Source: Dissertation Abstracts International, Volume: 54-06, Section: B, page: 2944.

Adviser: Donald M. Engelman.

The transmembrane regions of membrane proteins are thought to consist primarily of $\alpha$-helices whose axes are oriented roughly parallel with the alkane chains of the lipid bilayer. