles in the cell, especially in viral restriction, but also into A3's deleterious activity such as their role in the development of cancer. POS220 Elucidating the Mechanism of Lipid Binding Protein 8 Shuttling of Lysosomal Lipids into the Nucleus in Caenorhabditis Elegans Matthew Tillman 1, Meng Wang2, Eric Ortlund1 1Emory University, Georgia, USA, 2Baylor College of Medicine, Texas, USA Lipids not only play a vital role as an energy source and structural component in the cell, but also serve as signaling molecules. Many lipids have been identified as ligands for nuclear receptors, such as PPARs, to regulate transcription, however, it isn't understood how insoluble lipids derived from membranes and organelles are transported into the nucleus. Lipid binding proteins (LBPs) were discovered to solubilize lipids and transport them to the nucleus, serving an integral role in lipid‐signaling pathways. Lipid binding protein 8, LBP‐8, which is highly expressed in the fat storage tissue of Caenorhabditis elegans (C. elegans), was recently discovered to bind to lipids generated by lysosomal acid lipase (LIPL‐4) in lysosomes and shuttle them into the nucleus to serve as ligands for nuclear hormone receptor 49 and 80 (NHR‐49 and NHR‐80), homologs of PPARs, to prolong the lifespan of worms (Fig. 1). We recombinantly expressed LBP‐8 in Escherichia coli, purified, crystallized, and determined the first 1.3 Å high‐resolution structure of LBP‐8, which has allowed us to identify a structurally conserved nuclear localization signal and amino acids necessary for lipid binding. We have mutated these residues and confirmed a reduction in lipid binding to LBP‐8. We are currently transgenically expressing these mutant forms of LBP‐8 in C. elegans to observe effects on lifespan in worms in order to elucidate the mechanism in which LBP‐8 is shuttling lipids in the cell. POS226 Host Mimicry by Legionella Pneumophila Kathy Wong 1, Guennadi Kozlov1, John D. Perpich2, Miroslaw Cygler3, Yousef Abu Kwaik2, Kalle Gehring1 1McGill University, Canada, 2University of Louisville, USA, 3University of Saskatchewan, USA Ankyrin B (AnkB) is a bacterial protein that plays an essential role in the intracellular proliferation of Legionella pneumophila, the causative agent of Legionnaires’ disease. It collects proteins to target for degradation into free amino acids, generating a source of carbon and energy, and preventing a starvation response. AnkB contains two eukaryotic‐like domains, the combination of which have never been found in the same eukaryotic protein. The N‐terminal F‐box domain allows mimicry of host F‐box proteins for interaction with the host's ubiquitination pathway via Skp1 of the SCF (Skp1‐Cullin‐F‐box) E3 ubiquitin ligase complex. The C‐terminal ankyrin repeats allow AnkB to selectively bind targets for degradation. Here, we report the crystal structure of full length AnkB in complex with host Skp1. We found that AnkB contains an enlarged the substrate‐binding site with three ankyrin repeats rather than the two expected based on its sequence. Structural analyses and mutational studies have identified key residues responsible for decorating the Legionella‐containing vacuole with ubiquitin and for the replication of the pathogen during infection. Our study provides the first structural insights into the structural mimicry that allows the bacterial virulence factor, AnkB, to associate with the host ubiquitination complex and select substrates. POS232 Structural analysis of the tobramycin and gentamicin clinical resistome reveals limitations for next‐generation aminoglycoside design Angelia Bassenden 1, Albert Berghuis1, Dmitry Rodionov1, Shi Kun1 1McGill University, Montreal, Canada Since their discovery in the late 1920's, antibiotics have been extensively used and misused in humans and animal husbandry. Consequently, their effectiveness has waned as resistance has increased. Aminoglycosides are a broad‐spectrum class of antibiotics used in the treatment of infections caused by Gram‐negative and ‐positive bacteria. The most prevalent mode of aminoglycoside resistance is through covalent enzymatic modification. Two of the most medically important aminoglycosides, tobramycin and gentamicin, are targeted by several different resistance factors, including aminoglycoside 2”‐nucleotidyltrasferase [ANT(2”)]. Here we describe two crystal structures of one of the most widespread antibiotic resistance enzymes, ANT(2”) in complex with its nucleotide and two clinically relevant aminoglycosides, tobramycin or gentamicin. These structures complete the characterization of the medically important enzymes within the tobramycin and gentamicin resistome. Analysis of tobramycin and gentamicin binding to these resistome enzymes, as well as to their intended target, the ribosomal A‐site, reveals both extensive similarities and crucial differences. The observed differences in antibiotic binding interactions can, in principal, be exploited for the development of next‐generation aminoglycosides with selectively reduced affinities for resistome enzymes. We will present an analysis of viable avenues for next‐generation aminoglycoside antibiotic development based on the structural data for the tobramycin and gentamicin clinical resistome. POS236 Structural and mutational analysis of the nonribosomal peptide synthetase heterocyclization domain implicates a previously unexamined Asp‐Thr dyad in catalysis of the cyclodehydration reaction Kristjan Bloudoff 1, Martin Schmeing1, Christopher Fage2, Mohamed Marahiel2 1McGill University, Montreal, Canada, 2Philipps‐Universität Marburg, Germany Nonribosomal peptide synthetases (NRPSs) are a family of multimodular enzymes that synthesize structurally and functionally diverse peptides, many of which are interesting secondary metabolites. The central chemical step of peptide synthesis is amide bond formation, which is typically catalyzed by the condensation (C) domain. In some NRPSs, the C domain can be replaced by a heterocyclization (Cy) domain. The Cy domain performs amide bond formation as well as the intramolecular cyclization of serine, cysteine, or threonine sidechains, forming thiazoline, oxazoline or methyloxazoline rings. The rings are important for the form and function of the peptide product. We present the crystal structure of BmdB‐Cy2, an NRPS Cy domain, at a resolution of 2.3 Å. Despite sharing the same fold, the active sites of C and Cy domains have important differences. The structure allowed us to assess the roles of active site residues, which were subsequently probed in a bacillamide biosynthesis assay via mutational analyses. The drastically different effects, interpreted using our structural and bioinformatic results, provide insight into the catalytic mechanisms of the Cy domain, and suggest a critical role for a previously unexamined Asp‐Thr dyad in the cyclodehydration reaction. POS239 Developing Inhibitor Against Kinases Involved in Antibiotic Resistance Tolou Golkar 1, Albert Berghuis1 1McGill University, Montreal Canada The major mechanism of resistance in clinically relevant bacterial strains is through enzyme‐mediated alteration of antibiotics. Macrolide phosphotransferases I and II (MPH‐I and MPH‐II) are a group of enzymes that target macrolide antibiotics and inactivate them through the transfer of the α‐phosphate group of GTP. This research focuses on designing inhibitors against these two antibiotic resistance enzymes in order to restore antibiotic sensitivity. We used a 257‐compound library to identify 15 fragment binders using ligand‐based NMR methods—saturation transfer difference (STD) and WaterLOGSY (WL). Differential scanning fluorimetry (DSF) was used orthogonally to differentiate strong and weak binders from the 15 lead compounds. Competition assays were then used to preferentially select hits bound to the nucleotide‐binding pocket. The selected ligands were then subjected to isothermal titration calorimetry (ITC) studies to determine their binding affinities. Subsequently, these studies have established few hits that will be used for lead optimization. Structural studies of MPH‐I and MPH‐II can be utilized to establish the binding modes of these compounds in hopes of joining, merging or growing the initial hits into an effective inhibitor for these enzymes. POS242 Characterizing Proteins Using SAXS on a Hybrid Laboratory X‐ray Scattering Instrument Mike Hawkridge 1, Abraham Schierbeek1, Joerg Bolze1, Julie Quinn1 1PANalytical B.V., USA Small‐angle X‐ray scattering (SAXS) applied to protein solutions has become an accepted and rapidly growing structural biology technique. Measurements can be done under native conditions, while varying concentration, pH, ionic strength or temperature. The data provide information about molecular weight, size, shape and stability of the biomolecules and ultimately allow for a low resolution molecular shape envelope reconstruction. The information is complementary to that obtained from XRD, NMR or cryo‐EM. Although the setup for SAXS is easy in theory, it is in practice demanding with respect to the instrumentation and until recently it required dedicated, costly lab instruments or the usage of synchrotron beam lines, so the technique has not been readily available in the home laboratory. We recently developed an econmical solution that allows an easy configuring of a multi‐purpose XRD platform (Empyrean with ScatterX78, PANalytical) for protein SAXS measurements. The Empyrean is widely used in labs for general material research and characterization. Here we will show how this general XRD platform can be configured for SAXS experiments and demonstrate the performance on a number of proteins. POS257 New fast probing methodology for studying disordered protein regions using nuclear magnetic resonance spectroscopy Ivo Martins 1, Fábio Almeida2,3, Andre Faustino1, Glauce Barbosa2, Miguel Castanho1, Andrea Da Poian2, Nuno Santos1, Ivo Martins2 1Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Portugal, 2Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, RJ, Brazil, 3Instituto de Bioquímica Médica Leopoldo de Meis & Centro Nacional de Ressonância Magnética Nuclear, Universidade Federal do Rio de Janeiro and National Institute of Structural Biology and Bioimage, Rio de Janeiro, Brazil Understanding proteins structure and dynamic properties governing cellular processes is crucial for basic and applied research. Of particular interest are intrinsically disordered protein (IDPs) regions that display alternative transient conformations, enabling a multitude of functions. IDPs are particularly hard and time‐consuming to study by classical approaches developed for globular proteins with well‐defined conformations. New methods are required, given IDPs role in disease mechanisms. Here, employing dengue virus capsid protein (that possesses both structured and disordered regions), we developed a fast nuclear magnetic resonance (NMR) spectroscopy method to immediately determine N‐H groups’ solvent accessibility with amino acid residue resolution. The protein regions dynamics and the first residues of a‐helices are also readily determined. The method is based on minimal pH changes, using the well‐established 1H‐15N heteronuclear single quantum coherence (HSQC) pulse sequence in a simple to interpret manner. We use this technology to study and contrast the IDP regions of dengue virus capsid protein with its hydrophobic well‐structured section. This allowed gain insights into the flavivirus capsid protein biological function, of use not only for dengue but also for related flaviviruses, such as West‐Nile and Zika viruses. We show that dengue N‐terminal and specific residues within it, are key for the C protein biological activity, in line with previous findings. Therefore, with this approach we can complement other data. Importantly, the methodology