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Properties of the Mechanosensitive Channel MscS Pore Revealed by Tryptophan Scanning Mutagenesis

Rasmussen, Tim and Rasmussen, Akiko and Singh, Shivani and Galbiati, Heloisa and Edwards, Michelle D. and Miller, Samantha and Booth, Ian R. (2015) Properties of the Mechanosensitive Channel MscS Pore Revealed by Tryptophan Scanning Mutagenesis. Biochemistry, 54 (29). pp. 4519-4530. ISSN 0006-2960. PMCID PMC4519979. doi:10.1021/acs.biochem.5b00294.

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Bacterial mechanosensitive channels gate when the transmembrane turgor rises to levels that compromise the structural integrity of the cell wall. Gating creates a transient large diameter pore that allows hydrated solutes to pass from the cytoplasm at rates close to those of diffusion. In the closed conformation, the channel limits transmembrane solute movement, even that of protons. In the MscS crystal structure (Protein Data Bank entry 2oau), a narrow, hydrophobic opening is visible in the crystal structure, and it has been proposed that a vapor lock created by the hydrophobic seals, L105 and L109, is the barrier to water and ions. Tryptophan scanning mutagenesis has proven to be a highly valuable tool for the analysis of channel structure. Here Trp residues were introduced along the pore-forming TM3a helix and in selected other parts of the protein. Mutants were investigated for their expression, stability, and activity and as fluorescent probes of the physical properties along the length of the pore. Most Trp mutants were expressed at levels similar to that of the parent (MscS YFF) and were stable as heptamers in detergent in the presence and absence of urea. Fluorescence data suggest a long hydrophobic region with low accessibility to aqueous solvents, extending from L105/L109 to G90. Steady-state fluorescence anisotropy data are consistent with significant homo-Förster resonance energy transfer between tryptophan residues from different subunits within the narrow pore. The data provide new insights into MscS structure and gating.

Item Type:Article
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URLURL TypeDescription Information CentralArticle
Rasmussen, Tim0000-0003-1126-846X
Rasmussen, Akiko0000-0001-7796-1668
Miller, Samantha0000-0002-8223-9693
Additional Information:© 2015 American Chemical Society. This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Received: March 17, 2015; Revised: June 18, 2015; Published: July 1, 2015. This work was supported by a Wellcome Trust Programme grant [092552/A/10/Z awarded to I.R.B., S.M., J. H. Naismith (University of St Andrews, St Andrews, U.K.), and S. J. Conway (University of Oxford, Oxford, U.K.)] (T.R. and M.D.E.), by a BBSRC grant (A.R.) [BB/H017917/1 awarded to I.R.B., J. H. Naismith, and O. Schiemann (University of St Andrews)], by a Leverhulme Emeritus Fellowship (EM-2012-060\2), and by a CEMI grant to I.R.B. from the California Institute of Technology. The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007-2013 FP7/2007-2011) under Grant PITN-GA-2011-289384 (FP7-PEOPLE-2011-ITN NICHE) (H.G.) (awarded to S.M.). We thank Jim Naismith and his group (University of St Andrews) and Doug Rees (California Institute of Technology) for helpful discussions. Author Contributions: T.R. and A.R. contributed equally to this research. The authors declare no competing financial interest.
Group:Caltech Center for Environmental Microbial Interactions (CEMI)
Funding AgencyGrant Number
Wellcome Trust092552/A/10/Z
Biotechnology and Biological Sciences Research Council (BBSRC)BB/H017917/1
Leverhulme TrustEM-2012-060\2
Caltech Center for Environmental Microbial Interactions (CEMI)UNSPECIFIED
European Union Seventh Framework ProgrammePITN-GA-2011-289384
Issue or Number:29
PubMed Central ID:PMC4519979
Record Number:CaltechAUTHORS:20150827-104629563
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Official Citation:Properties of the Mechanosensitive Channel MscS Pore Revealed by Tryptophan Scanning Mutagenesis Tim Rasmussen, Akiko Rasmussen, Shivani Singh, Heloisa Galbiati, Michelle D. Edwards, Samantha Miller, and Ian R. Booth Biochemistry 2015 54 (29), 4519-4530 DOI: 10.1021/acs.biochem.5b00294
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:59925
Deposited By: Tony Diaz
Deposited On:11 Sep 2015 23:10
Last Modified:26 May 2022 16:45

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