10
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

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

      Read this article at

          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          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.

          Related collections

          Most cited references65

          • Record: found
          • Abstract: found
          • Article: not found

          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.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            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.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              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.
                Bookmark

                Author and article information

                Contributors
                (View ORCID Profile)
                (View ORCID Profile)
                Journal
                Communications Chemistry
                Commun Chem
                Springer Science and Business Media LLC
                2399-3669
                December 2020
                February 12 2020
                December 2020
                : 3
                : 1
                Article
                10.1038/s42004-020-0267-4
                08da3dc9-d0f7-4cd4-beea-2f7ab18545b0
                © 2020

                https://creativecommons.org/licenses/by/4.0

                https://creativecommons.org/licenses/by/4.0

                History

                Comments

                Comment on this article

                scite_
                0
                0
                0
                0
                Smart Citations
                0
                0
                0
                0
                Citing PublicationsSupportingMentioningContrasting
                View Citations

                See how this article has been cited at scite.ai

                scite shows how a scientific paper has been cited by providing the context of the citation, a classification describing whether it supports, mentions, or contrasts the cited claim, and a label indicating in which section the citation was made.

                Similar content86

                Cited by6

                Most referenced authors1,232