Measuring Transmembrane Helix Separation on the MscS Mechanosensitive Channel using Pulsed Electron-Electron Double Resonance (PELDOR) Spectroscopy

The heptameric mechanosensitive channel of small conductance (MscS) provides an essential function in Escherichia coli as it prevents cell lysis by opening in response to increased bilayer tension, during extreme osmotic shock. The gating of MscS has been extensively studied by three approaches: X-ray crystallography [1], cwEPR [2] spectroscopy and EMD simulations [3], but each of them has defined different closed and open structures of the channel. The proposed models report very different arrangements of the transmembrane helices. To resolve the discrepancy, we have attached spin labels to cysteine mutants on key structural elements within all three MscS transmembrane helices, specifically chosen to discriminate between the competing models. These distances from multiple mutants provide an independent assessment of the validity of the competing models. In addition, they offer the advantage of measurements on MscS in solution under physiological conditions. The resulting pulsed electron-electron double resonance (PELDOR) spectra allow the accurate measurement of transmembrane helix separation, offering a direct comparison for the different models. The distance distributions for the open crystal structure of MscS match the experimental data [4]. We also report two new crystal structures of spin labeled MscS which are in complete agreement with the PELDOR data and demonstrate the accuracy of PELDOR for complex ion channels. PELDOR is a powerful experimental tool which can be used for interrogating the conformation of transmembrane regions of multimeric membrane proteins.[1] Wang et al., Science 321, 1179 (2008).[2] Vasquez et al., Science 321, 1210 (2008).[3] Akitake et al., NSMB 14, 1141 (2007).[4] Pliotas et al., PNAS (doi/10.1073/pnas.1202286109) (2012)