is easily implemented in current protein NMR routines, providing structural and dynamics information in a fast manner, being of general interest for structural biology. POS263 Structural And Functional Studies Of Flavoenzymes Involved In Natural Product Biosynthesis Mahder Manenda 1, Julie Barma1, Marie‐Ève Picard1, Yaolong Chen2, Changsheng Zhang2, Rong Shi,1,3 1PROTEO, Université Laval, Canada, 2Chinese Academy of Sciences, China, 3IBIS, Université Laval, Canada Natural products have long been known to be sources of complex molecules. Most of these molecules exhibit medicinal benefits and have been developed into antimicrobial, anti‐tumor and anti‐viral drugs. Due to their versatility, flavin‐binding enzymes are ubiquitously found in numerous pathways that synthesize natural products of high interest. Examples include PieE, XiaI and Dbv29. PieE is involved the hydroxylation of the 3’ position of a piericidin pyridinol core. XiaI catalyzes the creation of the cyclic core structure of xiamycins. Dbv29 plays a catalytic role for a 4‐electron oxidation reaction on a hexose moiety necessary for glycopeptide maturation. In this project we have determined the structure of these enzymes using x‐ray crystallography. The enzymes belong to different classes of flavo‐proteins and structural insights from our study shed lights on their respective cofactor recruitment, cofactor/substrate binding and regio‐selective catalysis. These insights can contribute the potential use of such enzymes in semisynthetic strategies. POS264 Gathering Structural Information on a Depsipeptide Synthetase Initiation Module Diego A Alonzo 1, Clarisse Chiche‐Lapierre1, Diego A. Alonzo1, Clarisse E. Chiche‐Lapierre1, T. Martin Schmeing1 1McGill University, Montreal, Canada Cyclic depsipeptides are small bioactive molecules that consist of an alternating sequence of amino acids and hydroxyl acids. They are produced by massive enzymes called depsipeptide synthetases, which belong to the family of nonribosomal peptide synthetases. Depsipeptide synthetases employ up to sixty enzymatic reactions in a linear fashion to generate a single cyclic product. This work is focused on the first steps of the biosynthetic mechanism, which consist in the selection of alpha‐ketoacids and their further reduction to hydroxyl acids through the action of three protein domains: adenylation, ketoreduction and thiolation domains. In addition to catalyzing specific enzymatic reactions, these domains are also required to interact and communicate with each other for a successful synthetic cycle. To address how these interactions take place, we aim to obtain high‐resolution structures of a protein construct containing the three domains in several catalytic conformations. We were able to obtain crystals of an intact construct, which diffracted to a resolution of 4A. Furthermore, we evaluated the adenylation, thiolation and reduction activities of the construct through biochemical assays. Finally, we were able to perform intact protein LC‐MS analysis on this 150 kDa protein, and the data obtained has shed light onto several novel strategies to capture different depsipeptide synthetase conformations by X‐ray crystallography and improve the resolution of our current crystals. POS269 Towards The Structural Studies Of Bifunctional Aminoglycoside Resistance Enzyme AAC(6’)‐Ie‐APH(2’’)‐Ia Michal Zielinski 1, Albert Berghuis1 1McGill University, Montreal, Canada Since the introduction of aminoglycosides in 1940s, their extensive use in the clinic and agriculture has led to the selection of bacterial strains capable of evading the action of all aminoglycosides currently in use. Bacteria employ several mechanisms to confer aminoglycoside resistance, the most prevalent mode is through enzymatic modification of the antibiotic. AAC(6’)‐Ie‐APH(2’’)‐Ia is an enzyme capable of modifying virtually all aminoglycosides, and confers high levels of resistance in clinically relevant strains. Each domain is capable of actively modifying aminoglycosides, the AAC(6’)‐Ie domain by N‐acetylation and the APH(2”)‐Ia domain by O‐phosphorylation. Detailed structures exist for each of the domains separately, however the full‐length structure, despite extensive efforts is yet to be elucidated. This study describes the use of Pulsed Electron‐eLectron DOuble Resonance (PELDOR) technique in order to to obtain the orientation of two domains with respect to one another within AAC(6’)‐Ie‐APH(2’’)‐Ia. Observations obtained from this study may provide insight into this enzyme's bifuntional nature including: its mechanistic function, and potential conformational changes. POS270 Structural Study of Polyketide Fate Determination Frederik Teilfeldt Hansen 1, Graham Herberlig2, Yi Xiao Jiang1, Christopher Boddy2, T. Martin Schmeing1 1McGill University, Canada, 2University of Ottawa, Canada To survive in their challenging and complex environment, microbes rely on powerful bioactive compounds. Many of these compounds are synthesized using molecular machines called polyketide synthases (PKS), megaenzymes organized into repeating functional modules that work in an assembly line fashion. The repetitive assembly‐line synthesis in a PKS produces a full‐length polyketide attached to the transport domain of the PKS, the acyl carrier protein (ACP). The final step consists of the release of the polyketide by cyclization or hydrolysis, and is performed by an enzymatic domain of the PKS called a thioesterase (Te) domain. The action of the Te domain is extremely important, as hydrolysis produces a linear compound and cyclization produces a cyclic compound and the proper form of the compound is essential for its bioactivity. The cyclization vs hydrolysis decision depends on the individual Te domain and the nature of its presented substrate (polyketide‐ACP). The aim of this research is to elucidate the control mechanisms for the cyclization vs hydrolysis decision in fungal Te domains. Two Te domains, those found in the radicicol and dehydrocurvularin polyketide synthases, which differentially control cyclization and hydrolysis, have been heterologously expressed and purified. Protein crystals have been produced with the aim of achieving high resolution structures by x‐ray crystallography. These structures will provide enhanced knowledge of this crucial step in polyketide synthesis and could facilitate the ongoing effort to use PKSs for synthesis of novel designer chemicals. POS273 Legionella Effectors Interfering with Host Cell Phoshoproteome: Structural Insights into Host‐Pathogen Interactions Ksenia Beyrakhova 1, Miroslaw Cygler1, Alla Gagarinova1, Oleg Tyurin1, Caishuang Xu1, Lei Li1 1University of Saskatchewan, Canada Legionella pneumophila is a Gram‐negative pathogenic bacterium that causes severe pneumonia in humans. It establishes a replicative niche called Legionella containing vacuole (LCV) that allows bacteria to survive and replicate inside pulmonary macrophages. In order to hijack host cells defense systems Legionella injects more than 300 effector proteins into the host cell cytosol. Effector proteins interfere with signaling pathways and cause significant changes in host cell phosphoproteome. Here we aimed to decipher the structure and mechanisms of action of two effectors that directly interfere with cellular phosphorylation. LegK2, a bacterial protein kinase, is one of the several effectors that directly modify host cell phosphoproteome. We determined the structure of LegK2 non‐kinase domain and mapped its autophosphorylation sites. Moreover, we have identified a human protein that stimulates activity of LegK2. Based on the structural and functional data, we have found that another effector protein, Lem4, might have a potential to affect phosphorylation state of the host cell. Yeast two‐hybrid screening, MS‐based phosphoproteomics and in vitro phosphopeptide arrays were used to identify the interaction partners of LegK2 and Lem4, as well as phosphorylation sites modified by these effectors. POS324 NE‐CAT: Crystallography Beamlines for Challenging Structural Biology Research Kay Perry 1, Kanagalaghatta Rajashankar1, Malcolm Capel1, Igor Kourinov1, Anthony Lynch1, Frank Murphy1, David Neau1, Cynthia Salbego1, Jonathan Schuermann1 1NE‐CAT/Cornell University, New York, USA The NorthEastern Collaborative Access Team (NE‐CAT) focuses on the design and operation of synchrotron X‐ray beamlines for the solution of technically challenging structural biology problems and provides an important resource for the national and international research community. Currently, NE‐CAT operates two undulator beamlines: a 6 – 22 keV tunable energy beamline (24‐ID‐C) and a 12.662 keV single energy beamline (24‐ID‐E). Both beamlines are equipped with state‐of‐the‐art instrumentation. MD2 microdiffractometers installed at both beamlines provide very clean beams down to 5 microns in diameter and are capable of visualizing micron‐sized crystals. Large area pixel array detectors provide fast noiseless data collection and make possible it to resolve large unit cells. Both beamlines are equipped with custom‐built ALS‐style robotic sample automounters with dewars capable of holding 14 pucks. RAPD, our locally developed software suite RAPD provides data collection strategies, quasi‐real time data integration and scaling and simple automated MR/SAD pipeline through a 384‐core compute cluster. Users of the beamlines are supported 24/7 by experienced resident crystallographers. Funding for NE‐CAT is provided through P41 grant from the NIGMS and from the NE‐CAT member institutions. POS338 Structural Basis of Interactions Between PRL Phosphatases and CNNM Magnesium Transporters Guennadi Kozlov 1, Irina Gulerez1, Huizhi Zhang1, Howie Wu1, Kalle Gehring1 1McGill University, Montreal, Canada The phosphatases of regenerating liver (PRLs) are highly over‐expressed in metastatic cancers yet their mechanism of action is poorly understood. Recently, CNNM proteins, a family of membrane proteins involved in magnesium homeostasis, were identified as PRL‐binding partners. Disruption of the PRL‐CNNM interaction promotes tumor formation and invasiveness in animal and cellular models, strongly suggesting that the physiological function of PRLs is to regulate CNNM magnesium transport. Here, we determined crystal structures of PRL3 or PRL2 bound to the CBS‐pair domain of CNNM3. In the structures, the CBS‐pair domain is present as a dimer in the head‐to‐head arrangement that is typical for other CBS‐pair domains. The CNNM3 CBS‐pair domain contains a long loop that extends away from the dimerization interface and contacts the PRL catalytic site. The side chain of aspartic acid 426 sits in the pocket formed by the phosphatase P‐loop and WPFDD motif and likely mimics the negatively charged phosphate of a bound substrate. We used isothermal titration calorimetry (ITC) experiments and extensive mutagenesis to probe the importance of PRL residues for CNNM binding. Comparison of binding activity and in vitro phosphatase activity shows that they are strongly correlated. The results suggest that PRLs function as pseudophosphatases in regulating the action of CNNM proteins in cancer. POS348 Structural Insights Into the Replication Machinery of Menangle virus Melissa Webby 1, Richard Kingston1, Nicole Herr1, Jeremy Keown1, Michael Schmitz1, Esther Bulloch1 1University of Auckland, New Zealand Paramyxoviruses infect many organisms including mammals, birds, reptiles, and fish. Common human pathogens such as the measles, mumps, and parainfluenza viruses belong to this large family. Despite the prevalence of these viruses, structural understanding of the machinery responsible for RNA synthesis remains incomplete. We have therefore investigated the RNA‐dependent RNA polymerase (RdRP) of Menangle virus (MenV), a bat borne zoonotic pathogen, closely related to Mumps virus. Two