Incorporation of photosensitive fatty acids into phospholipids of Escherichia coli and irradiation-dependent cross-linking of phospholipids to membrane proteins.

Abstract

In an approach to the study of phospholipid-protein interactions in biological membranes, the photoactivable fatty acids, omega-(m-azidophenoxy)-undecanoic acid (I) and omega-(m-diazirinophenoxy)-hexadecanoic acid (II), were incorporated biosynthetically into the phospholipids of the Escherichia coli fatty acid auxotroph, strain K1060-B5. The extent of incorporation of the two fatty acids was 43% and 21%, respectively, of the total fatty acid content of the phospholipids. Membrane vesicles prepared from cells grown on the fatty acid supplements and [32P]H3PO4 were irradiated at suitable wavelengths to generate the reactive nitrene or carbene intermediates. Subsequent analysis of solubilized membrane proteins by two-dimensional isoelectric focusing-polyacrylamide gel electrophoresis indicated cross-linking between radioactive phospholipids and an number of proteins. A corresponding experiment with cells grown on oleic acid showed only trace amounts of covalently cross-linked phospholipid-protein adducts. While the extent of cross-linking in vesicles from cells grown on I was only 3 times the background level observed for oleic acid-grown cells, cells grown on II showed 30 times this amount. The present results, together with the previously observed nonreactivity of the nitrene generated from I to undergo C-H insertion, show that the use of carbene precursors such as II is promising for chemical analysis of specific phospholipid-protein interactions in bacterial membranes under biologically meaningful conditions.