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

A1 Journal article (refereed)


Internal Authors/Editors


Publication Details

List of Authors: Mattjus P, Molotkovsky JG, Smaby JM, Brown RE
Publication year: 1999
Journal: Analytical Biochemistry
Journal acronym: ANAL BIOCHEM
Volume number: 268
Issue number: 2
Start page: 297
End page: 304
Number of pages: 8
ISSN: 0003-2697
eISSN: 1096-0309


Abstract

A lipid transfer protein, purified from bovine brain (23.7 kDa, 208 amino acids) and specific for glycolipids, has been used to develop a fluorescence resonance energy transfer assay (anthrylvinyl-labeled lipids; energy donors and perylenoyl-labeled lipids; energy accepters) for monitoring the transfer of lipids between membranes. Small unilamellar vesicles composed of 1 mol% anthrylvinyl-galactosylceramide, 1.5 mol% perylenoyl-triglyceride, and 97.5% 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) served as donor membranes. Acceptor membranes were 100% POPC vesicles. Addition of glycolipid transfer protein to mixtures of donor and acceptor vesicles resulted in increasing emission intensity of anthrylvinyl-galactosylceramide and decreasing emission intensity of the nontransferable perylenoyl-triglyceride as a function of time. The behavior was consistent with anthrylvinyl-galactosylceramide being transferred from donor to acceptor vesicles. The anthrylvinyl and perylenoyl energy transfer pair offers advantages over frequently used energy transfer pairs such as NBD and rhodamine. The anthrylvinyl emission overlaps effectively the perylenoyl excitation spectrum and the fluorescence parameters of the anthrylvinyl fluorophore are nearly independent of the medium polarity. The non-polar fluorophores are localized in the hydrophobic region of the bilayer thus producing minimal disturbance of the bilayer polar region. Our results indicate that this method is suitable for assay of lipid transfer proteins including mechanistic studies of transfer protein function.


Keywords

anthrylvinyl, galactosylceramide, glycosphingolipid, lipid transfer protein, perylenoyl, phospholipid bilayers

Last updated on 2019-16-09 at 04:11