protein subunits comprise the viral RdRP; these are known as the large protein (L protein) and the phosphoprotein (P protein). All catalytic functions are housed in the L protein, while the P protein acts as a chaperone, facilitating binding of the L protein to the replication template. The P protein is known to self‐associate through a centrally located coiled‐coil (Figure 1). Using a range of biophysical techniques we have shown that self‐association of MenV P protein forms a tetrameric species. Structural analysis of the P Protein using Nuclear magnetic resonance (NMR) spectroscopy, X‐ray crystallography, and Small Angle X‐ray scattering (SAXS) shows that structured domains within the C‐terminal region are connected by highly dynamic sequences. Conformational transitions within the tetrameric P protein are likely to be important for facilitating translocation of the viral polymerase during replication without dissociation from the template. POS349 Structural and Biophysical Investigations on Sigma4 Domain to Reveal How RNA Polymerase Holoenzyme is Recruited to PmrA Box Promoters Chinpan Chen 1, Yuan‐Chao Lou1, Yi‐Fen Kao1 1Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan PmrA is a two‐component response regulator that manages genes for polymyxin resistance through a phosphorylation‐dependent regulation. Recently, we reported the 3.2 Å resolution crystal structure of phosphoryl analog BeF3–activated PmrA in complex with the promoter DNA, revealing that activation of PmrA induces the formation of a two‐fold symmetric dimer in the N‐terminal receive domain (REC), promoting 2 C‐terminal DNA‐binding domains (DBDs) to recognize the PmrA box located at the −35 position of the promoter. NMR dynamics experiments suggested that the REC and DBD domains tumble separately and have diverse orientations, which together with the DBD‐DBD interface may facilitate PmrA searching best interactions with RNA polymerase holoenzyme (RNAPH) for transcription activation. The primary sigma factor, controls the transcription by directing RNAPH to promoters composed of “−10” and “−35” elements recognized by sigma2 and sigma4 domains, respectively. It is hence possible that PmrA transcription activation is achieved by the interactions between PmrA and the sigma4 domain in the promoters, where the −35 sigma70‐recognition element is replaced by the PmrA box. A sigma4 chimera protein by fusing it with the short beta‐flap tip helix through an artificial linker was designed to improve its solubility and stability. Solution structure of the sigma4 chimera shows that it adopts a similar conformation as within RNAPH. The interactions among the sigma4 chimera protein, the PmrA box promoter DNA and the BeF3–activated PmrA are investigated to reveal how RNAPH is recruited to PmrA box promoters by PmrA. POS375 Structure of a Complex Between the Autoantigen GAD65 and a Human Autoantibody Liah Clark 1, James S. Green1, Benjamin T. Porebski1, Nicholas J. McKenzie1, Blake T. Riley1, Itamar Kass1, Nathan P. Cowieson2, David E. Hoke1, Ashley M. Buckle1 1Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Australia, 2Australian Synchrotron, Australia The human neuroendocrine enzyme glutamate decarboxylase (GAD) catalyses the synthesis of the inhibitory neurotransmitter GABA, using pyridoxal‐5’‐phosphate as cofactor. GAD exists as two isoforms named according to their respective molecular weights, GAD65 and GAD67. GAD65, but not GAD67, is a prevalent autoantigen, with autoantibodies to GAD65 being detected at high frequency in patients with autoimmune (type 1) diabetes and certain other autoimmune disorders. Using Small Angle X‐ray Scattering, mutagenesis and computational methods we describe the structure of a complex between GAD65 and a recombinant Fab fragment derived from the human monoclonal antibody b96.11. The structure provides insights into how a patient‐derived autoantibody engages with a “disease‐associated” epitope, and ultimately the molecular determinants of GAD autoantigenicity. POS378 A Closed Conformation of the Catalytic Domain of PHA Synthase from Chromobacterium Sp. USM2 Min Fey Chek1, Sun‐Yong Kim1, Tomoyuki Mori1, Mohd. Razip Samian2, Kumar Sudesh2, Toshio Hakoshima1 1Nara Institute of Science and Technology (NAIST), Japan, 2Universiti Sains Malaysia (USM), Malaysia PHA synthase (PhaC) is the key enzyme in the polymerization of polyhydroxyalkanoates (PHAs), a biodegradable polymer produced by a wide range of bacteria. Here we report the crystal structure of the catalytic domain of PhaC from Chromobacterium sp. USM2 (PhaCcs‐CAT) at atomic resolution (1.48Å). The crystal structure contains two molecules, which forms a dimer with pseudo dyad symmetry. The catalytic domain adopts an a/ß hydrolase fold comprising an a/ß core subdomain and a CAP subdomain. The dimer interface contains two patches of contacting residues. The first displays hydrophobic contacts of five residues (Leu‐369, Trp‐371, Pro‐386, Phe‐387, and Leu‐390) from each molecule, close to the N‐terminal end. The second comprises two salt bridging (between Glu‐329 and Arg‐365) and two aromatic stacking (between His‐448 and Phe‐333) interactions, near to the catalytic H477. The catalytic triad (Cys‐291, His‐477 and Asp‐447) is covered by the CAP subdomain and facing to a water channel buried inside the protein. Comparison with the recently‐reported structures of the catalytic domain of PhaCcn (Cupriavidus necator) reveals a sharp contrast in the conformations of the CAP subdomains and the dimer arrangements: PhaCcn displays a relatively loosen conformation allowing a narrow path to the active site, while PhaCcs has a tighter closed conformation. Both structures imply that the ping‐pong mechanism is unlikely because the active sites are too distant apart. No clear product egress observed in either structure inferring dynamic conformational changes is essential for the catalytic action. In conclusion, the three‐dimensional structure of the catalytic domain PhaC provides invaluable clues for understanding the catalytic mechanism of this industrially important enzyme. POS387 Killer protein and L‐type calcium channels: using a novel L‐type calcium channel inhibitor to characterize L‐type calcium channel structure, function, and voltage dependence. Alexis Williams 1, Thomas Smith1 1University of Texas Medical Branch, USA Voltage‐gated calcium channels (VGCC) are critical for maintaining the highly regulated intracellular calcium ion concentration that ensures the propagation of cellular signaling cascades, synaptic transmission, and gene regulation. Long‐lasting VGCC, also known as L‐type, are ubiquitously expressed throughout the body and can be found in cardiomyocytes, smooth muscle, brain, pancreas, and adrenal glands. The canonical structure of L‐type VGCCs consists of a transmembrane pore domain (a1C) through which calcium enters the cell, an extracellular regulator domain (a2d) responsible for the trafficking the a1C to the cell surface and ensuring the channel is sensitive to physiological membrane potentials, and a cytosolic kinetic modulator (ß) that interacts with intracellular intermediate messenger proteins such as calmodulin. Multiple inhibitors of L‐type calcium channels have been discovered and are inhibitory either via direct interaction with the a1C pore domain or through interaction with the a2d domain to regulate channel trafficking and stability. All previously known channel inhibitors operate through increasing the necessary voltage required to depolarize and open the channel. Recently, a novel L‐type inhibitor, KP4, was discovered. KP4 is a virally encoded secreted protein derived from a persistent infection of the P4 strain of the fungus Ustilago maydis. KP4 inhibits calcium entry into cells without altering the voltage required to open the channel across kingdoms. The following proposed mechanism is that KP4 interacts with a specific region of the extracellular a2d subunit containing a divalent cation binding site responsible for promoting protein‐protein interaction. Characterization of this interaction will result in a better understanding of calcium channel structure, function, and voltage dependence. POS392 Energetics of sheath contraction in Contractile injection systems Alec Fraser 1, Petr Leiman1 1University of Texas Medical Branch, USA The process of protein and DNA translocation across lipid membranes is central to the function of any organism. A large class of large multicomponent organelles, such as bacteriophage tails, Type VI Secretion System, R‐type pyocins, Serratia antifeeding prophage, and others, translocate their substrates using a rigid tube/contractile sheath mechanism. Functionally and structurally, these ‘contractile injection systems’ resemble a stretched spring (or sheath) wound around a non‐contractile tube. The system is locked in a high‐energy metastable state by a baseplate structure that plays an important role in sheath assembly and contraction triggering. Upon interaction of the baseplate with a target cell membrane, the sheath contracts and drives the tube through the cell envelope. A full atomic model of the sheath and tube in the extended and contracted state is available for R‐type pyocin, one of the simplest representatives of contractile injection systems. The structure suggests that the contraction is accomplished by rigid body rearrangement of sheath subunits and that the transformation is driven by the energy stored in the extended conformation of the sheath during assembly. Other data show that the contraction starts at the baseplate and propagates through the sheath as a wave. The individual subunits’ trajectories are however unknown. We will discuss how we can derive these trajectories from energetics considerations. These finding are important for understanding the substrates that can be translocated by contractile injection systems. POS403 Structural Basis of Selective Inhibition of PKG‐Ia by a New Balanol Derivative, N46 Liying Qin 1, Choel Kim1, Ying‐Ju Sung2, Darren Casteel3 1Baylor College of Medicine, Texas, USA, 2Geisinger Commonwealth Medical College, Pennsylvania, USA, 3University of California, San Diego, USA PKG‐Ia is a central regulator of smooth muscle tone and nociception. Activating PKG‐Ia in nociceptive neurons induces a long‐term hyperexcitability that causes chronic pain. A recent study shows that a balanol‐like compound, N46, inhibits PKG‐Ia with high potency and selectivity and attenuates thermal hyperalgesia and osteoarthritic pain in rats. To understand the molecular basis of the high potency and selectivity of N46, we measured its inhibition constants for PKG‐Ia catalytic domain and determined the structure of their complex at 2.2 Å resolution. Our structure reveals that N46 binds the active site with its external phenyl ring specifically interacting with the glycine‐rich loop and the αB helix. Structural alignment with cAMP‐dependent protein kinase (PKA) Cα shows that Gly369 in the glycine‐rich loop is replaced with Ser53 in PKA‐Cα and this may cause steric hindrance with N46 reducing PKA‐N46 interaction. Ile405 on the αB helix of PKG‐Ia interacts with N46 while an analogous residue in PKA‐Cα, Thr88, may not. In summary, our structure explains high potency and selectivity of N46 for PKG‐Ia and provides a starting point for structure‐guided design of selective inhibitors. POS416 The Incredible Stability of Postfusion HCMV Glycoprotein B Ellen White 1, Yuhang Liu2, Senguil Han2, Ekaterina Heldwein1 1Tufts University School of Medicine, Massachusetts, USA, 2Pfizer Inc., USA Human cytomegalovirus (HCMV) infects the majority of the population worldwide and commonly causes lifelong latent infections in individuals with healthy immune systems. However, HCMV can cause serious illness in individuals with compromised immune systems or developing fetus. There is currently no