Determining preferential paralipidomes of integral membrane proteins using copolymeric nanodiscs

Cellular membranes contain hundreds of different lipid species that solvate thousands of integral membrane proteins. While our understanding of overall cellular lipidomes has increased in recent years, the particular lipidomes surrounding distinct integral membrane proteins remain mysterious. This mystery remains, in part, due to the lack of robust experimental methods to directly evaluate the surrounding lipid environment of membrane proteins. In this work, the use of amphipathic copolymers is described for the study of the preferential surrounding lipid environment, termed paralipidome, of integral membrane proteins. A variety of amphipathic copolymers have now been found to be able to directly solubilize membrane proteins with a surrounding patch of lipids from biological membranes. Since copolymer solubilization does not require the use of detergents, copolymer stabilized nanoparticles have been advertised as maintaining a native membrane environment. A systematic study comparing lipid packing between copolymer-derived lipid nanoparticles and the membranes they were derived from revealed that copolymeric nanoparticles do not inherently retain native membrane properties. While copolymeric nanoparticles are not native membrane patches, they are still useful tools in the study of preferential membrane protein-lipid interactions. Copolymers were used to efficiently solubilize the adenosine 2A receptor (A2AR), a model mammalian integral membrane protein. Preliminary data on the preferential paralipidome of A2AR is reported by comparing the differences in lipid composition between A2AR containing nanoparticles, crude copolymer nanoparticles, and the source membranes from which they were solubilized. Lipids previously reported as positive allosteric modulators of A2AR, including cholesterol and unsaturated lipids, were found to be enriched in A2AR paralipidomes, among several other lipid preferences. While this work focuses on A2AR, the methods described here are applicable to study the preferential paralipidomes for any membrane protein of interest.