The influence of lipid binding on the oligomeric state of membrane protein complexes

A large fraction of membrane proteins assemble into long-lived, non-covalent complexes, in a process called oligomerisation. Oligomerisation confers many benefits on membrane proteins, from increased thermal stability and access to a greater diversity of potential functions, to greater opportunities for cellular regulation. Despite this ubiquity and importance, membrane protein oligomerisation, and the role of the lipid environment in determining oligomeric state are poorly understood. Non-denaturing mass spectrometry (MS) is a versatile method for probing both oligomeric state and protein-lipid interactions and hence is an ideal technique for investigating the relationship between these two phenomena. In this thesis, we report the application of a novel MS-based methodology to examine the oligomerisation of two dimeric membrane proteins: the bioamine transporter LeuT and the sugar transporter SemiSWEET. For SemiSWEET, we used both high-resolution and high-energy MS methods to characterise a small cohort of lipids which co-purify with the protein. Then, we performed subunit exchange experiments with dimeric SemiSWEET, establishing that it exists in a monomer-dimer equilibrium, the quaternary dynamics of which can be monitored in real time. Finally, by adding exogenous cardiolipin to SemiSWEET, we found that lipid binding shifts the monomer-dimer equilibrium towards the dimeric state. For LeuT, we observed by MS that the dimeric form, but not the monomeric form, of the protein co-purifies with a larger cohort of lipids, including diacylphospholipids and cardiolipin. Using biochemical experiments and molecular dynamics simulations we ascertained that these lipids bind specifically at the LeuT dimer interface, via a bridging interaction between cardiolipin phosphate groups and cationic residues on the protein subunits, and that these protein-lipid interactions are essential for LeuT dimerisation. Overall, the results show that the oligomeric states of membrane proteins can be strongly influenced by the local lipid environment, suggesting a potential mechanism for spatial and temporal regulation of protein function