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C.P. acknowledges the Biotechnology and Biological Sciences Research Council (BBSRC) (BB/S018069/1; BB/W019795/1) for funding. cwEPR data were collected at the Pliotas Laboratory (BB/S018069/1) and PELDOR data were collected at the University of St Andrews, supported by equipment funding from the Wellcome Trust (WT) (099149/Z/12/Z) and BBSRC (BB/R013780/1). CryoEM data were collected at the Astbury Biostructure Laboratory, funded by the University of Leeds and the WT (108466/Z/15/Z; 221524/Z/20/Z). J.E.H. and K.F. acknowledge funding from the Medical Research Council (MRC) (MR/P018491/1). S.F.H. is funded by the White Rose BBSRC DTP (BB/M011151/1). J.M. is funded by the WT (222373/Z/21/Z). SER holds a Royal Society Professorial Fellowship (RSRP/R1/211057). T.F.S and N.B. acknowledge funding from the German Federal Ministry of Education and Research (BMBF, via grant GBi2S and German Centrefor Infection Research (DZIF) 09.918) The Β-barrel assembly machinery (BAM complex) is essential for outer membrane protein (OMP) folding in Gram-negative bacteria, and represents a promising antimicrobial target. Several conformational states of BAM have been reported, but all have been obtained under conditions which lack the unique features and complexity of the outer membrane (OM). Here, we use Pulsed Electron-Electron Double Resonance (PELDOR, or DEER) spectroscopy distance measurements to interrogate the conformational ensemble of the BAM complex in E. coli cells. We show that BAM adopts a broad ensemble of conformations in the OM, while in the presence of the antibiotic darobactin B (DAR-B), BAM's conformational equilibrium shifts to a restricted ensemble consistent with the lateral closed state. Our in-cell PELDOR findings are supported by new cryoEM structures of BAM in the presence and absence of DAR-B. This work demonstrates the utility of PELDOR to map conformational changes in BAM within its native cellular environment. Publisher PDF

MscL was the first mechanosensitive ion channel identified in bacteria. The channel opens its large pore when the turgor pressure of the cytoplasm increases close to the lytic limit of the cellular membrane. Despite their ubiquity across organisms, their importance in biological processes, and the likelihood that they are one of the oldest mechanisms of sensory activation in cells, the exact molecular mechanism by which these channels sense changes in lateral tension is not fully understood. Modulation of the channel has been key to understanding important aspects of the structure and function of MscL, but a lack of molecular triggers of these channels hindered early developments in the field. Initial attempts to activate mechanosensitive channels and stabilize functionally relevant expanded or open states relied on mutations and associated post-translational modifications that were often cysteine reactive. These sulfhydryl reagents positioned at key residues have allowed the engineering of MscL channels for biotechnological purposes. Other studies have modulated MscL by altering membrane properties, such as lipid composition and physical properties. More recently, a variety of structurally distinct agonists have been shown bind to MscL directly, close to a transmembrane pocket that has been shown to have an important role in channel mechanical gating. These agonists have the potential to be developed further into antimicrobial therapies that target MscL, by considering the structural landscape and properties of these pockets.

Human equilibrative nucleoside transporters represent a major pharmaceutical target for cardiac, cancer and viral therapies. Understanding the molecular basis for transport is crucial for the development of improved therapeutics through structure-based drug design. ENTs have been proposed to utilise an alternating access mechanism of action, similar to that of the major facilitator superfamily. However, ENTs lack functionally-essential features of that superfamily, suggesting that they may use a different transport mechanism. Understanding the molecular basis of their transport requires insight into diverse conformational states. Differences between intermediate states may be discrete and mediated by subtle gating interactions, such as salt bridges. We identified four variants of human equilibrative nucleoside transporter isoform 1 (hENT1) at the large intracellular loop (ICL6) and transmembrane helix 7 (TM7) that stabilise the apo-state (∆Tm 0.7–1.5°C). Furthermore, we showed that variants K263A (ICL6) and I282V (TM7) specifically stabilise the inhibitor-bound state of hENT1 (∆∆Tm 5.0 ± 1.7°C and 3.0 ± 1.8°C), supporting the role of ICL6 in hENT1 gating. Finally, we showed that, in comparison with wild type, variant T336A is destabilised by nitrobenzylthioinosine (∆∆Tm -4.7 ± 1.1°C) and binds it seven times worse. This residue may help determine inhibitor and substrate sensitivity. Residue K263 is not present in the solved structures, highlighting the need for further structural data that include the loop regions.

