Hearing and touch, the Venus flytrap and osmotic pressure sensing are all consequences of the pervasive ability of ion channels to sense forces. Mechanosensitive ion channels occur in organisms as diverse as microbes and humans, and serve a range of vital functions, often enabling responses to adverse environmental stimuli. Although they are structurally diverse, they share a common mechanism, that of sensing and response to membrane tension. Several models have been proposed, but their underlying principle is unknown. A major hurdle in the study of mechanosensitive is the lack of molecular triggers for mimicking force generation in membranes to stabilise their functional states. Electron Paramagnetic Resonance (EPR) spectroscopy has emerged as a powerful tool for assessing protein conformation, folding, oligomerisation and dynamics. EPR distance measurements provide high resolution quantitative information on the equilibria of proteins, dovetailing well with techniques such as Xray crystallography and CryoEM. An inherent limit placed on membrane protein studies is the need to remove the protein from its natural membrane environment. However, recent developments provide lipid scaffolds that mimic that environment and offer flexibility of choice in lipid composition, while EPR measurements on proteins could also be performed in cells. Here, we will focus on the application of EPR spectroscopy in the study of integral membrane proteins, highlighting two recent case studies from our lab. The first explores an allosteric mechanism that regulates mechanosensitive ion channels1,2, and the second elucidates the effect antibiotics have on the conformation of the BAM complex, with measurements performed in intact cells3.
1. Kapsalis C, Wang B, El Mkami H, Pitt SJ, Schnell JR, Smith TK, Lippiat JD, Bode BE, Pliotas C (2019) Allosteric activation of an ion channel triggered by modification of mechanosensitive nano-pockets. Nature Communications 10: 46192
2. Wang B, Lane BJ, Kapsalis C, Ault JR, Sobott F, El Mkami H, Calabrese AN, Kalli AC, Pliotas C (2022) Pocket delipidation induced by membrane tension or modification leads to a structurally analogous mechanosensitive channel state. Structure 30: 608 622 e605
3. 3. Haysom SF, Machin J, Whitehouse JM, Horne JE, Fenn K, Ma Y, El Mkami H, Bohringer N, Schaberle TF, Ranson NA, Radford SE, Pliotas C (2023) Darobactin B Stabilises a Lateral-Closed Conformation of the BAM Complex in E. coli Cells. Angewandte Chemie International Edition 62: e202218783
Short Bio:
Christos Pliotas obtained a BSc (Hons) in Physics from the University of Athens, an MSc in Medical Physics and a PhD in Biochemistry from the University of Aberdeen. He did his postdoc at the University of St Andrews with James H. Naismith FRS focusing on the structural biology of membrane proteins and in 2016 he was awarded an independent 5-year Fellowship from the Royal Society of Edinburgh to start his own group at the Biomedical Sciences Research Complex, University of St Andrews. Christos moved to the Astbury Centre for Structural Molecular Biology, University of Leeds in October 2018, as a Lecturer in Integrative Membrane Biology. During his time in Leeds, he secured funding from BBSRC to lead multiple projects, including a New Investigator Award (2019), while he received the Sir Robin MacLellan Award for outstanding research by Tenovus (2022). As of June 2023, Christos moved to the Division of Molecular and Cellular Function, School of Biological Sciences, Faculty of Biology, Medicine and Health, at the University of Manchester, to take up a post as a Reader in Structural Biological EPR Spectroscopy. In Manchester, Christos will launch the new BioEmPiRe Centre for Structural Biological EPR Spectroscopy in the Michael Smith building, in early 2024. Christos is currently a committee member of the Royal Society Newton Fellowships for Biological Sciences, a committee member of the Royal Society of Chemistry ESR Group and a Fellow of the Royal Society of Biology (FRSB).
