A fluorescence resonance energy transfer approach for monitoring protein-mediated glycolipid transfer between vesicle membranes

A lipid transfer protein, purified from bovine brain (23.7 kDa, 208 amino a cids) and specific for glycolipids, has been used to develop a fluorescence resonance energy transfer assay (anthrylvinyl-labeled lipids; energy donor s and perylenoyl-labeled lipids; energy accepters) for monitoring the trans fer of lipids between membranes. Small unilamellar vesicles composed of 1 m ol% anthrylvinyl-galactosylceramide, 1.5 mol% perylenoyl-triglyceride, and 97.5% 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) served as donor membr anes. Acceptor membranes were 100% POPC vesicles. Addition of glycolipid tr ansfer protein to mixtures of donor and acceptor vesicles resulted in incre asing emission intensity of anthrylvinyl-galactosylceramide and decreasing emission intensity of the nontransferable perylenoyl-triglyceride as a func tion of time. The behavior was consistent with anthrylvinyl-galactosylceram ide being transferred from donor to acceptor vesicles. The anthrylvinyl and perylenoyl energy transfer pair offers advantages over frequently used ene rgy transfer pairs such as NBD and rhodamine. The anthrylvinyl emission ove rlaps effectively the perylenoyl excitation spectrum and the fluorescence p arameters of the anthrylvinyl fluorophore are nearly independent of the med ium polarity. The non-polar fluorophores are localized in the hydrophobic r egion of the bilayer thus producing minimal disturbance of the bilayer pola r region. Our results indicate that this method is suitable for assay of li pid transfer proteins including mechanistic studies of transfer protein fun ction. (C) 1999 Academic Press.