Mechanosensitive ion channels are integral membrane proteins ubiquitously present in bacteria, archaea, and eukarya. They act as molecular sensors of mechanical stress to serve vital functions such as touch, hearing, osmotic pressure, proprioception and balance, while their malfunction is often associated with pathologies. Amongst them, the structurally distinct MscL and MscS channels from bacteria are the most extensively studied. MscS-like channels have been found in plants and

Solvent accessibilities of and distances between protein residues measured by pulsed-EPR approaches provide high-resolution information on dynamic protein motions. We describe protocols for the purification and site-directed spin labeling of integral membrane proteins. In our protocol, peptide-level HDX-MS is used as a precursor to guide single-residue resolution ESEEM accessibility measurements and spin labeling strategies for EPR applications. Exploiting the pentameric MscL channel as a model, we discuss the use of cwEPR, DEER/PELDOR, and ESEEM spectroscopies to interrogate membrane protein dynamics. For complete details on the use and execution of this protocol, please refer to Wang et al. (2022).

The MscL channel gates in response to membrane tension changes to allow the exchange of molecules through its pore. Lipid removal from transmembrane pockets leads to a MscL response. However, it is unknown whether there is correlation between the tension mediated state and the state derived by pocket delipidation in the absence of tension. Transitions between MscL states may follow a similar pathway to cover the available conformational space but may not necessarily sample the same discrete intermediates. Here, we combined pulsed-EPR and HDX-MS measurements on MscL, coupled with molecular dynamics under membrane tension, to investigate the changes associated with the distinctively derived states. Whether it is tension or pocket delipidation, we find that MscL samples a similar expanded state, which is the final step of the delipidation pathway but only an intermediate stop of the tension mediated path. Our findings hint at synergistic modes of regulation in mechanosensitive channels.

Pulsed EPR methods, such as PELDOR (or DEER) and ESEEM have emerged as powerful tools for assessing membrane protein conformation, folding, oligomerisation and dynamics. PELDOR distance measurements can provide quantitative information on the conformational equilibria of membrane proteins, dovetailing well with techniques such as X-ray crystallography and cryo-EM. ESEEM spectroscopy can offer deuterium (or solvent) accessibility quantification at a single residue level. An inherent limit placed on membrane protein studies is the need to remove the protein from its natural membrane environment. However, recent developments in the membrane protein field provide lipid scaffolds that mimic that environment, to limit the impact of protein solubilisation and offer flexibility of choice in lipid composition, a key factor in the functional integrity of membrane proteins. Additionally, computational tools can be used to aid spin-labelling experiments, or tailored to incorporate experimentally derived PELDOR distance restraints to reveal the complete conformational ensemble of membrane proteins. In this chapter, we will focus on the application of pulsed EPR in the study of membrane proteins, highlighting case studies including our work on mechanosensitive ion channels. We will discuss experimental considerations, and introduce some of the computational tools that can be used in combination with pulsed EPR methods.

Membrane proteins (MPs) execute a wide variety of critical biological functions in all living organisms and constitute approximately half of current targets for drug discovery. As in the case of soluble proteins, the bacterium

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

This work was supported by the University of St Andrews and Nanomorphomics group funds. The work forms part of an International Patent Application No. PCT/GB2015/052482. Nanoparticles are of increasing importance in biomedicine but quantification is problematic because current methods depend on indirect measurements at low resolution. Here we describe a new high-resolution method for measuring and quantifying nanoparticles in suspension. It involves premixing nanoparticles in a hydrophilic support medium (methylcellulose) before introducing heavy metal stains for visualization in small air-dried droplets by transmission electron microscopy (TEM). The use of methylcellulose avoids artifacts of conventional negative stain-TEM by (1) restricting interactions between the nanoparticles, (2) inhibiting binding to the specimen support films and (3) reducing compression after drying. Methylcellulose embedment provides effective electron imaging of liposomes, nanodiscs and viruses as well as comprehensive visualization of nanoparticle populations in droplets of known size. These qualities facilitate unbiased sampling, rapid size measurement and estimation of nanoparticle numbers by means of ratio counting using a colloidal gold calibrant. Specimen preparation and quantification take minutes and require a few microliters of sample using only basic laboratory equipment and a standard TEM. Publisher PDF

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.

