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This project was supported by a BBSRC grant (BB/S018069/1) to C.P., who was supported by the Royal Society of Edinburgh, Tenovus (T15/41) and Carnegie Trust (OS000256), at the initial stages of this project. Further C.P. acknowledges support from the University of St Andrews for the C.K. studentship and the University of Leeds and the Chinese Scholarship Council for the Y.M. studentship. B.E.B. and C.P. acknowledge support by the Leverhulme Trust (RPG-2018–397). This work was also supported by previous Wellcome Trust [099149/Z/12/Z] and BBSRC equipment grants (BB/R013780/1). The mechanosensitive channel of large conductance (MscL) from Mycobacterium tuberculosis has been used as structural model for rationalizing functional observations in multiple MscL orthologues. Although these orthologues adopt similar structural architectures, they reportedly present significant functional differences. Subtle structural discrepancies on mechanosensitive channel nano-pockets are known to affect mechanical gating and may be linked to large variability in tension sensitivity among these membrane channels. Here we modify the nano-pocket regions of MscL from Escherichia coli and Mycobacterium tuberculosis and employ PELDOR/DEER distance and 3pESEEM deuterium accessibility measurements to interrogate channel structure within lipids, in which both channels adopt a closed conformation. Significant in-lipid structural differences between the two constructs suggest a more compact EcMscL at the membrane inner-leaflet, as a consequence of a rotated TM2 helix. Observed differences within lipids could explain EcMscL’s higher tension sensitivity and should be taken into account in extrapolated models used for MscL gating rationalization. Publisher PDF

Lipid availability within transmembrane nano-pockets of ion channels is linked with mechanosensation. However, the effect of hindering lipid-chain penetration into nano-pockets on channel structure has not been demonstrated. Here we identify nano-pockets on the large conductance mechanosensitive channel MscL, the high-pressure threshold channel. We restrict lipid-chain access to the nano-pockets by mutagenesis and sulfhydryl modification, and monitor channel conformation by PELDOR/DEER spectroscopy. For a single site located at the entrance of the nano-pockets and distal to the channel pore we generate an allosteric response in the absence of tension. Single-channel recordings reveal a significant decrease in the pressure activation threshold of the modified channel and a sub-conducting state in the absence of applied tension. Threshold is restored to wild-type levels upon reduction of the sulfhydryl modification. The modification associated with the conformational change restricts lipid access to the nano-pocket, interrupting the contact between the membrane and the channel that mediates mechanosensitivity., How mechanosensitive ion channels, such as MscL, are activated by lipids and physical properties of the membrane remains unclear. Here authors use PELDOR/DEER spectroscopy and identify a single site which generated an allosteric structural response in the absence of membrane tension.

This work was supported by Wellcome Trust grants WT092552MA (J.H.N. and I.R.B.), Senior Investigator Award WT100209MA (J.H.N.), 093228 (T.K.S.) and 092970 (M.S.P.S.), and Biotechnology and Biological Sciences Research Council grants BB/I019855/1 (M.S.P.S.), BB/H017917/1 (J.H.N. and I.R.B.) and BB/J009784/1 (H.B.). I.R.B. is supported as a Leverhulme Emeritus Fellow. J.H.N. is supported as a Royal Society Wolfson Merit Award holder and as a 1000 Talent Scholar at Sichuan University. A.C.E.D. was supported by an Engineering and Physical Sciences Research Council Systems Biology Doctoral Training Centre student fellowship. The ability of proteins to sense membrane tension is pervasive in biology. A higher-resolution structure of the Escherichia coli small-conductance mechanosensitive channel MscS identifies alkyl chains inside pockets formed by the transmembrane helices (TMs). Purified MscS contains E. coli lipids, and fluorescence quenching demonstrates that phospholipid acyl chains exchange between bilayer and TM pockets. Molecular dynamics and biophysical analyses show that the volume of the pockets and thus the number of lipid acyl chains within them decreases upon channel opening. Phospholipids with one acyl chain per head group (lysolipids) displace normal phospholipids (with two acyl chains) from MscS pockets and trigger channel opening. We propose that the extent of acyl-chain interdigitation in these pockets determines the conformation of MscS. When interdigitation is perturbed by increased membrane tension or by lysolipids, the closed state becomes unstable, and the channel gates. Postprint Postprint Postprint Postprint Postprint Postprint Postprint

