Use of small unilamellar vesicles to study the biochemical properties of the GRAM domain of GRAMD1.

Cellular membranes are composed of a diverse array of lipids, which are essential for maintaining membrane integrity and facilitating cellular signaling. While proteins often exhibit selective affinities for specific lipid species, the precise thresholds and determinants of these interactions need to be carefully studied to elucidate protein functions. Here, we employ an in vitro liposome-based assay to assess lipid-protein interactions using small unilamellar vesicles of defined lipid compositions. Specifically, we outline the methodology for purifying the GRAM domain of GRAMD1b/Aster-B protein, which has been previously characterized for its ability to detect accessible cholesterol and anionic lipids, including phosphatidylserine, at cellular membranes. We further detail the preparation of liposomes and demonstrate that the purified GRAM domain proteins bind preferentially to liposomes containing both cholesterol and anionic lipids, rather than to those containing either lipid alone, thereby confirming their role as co-incidence detectors. This finding highlights the utility of small unilamellar vesicles as a robust and versatile system to assess the lipid-binding preference of proteins, providing a complementary approach to cell-based assays.