Highlights • Uranyl acetate/tungsten double stains are proposed for imaging lipid rich nanoparticle in TEM. • Combined with methylcellulose embedment, the technique enhances membrane contrast. • The technique works for liposomes, nanodiscs and bicelles. • The double staining should improve quantification of lipid rich nanoparticles., Synthetic and naturally occurring lipid-rich nanoparticles are of wide ranging importance in biomedicine. They include liposomes, bicelles, nanodiscs, exosomes and virus particles. The quantitative study of these particles requires methods for high-resolution visualization of the whole population. One powerful imaging method is cryo-EM of vitrified samples, but this is technically demanding, requires specialized equipment, provides low contrast and does not reveal all particles present in a population. Another approach is classical negative stain-EM, which is more accessible but is difficult to standardize for larger lipidic structures, which are prone to artifacts of structure collapse and contrast variability. A third method uses embedment in methylcellulose films containing uranyl acetate as a contrasting agent. Methylcellulose embedment has been widely used for contrasting and supporting cryosections but only sporadically for visualizing lipid rich vesicular structures such as endosomes and exosomes. Here we present a simple methylcellulose-based method for routine and comprehensive visualization of synthetic lipid rich nanoparticles preparations, such as liposomes, bicelles and nanodiscs. It combines a novel double-staining mixture of uranyl acetate (UA) and tungsten-based electron stains (namely phosphotungstic acid (PTA) or sodium silicotungstate (STA)) with methylcellulose embedment. While the methylcellulose supports the delicate lipid structures during drying, the addition of PTA or STA to UA provides significant enhancement in lipid structure display and contrast as compared to UA alone. This double staining method should aid routine structural evaluation and quantification of lipid rich nanoparticles structures.

This work was supported by The Wellcome Trust (WT092552MA), The Leverhulme Trust (EM-2012-60\2), BBSRC SysMO (BB/F003455/1), a European Union Marie Curie ITN Award (NICHE; 289384) The potassium efflux system, Kef, protects bacteria against the detrimental effects of electrophilic compounds via acidification of the cytoplasm. Kef is inhibited by glutathione (GSH) but activated by glutathione-S-conjugates (GS-X) formed in the presence of electrophiles. GSH and GS-X bind to overlapping sites on Kef, which are located in a cytosolic regulatory domain. The central paradox of this activation mechanism is that GSH is abundant in cells (at concentrations of 10–20 mM), and thus, activating ligands must possess a high differential over GSH in their affinity for Kef. To investigate the structural requirements for binding of a ligand to Kef, a novel fluorescent reporter ligand, S-{[5-(dimethylamino)naphthalen-1-yl]sulfonylaminopropyl} glutathione (DNGSH), was synthesized. By competition assays using DNGSH, complemented by direct binding assays and thermal shift measurements, we show that the well-characterized Kef activator, N-ethylsuccinimido-S-glutathione, has a 10–20-fold higher affinity for Kef than GSH. In contrast, another native ligand that is a poor activator, S-lactoylglutathione, exhibits a similar Kef affinity to GSH. Synthetic ligands were synthesized to contain either rigid or flexible structures and investigated as ligands for Kef. Compounds with rigid structures and high affinity activated Kef. In contrast, flexible ligands with similar binding affinities did not activate Kef. These data provide insight into the structural requirements for Kef gating, paving the way for the development of a screen for potential therapeutic lead compounds targeting the Kef system. Publisher PDF

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

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.

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.

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)

Electrophiles react rapidly with DNA and proteins and are therefore toxic. Bacteria protect themselves from the immediate effects of electrophiles by lowering the cytosolic pH before the compounds are detoxified. This is achieved by the Kef systems, which mediate potassium efflux, in turn coupled to an influx of protons. We investigated the activation and complex regulation of this system. When electrophiles enter the cell, the most likely reaction partner is the nucleophile glutathione which is present at high concentration in the cytosol. The resulting glutathione adducts activate Kef while glutathione itself inhibits. The kinetics of activation was investigated in vivo using a potassium selective electrode. Activator and inhibitor bind to the same binding site on the regulatory KTN domain of Kef and cause differential structural changes, as shown by crystal structures of this domain [1]. We characterised the binding and these structural changes further using fluorescence and EPR spectroscopy in purified Kef proteins. A second binding site on the KTN domain binds nucleotides. Furthermore, Kef is regulated by a soluble protein which serves as activator [2]. We present data that this activator protein also has quinone oxidoreductase activity. The relationship between this enzymatic activity and its function as an activator of Kef was dissected using point mutations to disrupt enzyme activity but preserve structural integrity.[1] Rossild et al., Structure 17, 893 (2009).[2] Miller et al., J. Bac.182, 6536 (2000).