title: Conformational state of the MscS mechanosensitive channel in solution revealed by pulsed electron-electron double resonance (PELDOR) spectroscopy.
abstract: The heptameric mechanosensitive channel of small conductance (MscS) provides a critical function in Escherichia coli where it opens in response to increased bilayer tension. Three approaches have defined different closed and open structures of the channel, resulting in mutually incompatible models of gating. We have attached spin labels to cysteine mutants on key secondary structural elements specifically chosen to discriminate between the competing models. The resulting pulsed electron-electron double resonance (PELDOR) spectra matched predicted distance distributions for the open crystal structure of MscS. The fit for the predictions by structural models of MscS derived by other techniques was not convincing. The assignment of MscS as open in detergent by PELDOR was unexpected but is supported by two crystal structures of spin-labeled MscS. PELDOR is therefore shown to be a powerful experimental tool to interrogate the conformation of transmembrane regions of integral membrane proteins.
authors.affiliation: Biomedical Sciences Research Complex, University of St Andrews, North Haugh, St Andrews KY16 9ST, United Kingdom.
authors.forename: Christos
authors.initials: C
authors.lastname: Pliotas
authors.forename: Richard
authors.initials: R
authors.lastname: Ward
authors.forename: Emma
authors.initials: E
authors.lastname: Branigan
authors.forename: Akiko
authors.initials: A
authors.lastname: Rasmussen
authors.forename: Gregor
authors.initials: G
authors.lastname: Hagelueken
authors.forename: Hexian
authors.initials: H
authors.lastname: Huang
authors.forename: Susan S
authors.initials: SS
authors.lastname: Black
authors.forename: Ian R
authors.initials: IR
authors.lastname: Booth
authors.forename: Olav
authors.initials: O
authors.lastname: Schiemann
authors.forename: James H
authors.initials: JH
authors.lastname: Naismith
mesh_terms: D015153:Blotting, Western; D002850:Chromatography, Gel; D003461:Crystallography; D004578:Electron Spin Resonance Spectroscopy; D029968:Escherichia coli Proteins / Q000737:chemistry*; D007473:Ion Channels / Q000737:chemistry*; D008958:Models, Molecular; D016296:Mutagenesis; D018408:Patch-Clamp Techniques; D011487:Protein Conformation; D017422:Sequence Analysis, DNA; D013057:Spectrum Analysis / Q000379:methods*; D013113:Spin Labels
chemical_list: D029968:Escherichia coli Proteins; D007473:Ion Channels; C469542:MscS protein, E coli; D013113:Spin Labels