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      Acidic C-terminal domains autoregulate the RNA chaperone Hfq

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          Abstract

          The RNA chaperone Hfq is an Sm protein that facilitates base pairing between bacterial small RNAs (sRNAs) and mRNAs involved in stress response and pathogenesis. Hfq possesses an intrinsically disordered C-terminal domain (CTD) that may tune the function of the Sm domain in different organisms. In Escherichia coli, the Hfq CTD increases kinetic competition between sRNAs and recycles Hfq from the sRNA-mRNA duplex. Here, de novo Rosetta modeling and competitive binding experiments show that the acidic tip of the E. coli Hfq CTD transiently binds the basic Sm core residues necessary for RNA annealing. The CTD tip competes against non-specific RNA binding, facilitates dsRNA release, and prevents indiscriminate DNA aggregation, suggesting that this acidic peptide mimics nucleic acid to auto-regulate RNA binding to the Sm ring. The mechanism of CTD auto-inhibition predicts the chaperone function of Hfq in bacterial genera and illuminates how Sm proteins may evolve new functions.

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          Scalable web services for the PSIPRED Protein Analysis Workbench

          Here, we present the new UCL Bioinformatics Group’s PSIPRED Protein Analysis Workbench. The Workbench unites all of our previously available analysis methods into a single web-based framework. The new web portal provides a greatly streamlined user interface with a number of new features to allow users to better explore their results. We offer a number of additional services to enable computationally scalable execution of our prediction methods; these include SOAP and XML-RPC web server access and new HADOOP packages. All software and services are available via the UCL Bioinformatics Group website at http://bioinf.cs.ucl.ac.uk/.
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            Detergent binding explains anomalous SDS-PAGE migration of membrane proteins.

            Migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) that does not correlate with formula molecular weights, termed "gel shifting," appears to be common for membrane proteins but has yet to be conclusively explained. In the present work, we investigate the anomalous gel mobility of helical membrane proteins using a library of wild-type and mutant helix-loop-helix ("hairpin") sequences derived from transmembrane segments 3 and 4 of the human cystic fibrosis transmembrane conductance regulator (CFTR), including disease-phenotypic residue substitutions. We find that these hairpins migrate at rates of -10% to +30% vs. their actual formula weights on SDS-PAGE and load detergent at ratios ranging from 3.4-10 g SDS/g protein. We additionally demonstrate that mutant gel shifts strongly correlate with changes in hairpin SDS loading capacity (R(2) = 0.8), and with hairpin helicity (R(2) = 0.9), indicating that gel shift behavior originates in altered detergent binding. In some cases, this differential solvation by SDS may result from replacing protein-detergent contacts with protein-protein contacts, implying that detergent binding and folding are intimately linked. The CF-phenotypic V232D mutant included in our library may thus disrupt CFTR function via altered protein-lipid interactions. The observed interdependence between hairpin migration, SDS aggregation number, and conformation additionally suggests that detergent binding may provide a rapid and economical screen for identifying membrane proteins with robust tertiary and/or quaternary structures.
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              Folding-unfolding transitions in single titin molecules characterized with laser tweezers.

              Titin, a giant filamentous polypeptide, is believed to play a fundamental role in maintaining sarcomeric structural integrity and developing what is known as passive force in muscle. Measurements of the force required to stretch a single molecule revealed that titin behaves as a highly nonlinear entropic spring. The molecule unfolds in a high-force transition beginning at 20 to 30 piconewtons and refolds in a low-force transition at approximately 2.5 piconewtons. A fraction of the molecule (5 to 40 percent) remains permanently unfolded, behaving as a wormlike chain with a persistence length (a measure of the chain's bending rigidity) of 20 angstroms. Force hysteresis arises from a difference between the unfolding and refolding kinetics of the molecule relative to the stretch and release rates in the experiments, respectively. Scaling the molecular data up to sarcomeric dimensions reproduced many features of the passive force versus extension curve of muscle fibers.
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                Author and article information

                Contributors
                Role: Reviewing Editor
                Journal
                eLife
                Elife
                eLife
                eLife
                eLife Sciences Publications, Ltd
                2050-084X
                09 August 2017
                2017
                : 6
                : e27049
                Affiliations
                [1 ]deptCell, Molecular and Developmental Biology and Biophysics Program Johns Hopkins University BaltimoreUnited States
                [2 ]deptProgram in Molecular Biophysics Johns Hopkins University BaltimoreUnited States
                [3 ]deptDepartment of Chemical and Biomolecular Engineering Johns Hopkins University BaltimoreUnited States
                [4 ]deptT.C. Jenkins Department of Biophysics Johns Hopkins University BaltimoreUnited States
                University of Cambridge United Kingdom
                University of Cambridge United Kingdom
                Author information
                http://orcid.org/0000-0001-9615-065X
                http://orcid.org/0000-0003-4249-1955
                http://orcid.org/0000-0001-6380-2324
                http://orcid.org/0000-0003-0170-1987
                Article
                27049
                10.7554/eLife.27049
                5606850
                28826489
                acf5d615-3afd-4ce3-b0af-6b10702e4bd2
                © 2017, Santiago-Frangos et al

                This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

                History
                : 22 March 2017
                : 03 August 2017
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/100000057, National Institute of General Medical Sciences;
                Award ID: R01 GM120425-01
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000057, National Institute of General Medical Sciences;
                Award ID: R01 GM078221
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000057, National Institute of General Medical Sciences;
                Award ID: T32 GM008403-25
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000057, National Institute of General Medical Sciences;
                Award ID: T32 GM007231-40
                Award Recipient :
                The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
                Categories
                Research Article
                Biophysics and Structural Biology
                Custom metadata
                Modeling and biophysics show that the unstructured acidic tail of the Sm protein Hfq mimics nucleic acid to auto inhibit its chaperone activity, preventing Hfq from being sequestered by inauthentic substrates and providing insight into the evolution of Hfq's chaperone function among bacterial genera.

                Life sciences
                rna chaperone,small non-coding rna,sm protein,intrinsically disordered protein,rosetta,autoregulation,e. coli

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