Probing the Effect of Lipid Binding on the Monomer-Dimer Equilibrium of a Prokaryotic Sugar Transporter by Native Mass Spectrometry

Abstract

For many membrane proteins a specific oligomeric state is required for function as, for example, channels, transporters, and receptors. Oligomerisation of membrane proteins is complicated by the presence of the lipid bilayer, meaning that both protein-protein and protein-lipid interactions must be considered in order to understand this process fully. The contribution of protein-lipid interactions to oligomerisation is underreported, in part because X-ray crystallography often fails to unambiguously detect bound lipids - a consequence of their high conformational flexibility. Non-denaturing (native) mass spectrometry has emerged as a powerful method for probing the stoichiometry and lipid binding properties of membrane protein complexes. It is therefore an ideal method for studying the effect of lipid binding on membrane protein oligomerisation.We have developed a non-denaturing mass spectrometry approach which allows oligomerisation dynamics of detergent-solubilised membrane protein complexes to be observed in ‘real-time’. Using this method, we have identified and quantified a dynamic equilibrium between monomeric and dimeric forms of a bacterial sugar transporter. In addition, we characterised endogenous and exogenous lipid binding to the dimeric state of the protein at unprecedented mass resolution. Combining these approaches, we subsequently investigated the effect of lipid binding on the position of the monomer-dimer equilibrium and the oligomerisation kinetics in order to probe how the local lipid environment might influence oligomerisation in a biological membrane. Bacteria can modulate various properties of their lipid bilayers, in response to extracellular stimuli. Our characterisation of lipid-dependent oligomerisation could provide insight into a mechanism by which bacteria can regulate the function of membrane protein complexes, allowing them to adapt to their environment.