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      Simultaneous sensing and imaging of individual biomolecular complexes enabled by modular DNA–protein coupling

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          Abstract

          Many proteins form dynamic complexes with DNA, RNA, and other proteins, which often involves protein conformational changes that are key to function. Yet, methods to probe these critical dynamics are scarce. Here we combine optical tweezers with fluorescence imaging to simultaneously monitor the conformation of individual proteins and their binding to partner proteins. Central is a protein–DNA coupling strategy, which uses exonuclease digestion and partial re-synthesis to generate DNA overhangs of different lengths, and ligation to oligo-labeled proteins. It provides up to 40 times higher coupling yields than existing protocols and enables new fluorescence-tweezers assays, which require particularly long and strong DNA handles. We demonstrate the approach by detecting the emission of a tethered fluorescent protein and of a molecular chaperone (trigger factor) complexed with its client. We conjecture that our strategy will be an important tool to study conformational dynamics within larger biomolecular complexes.

          Abstract

          The folding and conformational dynamics of proteins can be studied using optical tweezers with the aid of DNA handles. Here this assay is extended to simultaneously visualize the binding of complexing partners while monitoring the induced conformational changes on the protein.

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          Molecular chaperones in protein folding and proteostasis.

          Most proteins must fold into defined three-dimensional structures to gain functional activity. But in the cellular environment, newly synthesized proteins are at great risk of aberrant folding and aggregation, potentially forming toxic species. To avoid these dangers, cells invest in a complex network of molecular chaperones, which use ingenious mechanisms to prevent aggregation and promote efficient folding. Because protein molecules are highly dynamic, constant chaperone surveillance is required to ensure protein homeostasis (proteostasis). Recent advances suggest that an age-related decline in proteostasis capacity allows the manifestation of various protein-aggregation diseases, including Alzheimer's disease and Parkinson's disease. Interventions in these and numerous other pathological states may spring from a detailed understanding of the pathways underlying proteome maintenance.
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            Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin.

            Protein interactions with peptides generally have low thermodynamic and mechanical stability. Streptococcus pyogenes fibronectin-binding protein FbaB contains a domain with a spontaneous isopeptide bond between Lys and Asp. By splitting this domain and rational engineering of the fragments, we obtained a peptide (SpyTag) which formed an amide bond to its protein partner (SpyCatcher) in minutes. Reaction occurred in high yield simply upon mixing and amidst diverse conditions of pH, temperature, and buffer. SpyTag could be fused at either terminus or internally and reacted specifically at the mammalian cell surface. Peptide binding was not reversed by boiling or competing peptide. Single-molecule dynamic force spectroscopy showed that SpyTag did not separate from SpyCatcher until the force exceeded 1 nN, where covalent bonds snap. The robust reaction conditions and irreversible linkage of SpyTag shed light on spontaneous isopeptide bond formation and should provide a targetable lock in cells and a stable module for new protein architectures.
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              Flexible nets. The roles of intrinsic disorder in protein interaction networks.

              Proteins participate in complex sets of interactions that represent the mechanistic foundation for much of the physiology and function of the cell. These protein-protein interactions are organized into exquisitely complex networks. The architecture of protein-protein interaction networks was recently proposed to be scale-free, with most of the proteins having only one or two connections but with relatively fewer 'hubs' possessing tens, hundreds or more links. The high level of hub connectivity must somehow be reflected in protein structure. What structural quality of hub proteins enables them to interact with large numbers of diverse targets? One possibility would be to employ binding regions that have the ability to bind multiple, structurally diverse partners. This trait can be imparted by the incorporation of intrinsic disorder in one or both partners. To illustrate the value of such contributions, this review examines the roles of intrinsic disorder in protein network architecture. We show that there are three general ways that intrinsic disorder can contribute: First, intrinsic disorder can serve as the structural basis for hub protein promiscuity; secondly, intrinsically disordered proteins can bind to structured hub proteins; and thirdly, intrinsic disorder can provide flexible linkers between functional domains with the linkers enabling mechanisms that facilitate binding diversity. An important research direction will be to determine what fraction of protein-protein interaction in regulatory networks relies on intrinsic disorder.
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                Author and article information

                Contributors
                s.tans@amolf.nl
                Journal
                Commun Chem
                Commun Chem
                Communications Chemistry
                Nature Publishing Group UK (London )
                2399-3669
                12 February 2020
                12 February 2020
                2020
                : 3
                : 20
                Affiliations
                [1 ]GRID grid.417889.b, ISNI 0000 0004 0646 2441, AMOLF, ; Amsterdam, 1098XG The Netherlands
                [2 ]GRID grid.5292.c, ISNI 0000 0001 2097 4740, Department of Bionanoscience, Kavli Institute of Nanoscience Delft, , Delft University of Technology, ; Van der Maasweg 9, 2629 HZ Delft, The Netherlands
                [3 ]GRID grid.5335.0, ISNI 0000000121885934, Present Address: Cambridge Centre for Proteomics, , University of Cambridge, ; Cambridge, CB2 1QR UK
                Author information
                http://orcid.org/0000-0001-6406-524X
                http://orcid.org/0000-0002-2985-622X
                Article
                267
                10.1038/s42004-020-0267-4
                9814868
                36703346
                08da3dc9-d0f7-4cd4-beea-2f7ab18545b0
                © The Author(s) 2020

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 25 June 2019
                : 17 January 2020
                Funding
                Funded by: FundRef https://doi.org/10.13039/501100003246, Nederlandse Organisatie voor Wetenschappelijk Onderzoek (Netherlands Organisation for Scientific Research);
                Categories
                Article
                Custom metadata
                © The Author(s) 2020

                chaperones,biophysical chemistry,proteins,dna
                chaperones, biophysical chemistry, proteins, dna

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