vaccine for HCMV. To enter a host cell, HCMV utilizes several conserved glycoproteins expressed on the virion surface, including glycoprotein B (gB), the viral fusogen. gB must undergo a conformational rearrangement from a metastable prefusion form to a stable postfusion form bringing together the viral envelope and the host cell membrane for fusion to occur. The structure of postfusion gB has been determined but the prefusion form remains elusive. Knowing the gB structure in a prefusion conformation will allow us to better understand the HCMV entry process. We have engineered gB constructs designed to stabilize prefusion conformation(s) and/or destabilize the postfusion conformation. Negative‐stain electron microscopy (EM) and small angle x‐ray scattering (SAXS) were used to determine the conformations of the constructs. Our results show that despite various modifications, all constructs so far adopt the postfusion conformation demonstrating the incredible stability of postfusion gB. We have also found the use of SAXS to be critical for a more accurate observation of the gB conformation. POS419 Discovering Quality Drug Seeds by Practical NMR‐based Fragment Screening Yann Ayotte 1, Jayadeepa Rajamani Murugesan1, Francois Bilodeau2, Sacha Larda2, Patricia Bouchard2, Nathalie Drouin2, Mélissa Morin2, Steven LaPlante1 1INRS ‐ Institut Armand‐Frappier, Canada, 2NMX Research and Solutions Inc, Canada Fragment‐based screening represents a potential means for smaller institutions to meet the needs for identifying the seeds for future medications. Fragment‐Based Lead Discovery (FBLD) is becoming a viable complement and alternative to traditional high‐throughput screens for discovering the seeds of future drugs. FBLD involves the screening of libraries of fragments to search for binders to target proteins. These binders can then be used as chemical biology probes, functional modulators or scaffolds to custom design potent inhibitors. Central to FBLD is the quality of the screening library. FBLD is a validated strategy which has led to compounds in the clinic/market, but serious issues remain that limit its practical application.This work describes the practical processes employed in creating a new fragment library where a combination of theoretical cheminformatics and experimental NMR filters were employed to remove undesirable compounds (reactive, toxic, unstable, aggregators, etc.,) and to prioritize desirable compounds (3D dimensionality, biocores, solubility, substructures).These approaches include the introduction of sensitive and simplified NMR‐based screening methods, software that assists in the identification of fragment binders, and follow‐up strategies that help to filter out problematic/promiscuous ligands. Overall, screening time is reduced, deconvolution efforts are semi‐automated, and medicinal chemists can focus on more promising drug seeds. Using these stringent criteria, a starting set of ∼7000 compounds was reduced to an enriched subset of 1604 compounds. As an ensemble, this new library is distinct from most commercial libraries, and individual compounds are readily available along with their 1H NMR spectra in buffer. Furthermore, an intelligent pooling strategy is introduced that enables higher‐throughput screening. POS464 Structural Basis for Amino‐Acids Recognition and Transmembrane Signaling by Double Cache Sensing Domain of P. florescence Chemoreceptor CtaA Abu Iftiaf MD Salah Ud‐Din 1, Anna Roujeinikova1 1Infection and Immunity Program, Monash Biomedicine Discovery Institute, Department of Microbiology, Monash University, Australia Chemotaxis towards environmental cues mediated by membrane‐embedded chemoreceptors is a fundamental phenomenon that plays a major role in root‐tip colonization by Pseudomonas fluorescens. The P. fluorescens chemotactic transducer of amino acid type A (CtaA) mediates taxis toward naturally occurring amino acids. The periplasmic ligand binding domain (LBD) of CtaA belongs to the Cache superfamily. Structural analysis of CtaA LBD in complex with its cognate ligands that CtaA adopts a double Cache_1 domain with a typical N‐terminal long a‐helix and two subdomains (membrane‐ distal and proximal). The biophysical, structural and bioinformatics analysis of CtaA allowed us to identify crucial residues that comprise ligand binding pocket at the membrane distal subdomain. Isothermal titration calorimetry analysis showed that the binding affinity of these ligands are at the micromolar range. This study highlights the importance of confirmation changes of the phenolic side chain of two aromatic residues (Y101 and Y109) that contribute to determining the substrate specificity, and molecular basis of signal transduction mechanism of the receptor. POS481 Protein dynamics and conformational disease: multi‐timescale characterisation of Alpha‐1‐Antitrypsin by NMR Alistair Jagger 1, Christopher Waudby1, Lisa Cabrita1, John Christodoulou1, James Irving1, David Lomas1 1University College London, UK Alpha‐1‐antitrypsin (a1AT) is a 52 kDa serine protease inhibitor found at high concentrations in human plasma. The Z mutation (Glu342Lys) occurs in 1 in 1700 Northern Europeans and causes the formation of long polymer chains that are retained at the site of synthesis in hepatocytes. Accumulation of polymers leads to liver cirrhosis and the reduced anti‐protease activity in the lung predisposes individuals to early onset emphysema. The rational design of therapeutics for the treatment of a1AT deficiency requires a detailed understanding of the polymerisation pathway. Polymerisation is thought to progress from a native, monomeric species of an unknown conformation. A recent crystal structure of this Z mutant revealed few differences to the wild‐type variant, suggesting that changes in aggregation behaviour are due to differences in structural dynamics between variants. To address this we have used solution state Nuclear Magnetic Resonance (NMR) together with biophysical and biochemical approaches to characterise the structure, dynamics and polymerisation of a1AT. In particular, methyl‐TROSY‐based NMR observations of [2H, 13CH3–ILV] labelled a1AT have allowed us to study dynamics in a1AT across a range of time scales, from ps to ms. Additionally, we show that it is possible to acquire high quality 1H‐13C NMR correlation spectra of patient‐derived WT and Z‐a1AT at natural isotopic abundance. These data begin to probe the impact of mammalian post‐translational modifications on the structure of a1AT and the structural and dynamic consequences of the Z mutation, and open up new possibilities for the rapid structural characterisation of other clinically relevant variants. POS485 Crystal Structure Of Acid Ceramidase Ahmad Gebai 1, Bhushan Nagar1, Alexei Gorelik1, Katalin Illes1 1McGill University, Montreal, Canada Acid ceramidase (aCDase) catalyzes the conversion of lysosomal membrane resident ceramide into sphingosine, the backbone of all other sphingolipids, to regulate diverse cellular processes. Because of its essential housekeeping role, abnormal function of aCDase lead to pathologies like Farber's disease and spinal muscular atrophy with progressive myoclonic epilepsy. Increased activity of aCDase or accumulation or ceramide have been linked to other diseases such as Alzheimer's disease, type 2 diabetes and cancer, making it an attractive target for therapeutic intervention. We present the first crystal structures of mammalian aCDases in both the proenzyme inactive form and the autocleaved active form. In the inactive form, the catalytic center is protected from solvent and slightly perturbed, which likely slows the autocleavage reaction but still allows it to take place under optimal conditions. Autocleavage of an internal peptide bond stably aligns the active site residues for catalysis and concomitantly creates a surface exposed binding pocket, allowing for entry of ceramide substrates. An unusually large hydrophobic surface adjacent to the active site suggests that it is the site of lysosomal membrane attachment where it can be positioned for optimal substrate delivery by the accessory protein, saposin D. Finally, mapping of Farber disease mutations onto the structure reveals that most variants likely destabilize the protein fold resulting in inactivation and disease onset. Our structural analysis clarifies the molecular mechanism of aCDase function and will inform the development of rationally designed aCDase inhibitors and the use of recombinant aCDase as disease therapeutics. POS501 Structural basis for identification of the interface of the N‐terminal domain of CoV N protein dimer as a target for drug development Ming‐Hon Hou 1, Chai‐Ning Hsu1 1Institute of Biotechnology and Institute of Genomics and Bioinformatics, National Chung Hsing University, Taiwan The nucleocapsid protein (N protein) of coronavirus (CoV) is essential for RNA‐binding in human cells and is required for the replication and transcription of viral RNA. Recent studies have suggested that human CoV N protein is a valid target for antiviral drug development. Recently, we demonstrated the crystal structure of the N‐terminal domain of MERS‐CoV N protein (N‐NTD) as a dimer. The dimer conformation was confirmed further in solution by cross‐link assay. The current study utilized structure‐based virtual screening against the interface of two MERS‐CoV N‐NTD dimers available in the Zinc database, through docking with varying precisions and computational intensities to identify several potential compounds. We also determined crystal structures of the N‐NTD‐ligand complex. Although the dimer interface site might not be directly involved in RNA binding, the binding of small molecule compounds to this site could promote oligomerization of the N protein through allostery, and could be alternative targets for antiviral drug development. Our findings provide a better insight into the development of new therapeutics that could potentially affect the interactions between two individual N‐NTDs in the Human CoV. The discovery of a new compound that could bind to the interface of the N‐NTD dimers would be imperative to design novel N protein inhibitors against human CoV in the future. POS515 Solution structure of the Arc capsid like domain Kaare Teilum 1, Lau Dalby Nielsen1, Simon Erlendsson1 1University of Copenhagen, Denmark Maintaining and controlling homeostatic plasticity and synaptic scaling require rapid synthesis and mobilization of receptors and other proteins involved in the underlying regulatory functions. Arc is an immediate early gene expressed directly in the dendritic compartments upon neuronal activation, and has apparent functions in regulating pathways controlling LTD and LTP in a bi‐directional manner. Arc displays sequential and partial structural similarity to retroviral gag proteins belonging to the retrotransposon Ty3/Gypsy family. Here we have determined the solution structure of the two‐lobe C‐terminal capsid like domain of Arc. The relative arrangement of the two lobes is similar to that seen for several other members of the Ty3/Gypsy family including the HIV‐1 capsid protein. We show that Arc at elevated temperatures transiently undergoes a structural transition to a well‐defined oligomeric state. Finally, Arc interacts with other synaptic proteins and we have extended the characterization of the ligand binding site and the consensus recognition motif and demonstrate binding to both the GluN2A and the GluN2B subunits of the NMDA receptor. POS523 High‐Pressure NMR Studies of HIF‐2/Ligand Interactions: A View of Small Molecule Regulation via Binding to Protein Cavities Donald Gagne 1, Kevin H. Gardner1, Donald Gagne1, James M. Aramini1, Bruce A. Johnson1, Kevin H. Gardner1 1CUNY ‐ Advanced Science Research Center, New York, USA Hypoxia Inducible Factors (HIFs) are heterodimers composed of pairs of