Please Note: The main page lists projects via BBSRC Research Theme(s) quoted and then relevant Topic(s)
Comparative investigation of lipid-protein interactions within the mechanosensitive MscS-like channel family
Secondary Supervisor(s): Professor Corinne Spickett, Dr Alice Rothnie
University of Registration: Aston University
BBSRC Research Themes: Understanding the Rules of Life (Microbiology, Structural Biology)
Project Outline
Membrane proteins have evolved alongside lipid molecules, and as result their structure and function are altered by the chemical and physical properties of the bilayer. In the case of mechanosensitive (MS) channels this lipid-protein relationship is even more intimate, as these systems sense and respond to tension changes in the bilayer. In bacteria, seven MS channels (MscL, MscS, YnaI, YbdG, MscK, MscM, YbiO) have been identified. These channels can be grouped into two major families, MscL and the rest under the umbrella of the MscS-like superfamily. MscS-like family members vary in the size of their transmembrane domain and the presence/absence of periplasmic domains, but all share the same MscS-like core. Bacterial MS channels sense changes in lateral tension generated in the membrane during rapid changes in osmolarity, allowing the bacteria to release solutes to prevent cell lysis during hypoosmotic shock. However, their redundancy implies these channels serve other fundamental physiological roles. Additionally, several of these channels have been implicated in bacterial pathogenicity and are potential drug targets.
Direct interactions with the membrane have been shown to be key in controlling the conformational states of several eukaryotic and prokaryotic mechanosensitive ion channels. In the case of MscS and MscL, interactions between lipid acyl chains and pressure-sensitive transmembrane pockets in the channels are negative modulators of channel gating, keeping them closed. Membrane tension reduces lipid packing within the bilayer which shifts the equilibrium causing lipids that stabilize the closed state of the channel to move out of these transmembrane pockets. Wang and Lane et al. showed that interrupting these interactions through a tryptophan mutation stabilized a biologically relevant subconducting state of MscL. However, whether specific lipid species are involved in these key protein-lipid interactions is not understood. We also have structural data indicating that sensing of curvature may play a role in the gating mechanism of MscS-like channels, in addition to lipids interacting with transmembrane pockets. There is no understanding of whether different members of the MscS-like channel family have preferences for specific membrane environments, which could be important for the regulation of these proteins. Understanding the differing lipid interactions and membrane environments of these different channels will form the basis of this project. The project will utilize co-polymer and amphipol based systems to extract the mechanosensitive channels in their native lipid environment. Following purification, the lipid environment associated with the channels will be examined using lipidomic approaches. Depending on the interests of the PhD student and project progression, there may also be the opportunity to use structural biology and native mass spectrometry approaches in the study of the MscS-like channels in collaboration with external collaborators. Additionally, students can also focus on early-lead development of small-molecules to target some of these channels by interrupting protein-lipid interactions.
Methods
Molecular biology (Aston): General molecular biology methods/skills such as site-directed mutagenesis, PCR, DNA purification, cell growth assays.
Protein expression, analysis and purification (Aston): SDS-PAGE, Western blotting, membrane isolation, detergent and polymer solubilization/extractions, protein purification (affinity chromatography, size-exclusion chromatography).
Biophysical: lipid analysis using LC-MS/MS (Aston), EPR spectroscopy (Manchester).
References
Teo, A. C. K. et al. Analysis of SMALP co-extracted phospholipids shows distinct membrane environments for three classes of bacterial membrane protein. Scientific Reports 1813, (2019).
Flegler, V. J. et al. The MscS-like channel YnaI has a gating mechanism based on flexible pore helices. PNAS 117, 28754–28762 (2020).
Kefauver, J. M. et al. Discoveries in structure and physiology of mechanically activated ion channels. Nature 587, 567–576 (2020).
