Maleimide-functionalized lipids that anchor polypeptides to lipid bilayersand membranes

Two maleimide-containing diacylglycerol derivatives were synthesized to per mit the anchoring of short peptides and longer polypeptides to phospholipid bilayers and membranes. The maleimide was introduced at the site normally occupied by a phospholipid headgroup. The first lipid, the dipalmitoyl este r of 1-maleimido-2,3-pmpanediol, was developed as a membrane anchor for ext racellular domains of transmembrane proteins. The second anchoring lipid, i n which the 3-position contained a 6-aminohexanoate, was designed for conve nient modification with amine-reactive reporter groups. Specifically, the N BD fluorophore, 7-nitrobenzo-2-oxa-1,3-diazale-aminohexanoic-N-hydroxysucci nimide ester, was attached to give an fluorescent anchoring reagent. Next, these reagents were applied to the anchoring of a C-terminally cysteamine-m odified 8 kDa polypeptide that comprises the extracellular N-terminal domai n of the human thrombin receptor, a transmembrane protease-activated recept or (PAR-1). Gel filtration and fluorescence analysis showed that the fluore scent lipopolypeptide spontaneously inserted into preformed phospholipid ve sicles, but it did not insert into whole cell membranes. In contrast, the d ipalmitoyl derivative could only be reconstituted into artificial membranes by mixing the lipopolypeptide and phospholipid before vesicle formation. T hese results suggest that biophysical interactions governing the lipopolype ptide insertion into artificial and cellular membranes may differ. The thio l-reactive lipidating reagents should be valuable materials for studying th e structure and function of peptides and polypeptides at phospholipid bilay er surfaces.