regulated (HIF‐alpha) and constitutive (ARNT) subunits, both of which are bHLH/PAS (basic Helix‐Loop‐Helix/Period‐ARNT‐Single minded) transcriptional regulators. While HIFs are essential for normal physiological responses to low oxygen levels in higher eukaryotes, misregulation of HIF abundance or activity correlates with the onset and progression of several cancers. Protein/protein interactions between HIFs and ARNT, as well as various downstream components, are important in the proper function and dysfunction of the pathway. Small molecule inhibitors of such interactions can serve as potential new anticancer therapies, as we previously demonstrated by the discovery of nanomolar affinity artificial ligands which bind to a large 290 Å3 hydrated cavity buried within the HIF‐2alpha PAS‐B domain. To address the question of how small molecules access this cavity and affect HIF function, we used biophysical and biochemical approaches to examine the linkage between protein dynamics and internal ligand binding. Current data using high‐pressure NMR and CPMG (Carr‐Purcell‐Meiboom‐Gill) relaxation dispersion experiments suggest how small molecules can access the internal, regulatory cavity through rapid protein dynamics. We propose that the presence of the ligand within the cavity perturbs the beta‐sheet surface, leading to a destabilization of its interaction with ARNT. A better understanding of how protein and ligand interact and regulate protein function is critical to deciphering the root causes of disease and developing novel research tools and pharmacological leads. POS533 Structural insights into the catalytic mechanism of Phenolic acid decarboxylase Marie‐Ève Picard 1, Rong Shi1 1Université Laval, Canada Phenolic acid decarboxylase (PAD) is an enzyme present in bacteria, notably Bacillus pumilus. This protein is characterized by a ten strand beta‐barrel adopting a lipocalin‐like fold flanked with three alpha helices. This fold is mainly known for binding small hydrophobic molecules. PAD shares a high similarity with the Ferulic Acid Decarboxylase (FAD), where both are involved in detoxification processes through the decarboxylation of small aromatic molecules such as ferulic, p‐coumaric and caffeic acids. In organic chemistry, decarboxylation is an important process for the elimination of carboxylic acids, mostly achieved through the use of progressive heating up to 200 degree C. While this process is simple, high temperature may be detrimental for more complex molecules. Achieving decarboxylation through biocatalysis is thus an interesting goal. For example, this was previously fulfilled for one substrate, the sinapic acid [Green Chem., 2013, 15, 3312] using an engineered PAD protein. Further development of a larger variety of substrates relies on an ultimate understanding of the enzymatic mechanism. Here, we report the structure of PAD mutant with an analogue of ferulic acid present in the catalytic site. Interestingly, its binding mode in the pocket is different from those previously reported in the literature. Based on these results, we hereby suggest an alternative mechanism for the decarboxylation reaction by these enzymes. 22. SYNTHETIC BIOLOGY POS108 A Bacterial Bandpass Assay for Protein‐Protein Interactions Katherine Brechun 1,2, Andrew Woolley1, Katja Arndt2 1University of Toronto, Canada, 2Universität Potsdam, Germany We have developed an E. coli bandpass assay for protein‐protein interactions that can be used to discover proteins with tuned interaction strength. The assay is based on a genetic circuit that links protein‐protein interaction strength to beta‐lactamase activity. Interactions that are too weak are selected against because they do not show resistance to a given concentration of ampicillin. Interactions that are too strong lead to excessive beta‐lactamase activity, which causes repression of an essential gene for growth. This combined positive and negative selection pressure limits cell growth to a concentration range of ampicillin where both types of selection pressure are satisfied by the beta‐lactamase activity level. In turn, the beta‐lactamase activity level is dictated by the interaction strength of the expressed protein pair, resulting in a bandpass indicating the strength of the protein‐protein interaction. The function of this assay was demonstrated by analyzing a series of synthetic coiled‐coil interaction partners. The bandpass function enabled pure cultures to be separated from a mixture of cells expressing different coiled‐coils, based on the interaction strength of each protein pair. This system can be applied in the development of protein inhibitors by co‐expressing an inhibitor that impedes the interaction of the protein pair in the system. This was demonstrated by analyzing a series of inhibitors targeting the bZIP (basic leucine zipper) proteins CREB and AP‐1. It is expected that this bandpass assay will facilitate the evaluation of inhibitor libraries designed to develop inhibitors with tuned interaction strength. POS142 Design of Optogenetic Tools for the Control of Protein Synthesis Huixin (Lulu) Lu 1, Andrew Woolley1, Mostafizur Mazumder1, Anil Kumar1, Xiuling Xu1, Anna Jaikaran1 1University of Toronto, Canada The precise spatiotemporal regulation of protein synthesis is essential for many cellular processes including synaptic firing, embryonic development and tumour formation. The traditional methods used to study protein synthesis offer only crude spatiotemporal control of protein expression, limited to timescales of days or weeks. Optogenetic methods, in contrast, could control protein synthesis non‐invasively within minutes and with a spatial scale as small as a single synapse. Here, we present the development of new optogenetic tools for the down‐regulation of protein synthesis: fusions of a circularly permutated LOV2 from Avena sativa with varying lengths of human 4E‐BP2 (opto‐4EBP2), an inhibitor of translation. To identify active constructs, we designed a yeast growth assay to screen structure‐based designs in vivo. In the screen, opto‐4EBP2 slowed the growth of yeast that expressed human eIF4E (a key translation initiation factor inhibited by 4EBP2) under blue light but not in the dark. With a non‐binding opto‐4EBP2, growth was restored to wild‐type levels. In vitro studies using SEC showed that light‐state opto‐4EBP2 bound eIF4E under blue light, while dark‐state opto‐4EBP2 did not bind eIF4E. This binding was reversible and repeatable. Using the yeast growth assay, libraries and structure‐based designs of opto‐4EBP2 were screened for increased light‐dark differences in activity. Several candidates showed stronger inhibition under low levels of blue light. Thus, we have developed a new set of optogenetic tools, opto‐4EBP2s, which down‐regulate protein synthesis under blue light. These optogenetic tools will advance the study of protein synthesis, for example in neurodevelopmental disorders or embryo morphogenesis. POS228 Development of Small‐Molecule‐Binding Synthetic Notch Receptors Remy Peace 1, John Ngo1 1Boston University, Massachusetts, USA Notch is a cell‐surface receptor that facilitates cell‐to‐cell signaling through a mechanism requiring mechanical force. The receptor is activated upon trans‐cellular binding to target ligands, which induces to successive proteolytic cleavages to liberate an intracellular transcriptional effector such that it can translocate to the nucleus. Here we describe the development of a set of synthetic Notch (“SynNotch”) receptors that have been engineered to bind and recognize small molecules, including fluorescein and derivatives of O6‐benzylguanine. We show that these engineered receptors are proteolytically cleaved upon binding to surface‐immobilized target ligands, and that receptor activation leads to the induction of a specified target gene by over 100‐fold. Overall these systems will serve as powerful probes for studying the natural mechanisms of Notch activation, but in addition will serve as powerful synthetic biology tools for re‐programming sensing capabilities of mammalian cells. In future work we will apply these receptors to detect specific cell types labelled with cell‐surface binding ligands. POS231 Designer Biosensors for Engineered Metabolic Pathways and Enzyme Evolution Mohamed Nasr 1, David Kwan1, Vincent Martin1 1Centre for Applied Synthetic Biology, Department of Biology, Concordia University, Canada Synthetic biology techniques aimed at constructing artificial metabolic pathways in genetically modified microorganisms are emerging as important sustainable methods for the production of biofuels, pharmaceuticals and commodity chemicals. To reach industrially relevant scales however, challenges related to bottlenecks and system optimization must be addressed. Directed evolution offers a solution to these limitations, yet the lack of high‐throughput detection methods for the products of these reactions remains a disadvantage. The purpose of this work is to utilize transcriptional factor‐based biosensors, particularly from the TetR family of repressors, to link the production of these substances to a signal such as fluorescence or antibiotic resistance. Transcriptional repressors are proteins that regulate genes by binding specific effector molecules, and conditionally binding to DNA. This project aims at expanding the toolbox of repressors available by rationally engineering their effector‐binding domains to respond to alternative effector molecules. As a proof of principle, using a combined computational and directed evolution approach, we will engineer biosensors from these proteins to respond to intermediates of an engineered metabolic pathway to adipic acid that has been derived from the shikimate pathway. Adipic acid is a precursor of nylon and plastics and is currently produced unsustainably from petrochemicals, with worldwide annual demands of over 2 million tonnes. In optimizing a biorenewable alternative for the production of adipic acid, our “designer” biosensors will be used as parts within genetic circuits for pathway dynamic control and as tools for the directed evolution of pathway enzymes to ultimately improve yields. POS248 Inducible gene expression control using CRISPR/dCas9 and antiviral protease inhibitors Elliot Tague 1, John Ngo1 1Boston University, Massachusetts, USA Drug inducible gene expression has been widely used to study and control biological functions—however, many of the drugs used in presently available systems possess endogenous cellular targets, which can cause undesirable side‐effects that make them incompatible for use in therapeutic applications. With the increasing prospect of the use of gene modulation in human therapies (i.e., gene therapy, cell‐based therapies, etc.), orthogonal drug‐inducible systems that use safe ligand molecules will be needed. Here we present a novel method for drug‐inducible gene expression control using existing (FDA‐approved) anti‐viral drug compounds that are able to bind and inhibit the cis‐proteolytic activity of the Hepatitis C virus (HCV) protease NS3/4a. We show that the protease can be used to render an artificial transcription factor based on dCas9 subject to drug control via insertion of the viral enzyme between the dCas9 scaffold and a C‐terminal transactivation domain. In the absence of drug, the protease serves as a self‐immolating linker that leads to dismemberment of the chimera. Upon exposure to drug, intact copies of the protein are able to enter the nucleus to activate the expression of sgRNA‐specified target genes. Overall, these results demonstrate the versatility of using the HCV NS3/4a domain as a drug‐sensitive module for regulating the activity and localization of engineered transcription factors. POS259 Function And Engineering of Enzymes Involved in The Glycosylation of Natural Products Fathima