The mechanisms by which proteins are integrated into or translocated across cell membranes are subjects of major interest in modern molecular and cell biology. Studies on a number of integral membrane proteins have shown that they can spontaneously incorporate into phospholipid bilayers without the presence of any other proteins to mediate this insertion. The object of the proposed research is to determine which features of the primary and secondary structure of an integral membrane protein allow it to insert into a phospholipid bilayer. This will be investigated using a model the spontaneous insertion of bacteriorhodopsin fragments and mutants thereof into performed unilamellar vesicles of dimyristoylphosphatidylcholine. Bacteriorhodopsin, which has the most well-studied structure of any membrane protein, consists seven helical domains crossing back and forth across the membrane bilayer connected by hydrophlic sequences of varying length. Preliminary studies have shown that when the protein is cleaved with chymotrypsin between domains 2 and 3, the larger fragment which results is still capable of membrane insertion. Additional fragments containing various combinations of domains and mutants thereof will now be generated both by proteolysis and by manipulation of the cloned gene in an expression vector using site-specific mutagensis. Incorporation experiments using these polypeptides and phospholipid vesicles will be used to narrow down which sequences in bacteriorhodoposin are necessary and sufficient for insertion into a phospholipid bilayer. Results from these experiments will form the basis for studies using additional mutations to determine what structures mediate membrane insertion and which determine the orientation in the membrane. Finally the sequences identified as mediating insertion of an integral membrane protein will be tested to determine whether they can also anchor or translocate an otherwise hydrophilic protein attached to them.
| Xu, Z J; Moffett, D B; Peters, T R et al. (1995) The role of the leader sequence coding region in expression and assembly of bacteriorhodopsin. J Biol Chem 270:24858-63 |
| Teintze, M; Xu, Z J (1992) Membrane assembly of bacterio-opsin mutants expressed in halobacteria and incorporation of the proteins into phospholipid bilayers. Biophys J 62:54-5 |