Mechanosensitive (MS) ion channels are multimeric integral membrane proteins that respond to increased lipid bilayer tension by opening their nonselective pores to release solutes and relieve increased cytoplasmic pressure. These systems undergo major conformational changes during gating and the elucidation of their mechanism requires a deep understanding of the interplay between lipids and proteins. Lipids are responsible for transmitting lateral tension to MS channels and therefore play a key role in obtaining a molecular-detail model for mechanosensation. Site-directed spin labeling combined with electron paramagnetic resonance (EPR) spectroscopy is a powerful spectroscopic tool in the study of proteins. The main bottleneck for its use relates to challenges associated with successful isolation of the protein of interest, introduction of paramagnetic labels on desired sites, and access to specialized instrumentation and expertise. The design of sophisticated experiments, which combine a variety of existing EPR methodologies to address a diversity of specific questions, require knowledge of the limitations and strengths, characteristic of each particular EPR method. This chapter is using the MS ion channels as paradigms and focuses on the application of different EPR techniques to ion channels, in order to investigate oligomerization, conformation, and the effect of lipids on their regulation. The methodology we followed, from the initial strategic selection of mutants and sample preparation, including protein purification, spin labeling, reconstitution into lipid mimics to the complete set-up of the pulsed-EPR experiments, is described in detail.

Funding: EaStCHEM studentship to E.B. The work was funded by BBSRC grant BB/H017917/1 to JNH, OS & IRB, The Leverhulme Foundation (EM-2012-60\2) and equipment from a Wellcome Trust Capital Award. Mechanosensitive channel proteins are important safety valves against osmotic shock in bacteria, and are involved in sensing touch and sound waves in higher organisms. The mechanosensitive channel of small conductance (MscS) has been extensively studied. Pulsed electron-electron double resonance (PELDOR or DEER) of detergent-solubilized protein confirms that as seen in the crystal structure, the outer ring of transmembrane helices do not pack against the pore- forming helices, creating an apparent void. The relevance of this void to the functional form of MscS in the bilayer is the subject of debate. Here, we report PELDOR measurements of MscS reconstituted into two lipid bilayer systems: nanodiscs and bicelles. The distance measurements from multiple mutants derived from the PELDOR data are consistent with the detergent-solution arrangement of the protein. We conclude, therefore, that the relative positioning of the transmembrane helices is preserved in mimics of the cell bilayer, and that the apparent voids are not an artifact of detergent solution but a property of the protein that will have to be accounted for in any molecular mechanism of gating. Publisher PDF

A pressure gradient across a curved lipid bilayer leads to a lateral force within the bilayer. Following ground breaking work on eukaryotic ion channels, it is now known that many proteins sense this change in the lateral tension and alter their functions in response. It has been proposed that responding to pressure differentials may be one of the oldest signaling mechanisms in biology. The most well characterized mechanosensing ion channels are the bacterial ones which open when the pressure differential hits a threshold. Recent studies on one of these channels, MscS, have developed a simple molecular model for how they sense and adapt to pressure. Biochemical and structural studies on mechanosensitive channels from eukaryotes have disclosed pressure sensing mechanisms. In this review, we highlight these findings and discuss the potential for a general model for pressure sensing.

Pulse electron paramagnetic resonance (EPR) is gaining increasing importance in structural biology. The PELDOR (pulsed electron–electron double resonance) method allows extracting distance information on the nanometer scale. Here, we demonstrate the efficient extraction of distances from multimeric systems such as membrane‐embedded ion channels where data analysis is commonly hindered by multi‐spin effects.