Mohideen 1, David Kwan1 1Department of Biology, Centre for Applied Synthetic Biology, Concordia University, Canada Many pharmaceutically active small molecule natural products contain sugar moieties that play an important role in their bioactivity. An example of one class of such molecules is the anthracyclines which include the anticancer doxorubicin. These natural product glycosides are biosynthesized by action of glycosyltransferases (GTs). To modify or improve the bioactivity of these molecules by altering glycosylation, in vitro enzymatic methods could circumvent multistep, labor‐intensive routes in organic synthesis. This aim is facilitated by screening and engineering GTs to produce modified glycosides. Thus, I have developed a high‐throughput screen for assaying GTs enabled by rapid isolation and detection of chromophoric or fluorescent glycosylated natural products. This will be a valuable tool for discovering and engineering GTs through directed evolution. Epirubicin, a semisynthetic derivative of doxorubicin, is a high value anticancer drug with fewer side effects than its parent. It is conventionally produced by replacement of the sugar moiety of doxorubicin through several organic synthetic steps. In genetically modified bacteria, engineered biosynthesis has been demonstrated to produce limited amounts of epirubicin. Low yields from these efforts are likely due to poor activity of exogenous enzymes in this artificial biosynthetic pathway. To address this, we will engineer improved GT enzymes by directed evolution. Towards this aim, using a novel in vitro enzymatic synthesis we have produced modified sugar donor substrates for the GT‐catalyzed synthesis of anthracyclines. Our resulting library of sugar donors will be used in high‐throughput screens to engineer GTs by directed evolution, including those for the production of epirubicin. POS292 Engineering the Mechanical Sensitivity of the Notch Receptor David Sloas 1, John Ngo1 1Boston University, Massachusetts, USA Cells are able to sense mechanical cues via transmembrane receptors that can directly affect changes in gene expression. One such receptor is the Notch protein, which regulates a variety of cell fate decisions and is activated through a mechanism involving mechanical force. Notch receptors contain a “Negative Regulatory Region (NRR),” which serves as a force‐activated mechanical switch to regulate the localization of the Notch intracellular domain (NICD), a transcriptional effector that is cleaved from the plasma membrane and transported to the nucleus upon receptor activation. In the receptor's resting state, the NRR adopts an autoinhibited conformation in which the activating cleavage site (S2) is sterically blocked; the receptor is activated through force‐mediated unraveling of the NRR to reveal the S2 site, which occurs in response to = 5 pN of pulling force. Our goal is to use Notch as a scaffold to engineer new mechanoreceptors that respond to varying degrees of force. In an initial design strategy, we have integrated NRR‐binding domains into the receptor such that they stabilize the NRR and increase the force required to expose S2. We present data demonstrating that these chimeric receptors exhibit increased force resistivity in cell‐based assays. Furthermore, we show these receptors can be used to program cells with the ability to discriminate between ligands bound to stiff versus soft surfaces. We also show that the NRR‐binding domains are susceptible to engineering for tunable mechanical sensitivity. In future work, our mechanoreceptors will be invaluable in studying how proteins are able to transmit information regarding mechanical properties in their microenvironment to the nucleus. POS371 A synthetic two‐component system redirects oncogenic signaling to therapeutic outputs Hokyung Kay Chung 1, Michael Lin1 1Stanford University, California, USA Many cancers are driven by constitutively active signaling that promote cell growth, proliferation, or survival. Pharmacological approaches aim at blocking aberrant signaling often suffer from resistance or narrow therapeutic window. Here, we present a novel approach where signals driving oncogenesis are instead co‐opted to trigger therapeutic responses via rewiring by synthetic signal transduction pathways. This system queries whether a specific oncogenic signal exists to selectively trigger a therapeutic program. For this purpose, we conceived the idea of oncogenic signal‐induced proteolysis, where inhibitory motif‐tethered effectors are liberated upon activation. We term this general approach as rewiring of aberrant signaling to effector release (RASER). In this study, we describe the engineering and application of a compact two‐component system to sense constitutive ErbB phosphorylation and trigger therapeutic responses. Modular sensing and actuation domains of the RASER system allow facile optimization of the sensing and versatile programming of therapeutic outputs. Mathematical modeling of the entire system enables in silico optimization of several biochemical parameters to further enhance system responsivity. The resulting system, ErbB‐RASER responds specifically to constitutively active ErbB, is as sensitive to constitutive ErbB signaling as native growth‐ and survival‐promoting pathways, and can be programmed to induce a variety of outputs including direct induction of apoptosis and transcription of apoptosis‐inducing genes. These results represent, to our knowledge, the first successful attempt to use computational modeling to design synthetic pathways in mammalian cells for therapeutic effects. The RASER system should be generalizable to various cancers by customizing sensor‐actuator modules to specific oncogenic signals, and holds potential as a novel synthetic approach for cancer treatment. 23. SYSTEMS BIOLOGY POS241 Functional CRIPSR Screen Identifies Regulator Networks of Met RTK Bruce Huang 1,2, Morag Park1,2, Elena Kuzmin2, Genevieve Morin1,2, Sidong Huang1 1Department of Biochemistry, McGill University, Canada, 2Goodman Cancer Research Centre, McGill University, Canada Activation of the Met receptor tyrosine kinase (RTK) is linked to tumor growth, survival, metastasis and drug resistance, resulting in poor patient prognosis. Met overexpression triggers ligand‐independent self‐activation, most commonly caused by transcriptional upregulation of Met. However, the mechanism of Met regulation remains unclear. Using phenotypic selection by fluorescence activated cell sorting (FACS) coupled with a functional genetic screen using a pooled CRISPR library, we have identified bona fide modulators of the Met RTK. We use the Met expressing colorectal cancer cell line, DLD‐1, sorts into two sub‐populations based on Met intensity. DLD‐1 cells were infected with a pooled CRISPR library and CRISPR targets in each population identified using next generation sequencing. Model‐based Analysis of Genome‐wide CRISPR‐Cas9 Knockout (MAGeCK) was then used to rank genes by comparing the relative abundance of Met‐low and Met‐high population of each CRISPR target. The top ranked genes cause the strongest shift of cells towards a Met‐low or Met‐high population. Our results identify both essential proteins for transcription, translation and peptide processing, as well as novel regulators including transcription factors and components of E3 ubiquitin kinase complex. We conclude that the combination of a pooled CRISPR library and FACS is a robust tool to identify regulators of Met protein abundance. Identification of novel regulators of Met may lead to novel targets and companion diagnostics in several cancers. 24. THERAPEUTICS AND ANTIBODIES POS011 Generation of Allosteric Chaperones to Treat G6PD (Glucose‐6‐Phosphate Dehydrogenase) Deficiency Sunhee Hwang 1 1Stanford University School Of Medicine, California, USA Glucose‐6‐phosphate dehydrogenase (G6PD) catalyzes the first step of the pentose phosphate pathway, in which reduced NADPH (nicotinamide adenine dinucleotide phosphate) is generated. NADPH is used to maintain the reduced state of glutathione (GSH), which plays a critical role in regulating antioxidant balance and thus protecting cells from oxidative damage. Particularly, erythrocytes, which lack mitochondria, rely on G6PD for the generation of antioxidants. G6PD deficiency, caused by a loss of enzymatic activity and structural integrity due to point mutations in G6PD, disrupts the physiological antioxidant balance with significant decreases in NADPH and GSH levels, and thus increases the vulnerability of cells to oxidative stress. Currently there are no treatments available for G6PD deficiency. Given that G6PD deficiency can lead to hemolytic crisis and following sequalae, there is a pressing need to develop a therapeutic plan correcting G6PD deficiency. Towards this end, we characterized the most common G6PD mutant enzyme, Canton G6PD (R459L), by X‐ray crystallography. We identified structurally distorted areas in the enzyme leading to the decreased enzyme activity. Using this enzyme, we screened over 100,000 molecules for chaperones (activators) and identified a potential molecule (referred to as AG1 hereafter) that activated the enzyme by up to 2‐fold and significantly increased the enzyme's stability in cells. AG1 activated other G6PD mutant enzymes as well, suggesting that it can be a general treatment for G6PD deficiency. Furthermore, AG1 alleviated loss‐of‐function phenotypes in a G6PD‐deficient zebrafish model. Taken together, our study provides novel insights into developing a therapeutic strategy to G6PD deficiency that reduces G6PD deficiency‐associated pathologies that affect 7–8% of world population. POS085 Effects of Antibodies on Tau Phosphorylation and Tubulin Polymerization Sanela Martic 1 1Oakland University, Michigan, USA Tau protein regulates neuronal cell function. The post‐translational modifications of tau lead to microtubule instability, cell death, and neurodegeneration. Currently, neurodegeneration remains without a cure, but immunotherapies in animal models have shown reduction or clearance of tau pathology. The mechanism of antibody‐based inhibition is currently unclear. Our main objective is to determine the role of anti‐tau antibodies to nonphosphorylated and phosphorylated tau on phosphorylation of tau441 and tubulin polymerization. In vitro phosphorylation of tau441 was carried out in the presence of glycogen synthase kinase or microtubule‐affinity regulating kinase. Specific antibodies were added during phosphorylation to evaluate their role on phosphorylation at Ser199, Thr231 and Ser262 residues. The extent of phosphosphorylation was determined using Western blot. Tubulin polymerization into microtubules was measured using fluorescence spectroscopy. Tau, antibodies or tau/antibodies were introduced prior to polymerization of tubulin. Antibodies targeting R4 domain of tau441 induced a “turn‐on” phosphorylation by microtubule‐affinity regulating kinase. “Turn‐off” phosphorylation was observed with antibodies to pThr231 and glycogen synthase kinase. The tubulin polymerization into microtubules was inhibited by all antibodies tested. However, tau effectively rescued microtubules even in the presence of antibodies. By a substrate epitope‐targeting, the phosphorylation and protein‐protein interactions may be regulated. POS116 Co‐Crystal Structure of Tubulin with PF‐06380101, a Novel Auristatin Analogue with Improved Cell Potencies Alison Varghese 1, Kevin Parris1, Jayvarthan Pandit1, Suman Shanker1, Cynthia Song1, Andreas Madernas1 1Pfizer, Inc., USA Antibody‐drug conjugates (ADC's) combine the potency of a highly cytotoxic small molecule (payload) conjugated to a highly specific monoclonal antibody (mAb) resulting in cancer therapy that targets the diseased cells while leaving healthy cells intact. To date, there are two FDA‐approved ADC's on the