Membrane proteins reside in complex lipid environments composed of hundreds of lipid species. The function of these proteins depends on both their surrounding lipid composition and the biophysical properties of the lipid collectives. While some proteins bind specific lipid species, the distinct local membrane environments—termed paralipidomes—assembled by specific membrane proteins remain poorly defined. Here, we apply two complementary technologies to define protein paralipidomes. To probe local biophysical environments, we use HaloTags to covalently label membrane proteins with membrane-sensitive fluorescent probes, enabling in situ measurements of lipid packing in live cells. Using this strategy, we quantify differences in packing between the inner and outer plasma membrane leaflets and directly observe nanoscopic heterogeneities associated with raft vs. non-raft proteins. To determine paralipidome composition, we use synthetic amphipathic copolymers to solubilize membrane proteins from native membranes without detergents, preserving a solvating lipid bilayer. We then performed MS-MS to quantify the lipidome surrounding a given protein as compared to the bulk lipidome of the cell. Using this approach, we characterized the paralipidome of the G-protein coupled receptor adenosine 2A receptor (A2AR). Distinct lipid profiles were identified for apo-, agonist-, and antagonist-bound A2AR, suggesting conformation-dependent lipid selection. We are currently investigating how lipid composition affects ligand binding and receptor activation and integrating both approaches to comprehensively map biophysical and compositional paralipidomes. Together, these methods define a methodological framework for studying how the function of membrane proteins is regulated by their local lipid nano-environment.
Levental et al. (Sun,) studied this question.