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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.

SUBMITTER: Pliotas C

PROVIDER: S-EPMC3479538 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Conformational state of the MscS mechanosensitive channel in solution revealed by pulsed electron-electron double resonance (PELDOR) spectroscopy.

Pliotas Christos C Ward Richard R Branigan Emma E Rasmussen Akiko A Hagelueken Gregor G Huang Hexian H Black Susan S SS Booth Ian R IR Schiemann Olav O Naismith James H JH

Proceedings of the National Academy of Sciences of the United States of America 20120910 40

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 ...[more]

PMID: 23012406

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Project description:Pulse electron paramagnetic resonance (EPR) is being applied to ever more complex biological systems comprising multiple subunits. Membrane channel proteins are of great interest as pulse EPR reports on functionally significant but distinct conformational states in a native environment without the need for crystallization. Pulse EPR, in the form of pulsed electron-electron double resonance (PELDOR), using site-directed spin labeling, is most commonly employed to accurately determine distances (in the nanometer range) between different regions of the structure. However, PELDOR data analysis is more challenging in systems containing more than two spins (e.g., homomultimers) due to distorting multispin effects. Without suppression of these effects, much of the information contained in PELDOR data cannot be reliably retrieved. Thus, it is of utmost importance for future PELDOR applications in structural biology to develop suitable approaches that can overcome the multispin problem. Here, two different approaches for suppressing multispin effects in PELDOR, sparse labeling of the protein (reducing the labeling efficiency f) and reducing the excitation probability of spins (?), are compared on two distinct bacterial mechanosensitive channels. For both the pentameric channel of large conductance (MscL) and the heptameric channel of small conductance (MscS) of Escherichia coli, mutants containing a spin label in the cytosolic or the transmembrane region were tested. Data demonstrate that distance distributions can be significantly improved with either approach compared to the standard PELDOR measurement, and confirm that ? < 1/(n-1) is needed to sufficiently suppress multispin effects (with n being the number of spins in the system). A clear advantage of the sparse labeling approach is demonstrated for the cytosolic mutants due to a significantly smaller loss in sensitivity. For the transmembrane mutants, this advantage is less pronounced but still useful for MscS, but performance is inferior for MscL possibly due to structural perturbations by the bulkier diamagnetic spin label analog.

Dataset Information

Conformational state of the MscS mechanosensitive channel in solution revealed by pulsed electron-electron double resonance (PELDOR) spectroscopy.

Publications

Conformational state of the MscS mechanosensitive channel in solution revealed by pulsed electron-electron double resonance (PELDOR) spectroscopy.

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