market (Kadcyla® and Adcetris®) and over 30 are currently in clinical trials. Many payloads in development are antimitotic agents that disable mitosis in addition to altering the cytoskeletal structure leading to cell death. One series of the most potent antimitotic agents, auristatins, act by inhibiting the polymerization of tubulin. Protein crystal structures of tubulin bound to auristatins have previously been reported at 3.8 Å resolution with disordered residues at the C‐terminus. Here, we present the crystallization and structure of tubulin bound to a novel auristatin, PF‐06380101, at a significantly improved 3.1 Å resolution providing a more detailed examination of auristatin in its preferred binding mode. Along with excellent potency and improved ADME properties, this structural information serves as a tool to enable the design and development of safer, effective, ADC's for chemotherapeutic use. POS149 Antibody fragment production in Pichia pastoris with highly attenuated O‐glycosylation patterns and without addition of pure O2 Alexandre Di Paolo 1, Nathalie Pirlot1, Laurent Jost1, Rudi Piedboeuf1, Jean Gudas2, David T. Ho2, Green Zhang2 1Kaneka Eurogentec, Belgium, 2ImaginAb, USA Kaneka Eurogentec has developed an efficient Pichia pastoris expression platform for the production of antibody fragments. The proteins are produced in high titers (up to 1 g/L, single copy clones) without addition of pure oxygen during fermentation and the products show very limited O‐glycosylation patterns. In the course of this study we have developed antibody fragment production conditions (Eurogentec) and tested binding properties (ImaginAb) of various types of fragments (Fab, cys‐diabodies, minibodies) derived from an anti‐IAb20 antibody (“H8, Oxford Biomedica PLC”) that has been humanized and engineered for imaging and therapeutic purposes. In total 5 diabodies, 1 minibody and 1 Fab were studied. Here we present the fermentation and O‐glycosylation optimization results obtained for two cys‐diabodies, namely IAb20C3 and IAb20C4. These cys‐diabodies not only display a low level of O‐glycosylation but also share identical binding properties as their equivalent produced in mammalian cells. POS201 Dissecting the catalytic fragment of Pseudomonas exotoxin A John Weldon 1, Earl Brooks1, Kaleem Coleman1, Victor Eromosele1, Olubunmi Olakunle1, Kavisha Schroff1, Alec Ahearn1, Jack Sanford1, Rodrigo Montoro1 1Towson University, Maryland, USA Pseudomonas exotoxin A (PE) is a bacterial toxin that halts protein synthesis in eukaryotes and archaea by inhibiting the action of translation elongation factor 2 (EF2). Mature native PE has functional domains responsible for receptor binding, intracellular trafficking, and catalysis. The catalytic fragment of the toxin is a mono‐ADP‐ribosyltransferase that exerts its toxic effect in the cytosol by attaching an ADP‐ribosyl group, derived from NAD, to a specific residue in EF2. PE has been adapted for use as a cancer therapeutic through the development of recombinant immunotoxins (RITs), fusion proteins produced by combining antibodies with protein toxins. RITs show promise as treatments for cancers, but suffer from several limitations that include patient immunogenicity, nonspecific toxicity against untargeted cells, and inefficient cell killing of some cell types. In an effort to improve the therapeutic potential of RITs, we are studying the catalytic domain of PE to determine its minimum essential sequence. Using a series of deletion and substitution mutations in the catalytic domain of PE, we are purifying and studying the structure and function of these variants and observing the changes that occur. This work was performed primarily by students as part of the Biochemistry Lab course at Towson University and was designed to give them experience in common biochemistry laboratory techniques. POS336 Improving the Stability of a Bovine Secretory IgA Nanobody by Rational Design of the Fragment Crystallisable Chain Adam Chin‐Fatt 1, Rima Menassa2 Western University1, Agriculture and Agri‐Food Canada2 The secretory IgA nanobody is a modular molecule comprising two highly ordered constant domains, collectively termed the fragment crystallisable (Fc), that are significantly more conserved than their fused antigen binding partner, the variable heavy chain fragment (VHH), which comprises a spectrum of sequences depending on their corresponding binding partners. Considering that the Fc may confer stability to its various VHH partners, rational design of the Fc was enabled by bioinformatic analysis and molecular modeling to predict key amino acid substitutions that may induce the formation of either surface salt bridges or disulfide bonds. These predicted stabilizing mutations were then enabled by site directed mutagenesis of the native bovine Fc sequence and screened for protein accumulation and thermostability following transient expression into leaves of Nicotiana benthamiana. Our results so far have identified seven candidates that show better accumulation and enhanced thermostability. To determine if these seven Fc mutants can confer their enhanced stability to the whole secretory IgA molecule, they are currently being fused to a variable heavy chain (VHH) that binds Clostridium difficile and will be co‐expressed with the joining chain, that binds together VHH‐Fc dimer pairs, and the secretory component, that wraps around the joined tetramer. The goal of this project is to develop a stabilized bovine Fc chain that could potentially be a generic stabilizing scaffold for various VHH's and the corresponding secretory IgA. POS365 Monoclonal antibodies specifically targeting amyloidogenic forms of transthyretin (TTR) with potential to treat TTR‐related cardiomyopathy and polyneuropathy Natalie J. Galant 1, Natalie J. Galant1, Jeffrey N. Higaki1, Punam Ghosh1, Kevin C. Hadley1, Stephen J. Tam2, Ken Flanagan2, Tarlochan Nijjar2, Ronald Torres2 1University Health Network, University of Toronto, Canada, 2Prothena Biosciences Inc., USA Transthyretin (TTR, or prealbumin) is an abundant serum protein which normally forms soluble, stable homotetrameric complexes. Point mutations and unknown pathological conditions can favour the dissociation of the TTR tetramer into non‐native monomers. These monomers aggregate and accumulate as amyloid throughout the body, particularly in the heart and peripheral nerves. This deposition of TTR amyloid (ATTR) in cardiac tissue and nerves results in the development of cardiomyopathy and polyneuropathy, respectively. We have recently developed conformation‐specific polyclonal and monoclonal antibodies (mAbs) which can potentially treat both of these diseases via their ability to specifically recognize and bind to the disease‐associated forms of TTR via a cryptotope (an epitope normally buried and inaccessible in the native protein, but exposed in its altered conformation). These mAbs were demonstrated in vitro to specifically binding to misfolded TTR, inhibit fibril formation, induce phagocytic clearance of non‐native and aggregated TTR, and immunoreact with TTR amyloid in diseased heart tissue (Galant et al., 2016; Higaki et al., Amyloid, 2016). We further investigated the mechanism of mAb‐mediated inhibition of fibrillogenesis using immunogold transmission electron microscopy (TEM). This high resolution imaging technique has confirmed the cryptotope as an effective mAb target due to its exposure within both pathological TTR misfolding intermediates and end‐point insoluble TTR fibrils. These results further support the use of monoclonal antibodies to target pathological protein conformations as potentially effective immunotherapies for ATTR amyloidosis. Online video poster: http://tinyurl.com/llqq6kn References: Galant, N.J., et al. (2016). Scientific Reports. 6, 25080: 1–11. Higaki, J.N., et al. (2016). Amyloid. 23(2): 86–97. POS459 NIH funding opportunities and resources Aaron Pawlyk 1 1 NIDDK/NIH, USA This poster will highlight current funding opportunities and resources available at the NIDDK and the NIH as a whole. Research resources available to the scientific community will also be highlighted. This poster will explain the science supported by the NIDDK, especially as it pertains to protein structure, function, and signaling. Funding opportunities such as R01s, large collaborative projects, biomarkers, and therapeutics discovery and development will be presented. NIH resources, and additional opportunities, such as the Nuclear Receptor Signaling Atlas, Diabetic Complications Consortium, NIDDK Central Repository, Diabetes Research Centers, Illuminating the Druggable Genome, and KO Mouse Phenotyping project will be discussed. NIH Program Staff will be available at the poster to discuss these opportunities and resources. POS460 Inhibition of Protein Expressions (5‐Lipoxygenase) by Zileuton in Cisplatin‐Induced Renal Toxicity Salim Alrejaie 1, Osama Alkhamees2 1KSU, Saudi Arabi, 2Al Imam University, Saudi Arabia Despite its well documented effectiveness as an anticancer therapy, cisplatin (CPT) exerts several unwanted dose‐related adverse effects including nephrotoxicity, ototoxicity, hepatotoxicity and allergic reactions. Present study was to evaluate the protein expressions of lipoxygenase (LOX) signaling following CPT challenge and the possible preservative role of the 5‐LOX inhibitor, Zileuton (ZT), in renal tissue of Wistar rats. Animals were challenged with CPT (7.5 mg/kg IP), while treated with ZT (25 mg/kg/day PO) for 3weeks, starting 2 weeks before CPT challenge. The protein expressions of LOX signaling enzymes and products were quantified by western blot including LOX enzymes, 5‐LOX activating protein (FLAP), leukotriene‐A4 (LT‐A4) hydrolase, LT‐C4 synthase, LTB4 and cysteinyl (cys) LTs receptors type 1 and 2 in kidney. CPT significantly provoked the expression of LOX signaling, while 5‐LOX inhibition by ZT attenuated these alterations and improved the expressions of LOX enzymes and products. In conclusion, this primary study indicates the contribution of LOX signaling in CPT mediated‐nephrotoxicity. Inhibition of 5‐LOX by ZT could establish a novel therapeutic approach. POS487 Drug Delivery System for Anti‐cancer Drug Paclitaxel Using Human Lipocalin‐type Prostaglandin D Synthase Kosuke Furuta 1, Masatoshi Nakatsuji1, Yoshiaki Teraoka1, Yuya Sano1, Takashi Inui1 1Graduate School of Life & Environmental Sciences, Osaka Prefecture University, Japan Paclitaxel (PTX) is one of the most effective anti‐cancer agents for treating various cancers including breast cancer. However, the clinical use of PTX is limited by its poor solubility in aqueous solutions. Hence, the clinical formulation of PTX (Taxol) contains Cremophor EL® to improve the solubility of PTX, which causes serious side effects such as hypersensitivity reactions, neurotoxicity, and nephrotoxicity. We previously reported that lipocalin‐type prostaglandin D synthase (L‐PGDS) could bind to and solubilize 7‐ethyl‐10‐hydroxy‐camptothecin (SN‐38), a poorly water‐soluble anti‐cancer drug, and SN‐38/L‐PGDS complex showed high anti‐tumor activity in vitro and in vivo. In this study, we attempted to develop a safe drug delivery system for PTX using L‐PGDS. To estimate the binding capability of L‐PGDS to PTX, we performed docking simulations using AutoDock Vina. In the docking model, the PTX molecule was located into a hydrophobic cavity of L‐PGDS with the predicted free energy change of 58.2 kJ/mol. Next, we investigated the effect of L‐PGDS on the solubility of PTX. PTX was insoluble in 5 mM Tris‐HCl buffer (pH 8.0), while it was solubilized to be 290 ± 42 µM by 1 mM L‐PGDS solution. Then, we investigated the cytotoxic activity of PTX/L‐PGDS complex against human breast cancer cell lines MDA‐MB‐231, and MDA‐MB‐468 by the WST‐8 assay. The IC50 values of PTX/L‐PGDS complex on the growth of MDA‐MB‐231 