Membrane proteins constitute ~30% of the mammalian proteome and 60% of all drug targets, localizing a major fraction of cellular bioactivity at membrane interfaces. Mammalian membrane proteins are solvated by hundreds of distinct lipid species that are actively turned over and sorted. Membrane protein function is influenced by the biophysical phenotypes arising from lipid collective behaviors such as membrane tension, packing, and fluidity. Further, proteins can selectively recruit local lipid environments, called paralipidomes, that are distinct from the bulk membrane, with varying preferences for lipid headgroups, saturation, and sterols. Experimental studies of cell membrane biophysics rely largely on synthetic fluorescent reporters whose photophysical properties are sensitive to their local environment, which have almost exclusively been applied for measuring bulk membrane properties. Thus, the biophysical properties of local protein paralipidomes remain elusive. To address this technology gap, we have deployed HaloTags to covalently modify membrane proteins with membrane-sensitive probes to measure the biophysical properties of a protein’s local, native paralipidome in living cells. We show the utility of this technology to characterize the differences in local lipid packing between the inner and outer plasma membrane leaflets. Next, we identify differences in local lipid packing between established raft and non-raft transmembrane proteins, representing direct measurements of nanoscopic domains in the living plasma membrane. Future work will further utilize these probes to track lipid packing through the protein secretory pathway, during immune signaling events, and during functional plasma membrane scrambling.