and MDA‐MB‐468 cells were 8.1 ± 0.64 and 6.4 ± 0.47 nM, respectively, indicating that PTX/L‐PGDS complex had high anti‐cancer activity. These results, taken together, demonstrated that L‐PGDS is a suitable drug delivery vehicle for PTX. POS524 New Concepts for Designing Novel Multi‐Functional Anti‐Sickling Hemoglobins for Gene Therapy of Sickle Cell Disease Savita Bhutoria 1, Craig Branch2, Seetharama A Acharya1 1Division of Hematology, Department of Medicine and of Physiology and Biophysics, Albert Einstein College of Medicine, USA, 2Gruss Magnetic Resonance and Imaging Center, Albert Einstein College of Medicine, USA The sickle cell disease (SCD) is a monogenic hereditary disease. Gene therapy of SCD by introducing anti‐sickling Hb inside the RBC is considered the most promising approach to curing the disease. The globin chains chosen for gene therapy at present include the mutant ß‐chains or α‐chains that inhibit polymerization from trans‐dimer location. Even though these do not inhibit the polymerization completely it has been suggested that these increase the delay time of polymerization enough to afford therapeutic benefits. However, our recent studies in transgenic mouse models have demonstrated that 15–20% of anti‐sickling Hbs considered adequate is not sufficient to normalize the high cerebral blood flow (CBF) present in SCD. Besides we have seen that at least some of the SCD therapeutic approaches induce oxygen debt in the brain by reducing CBF. On the other hand an anti‐anemia therapeutic, high oxygen affinity PEG‐Hb, afford good SCD therapeutic activity and protect the brain against oxygen debt and normalize CBF. These new insights into the cerebral pathology of the SCD have prompted us to advance a combination of anti‐sickling and anti‐anemia therapy against SCD. Anti‐anemia therapeutics generally have higher nitrite reductase activity and thus can be vaso‐dilatory. Based on our experimental and structure modeling results, here we suggest that swine, human‐swine chimeric and canine a‐chains that completely neutralize the polymerization, exerting the activity from both cis‐ and trans‐dimer locations with anti‐sickling, anti‐anemia and anti‐ischemic activities. The therapeutic activity of these a‐chains can be synergized with α‐chains to further enhance the therapeutic activities and protect against possible a‐thalassemia. 25. TRANSCRIPTION/TRANSLATION/POST‐TRANSLATIONAL MODIFICATIONS POS244 Investigating resistance to Pseudomonas exotoxin A in diphthamide‐deficient mutants of HEK293 cells Benjamin Atha 1, John Weldon1, Jack Sanford1, Lauren Russell1 1Towson University, Maryland, USA Elongation factor 2 (EF2), is a GTPase required during translation for the translocation of ribosomes along mRNA. EF2 contains a unique post‐translational modification, called diphthamide, not found on any other protein. Diphthamide is a single post‐translationally modified histidine (His‐715 in mammals), and is a central target for several bacterial toxins. Diphtheria toxin, Pseudomonas exotoxin A (PE), and cholix toxin can all arrest EF2 and inhibit translation by transferring an ADP‐ribosyl group from NAD to the EF2 diphthamide residue. These toxins have been utilized in antibody‐toxin conjugated cancer therapeutics termed recombinant immunotoxins. We have investigated PE resistance in HEK293 cells expressing EF2 with mutations at the diphthamide histidine. PE resistance was determined by treating cells with the PE‐based recombinant immunotoxin HB21. Preliminary data indicate that none of the mutants tested have toxin resistance. Future experiments will explore toxin resistance to endogenous His‐715 substitution mutations using CRISPR/Cas9‐mediated genome editing, and will evaluate additional cell lines. POS431 The structural basis for Parkin‐mediated mitochondrial quality control Marta Vranas 1, Jean‐François Trempe1 1McGill University, Montreal, Canada Mutations in the Parkin and PINK1 genes cause familial forms of Parkinson's disease (PD). Parkin and PINK1 work together in mitochondrial quality control pathway essential to prevent neurodegeneration. The kinase PINK1 senses damaged mitochondria by accumulating at depolarized membranes and phosphorylating ubiquitin. Phospho‐ubiquitin (pUb) then recruits and activates the E3 ubiquitin ligase Parkin. Parkin ubiquitinates outer mitochondrial membrane proteins, marking them for proteasomal degradation and recruiting the autophagy machinery. We along with others have previously shown that Parkin adopts an auto‐inhibited conformation (Trempe et al. 2013). The release of inhibition is initiated by pUb binding to the RING1 domain of Parkin, which allosterically displaces its Ubl domain (Sauvé et al., 2015). This promotes phosphorylation of the Ubl at Ser65 by PINK1 and increases ubiquitin ligase activity. However, the molecular mechanisms underlying the conformational changes and substrate specificity on mitochondria remain unclear. Here, we dissect the mechanism of Parkin activation through a combination of biophysical measurements, mitochondrial ubiquitination and cellular mitophagy assays. Mutation of Trp403, which anchors the Repressor Element of Parkin, rescues the phospho‐dead mutant S65A, indicating that S65 phosphorylation releases the REP and enables E2‐binding (Tang & Vranas et al., 2017). After transthiolation of ubiquitin from E2 to Parkin, effective and specific substrate ubiquitination is orchestrated by His433. These experiments pave the way for novel therapeutic approaches that could restore activity of impaired Parkin or PINK1. POS449 Structural and Functional Insight into the Epigenetic Regulator SMCHD1 Kelan Chen 1, Richard Birkinshaw1, Alexandra Gurzau1, Jarrod Sandow1, Peter Czabotar1, Renwick Dobson2, Grant Pearce2, Isabelle Lucet1, Marnie Blewitt1 1The Walter and Eliza Hall Institute of Medical Research, Australia, 2University of Canterbury, New Zealand Structural Maintenance of Chromosomes flexible Hinge Domain‐containing 1 (Smchd1) is a non‐canonical SMC protein that plays critical roles in epigenetic regulation including X chromosome inactivation, genomic imprinting and regulation of autosomal gene expression. Recently, mutations in SMCHD1 have been implicated in facioscapulohumeral muscular dystrophy (FSHD) and a rare craniofacial disorder called Bosma arhinia microphthalmia syndrome (BAM). While the importance of SMCHD1 is well‐described, how SMCHD1 protein functions at the molecular level to mediate epigenetic control is still unclear. We have performed structural‐functional characterisation of the two recognisable domains of Smchd1, namely the SMC hinge domain that is responsible for nucleic acid binding and the putative GHKL ATPase domain. We demonstrated that the hinge domain of Smchd1 assembles into an unconventional dimeric arrangement flanked by intermolecular coiled‐coils. We solved the crystal structure of the core hinge domain and investigated the structural basis for nucleic acid interaction. Furthermore, we showed the N‐terminal region of Smchd1 that encapsulates the ATPase domain grossly resembles the crystal structure of full‐length Hsp90 protein. Importantly, we found the ATPase domain of Smchd1 is catalytically active. Therefore, similar to Hsp90's ATP‐binding dependent conformational changes, we envisage that Smchd1 dimer may undergo energy‐dependent conformational changes to engage with chromatin. Additionally, ongoing characterisation of recombinant proteins incorporating patient‐derived SMCHD1 mutations have provided potential explanations for the underlying pathogenesis. Our study has provided important insight into understanding how SMCHD1 protein elicits epigenetic control at the molecular level and formed the basis of exploring activation of SMCHD1 as a potential therapeutic treatment for FSHD. POS520 Improvement relative quantitation of the a‐ and ß‐ aspartic acid isoform of amyloid‐ß peptide using MALDI‐CID‐TOF/TOF MS Igor Popov1, Stanislav Pekov1, Maria Indeykina2, Alexey Kononikhin1, Roman Levin1, Ilyas Khaliullin1, Evgeny Nikolaev1 1Moscow Institute of Physics and Technology, Russia, 2Emanuel Institute of Biochemical Physics, Russia The a‐aspartic acid isomerization to ß‐amino acid is well‐known peptide modification. Isoaspartate containing proteins playing a significant role in aging processes and could lose their structure becoming biologically inactive or even harmful. Thus high sensitive method of MALDI‐TOF MS could be applied for determination a‐ and ß‐ isoforms ratio of aspartic acid in peptides. Amyloid‐ß peptide contains ß‐Asp7 could be noted as a biomarker for Alzheimer's disease diagnostic and amyloid‐ß peptide is a good model system for improvement isoaspartic acid detection method. Experiments were performed on model systems containing normal and ß‐Asp7 isoform of synthetic peptide using Bruker UltrafleXtreme mass spectrometer was used and fragmentation spectra were obtained using collision‐induced dissociation with different collision gases. The binary mixtures of synthetic 1–16 amyloid‐ß fragments were investigated and fragmentation spectra were analyzed to find marker ions for normal and isoform of target peptide and their relative intensities ratios. It is shown that fragmentation spectra have differences in intensities of fragments formed during dissociation bonds near aspartic/isoaspartic acid group. A correlation between relative intensities of marker ions and isoform percentage in mixture allows to determine amount of ßAsp7‐containing peptide referred to normal one. MALDI‐TOF measurements allows to use small amounts of sample or less concentrate sample without additional sample preparation steps and is shown that detection limit is below 1 nmol and could be improved through concentration of sample directly on target plate. This simple method could also being adapted to other peptides and proteins prone to aspartic acid isomerization processes. The work was supported by the Russian Science Foundation grant no. 16‐14‐00181. POS531 Discovering Histone Deacetylase Substrates Using the HDAC Toolbox Katherine Leng 1, Carol Fierke1, Noah Wolfson1, Carol Ann Castañeda1, Eric Sullivan1, Jeffrey Lopez1 1University of Michigan, USA Lysine acetylation is an important, post‐translational modification found ubiquitously throughout the cell. Lysine acetylation occurs on thousands of human nuclear and non‐nuclear proteins and regulates a wide variety of dynamic cellular processes. Acetylation erasers or histone deacetylases (HDACs) are attractive therapeutic targets due to their aberrant activity in diseases ranging from cancer to neurodegenerative disorders. HDACs comprise a family of 18 enzymes, and while most HDACs have been well‐studied and their crystal structures solved, little is known about the division of deacetylation roles among the isozymes. We have created an HDAC toolbox containing a variety of methodologies that, when used in combination, allow us to unravel the complexity of HDAC substrate specificity. The toolbox includes computational substrate predictions, in vitro peptide and protein deacetylation assays, protein library deacetylation screening, HDAC‐interactor photocrosslinking in cell lysate, and in vivo acetylation analysis after HDAC knockout. We have used HDAC8, the simplest and best‐studied HDAC, to develop the toolbox, but the toolbox is compatible with all HDACs. We have identified several dozen novel, putative HDAC8 substrates including heat shock protein 90 beta (Hsp90) and isocitrate dehydrogenase 1 (IDH1) as well as previously identified HDAC8 substrates such as structural maintenance of chromosomes 3 (SMC3). We have used these methods to gain a better understanding of HDAC8 substrate specificity. Moreover, we have identified potentially physiologically relevant HDAC8 substrates that can inform rational drug design and allow for more effective drug development.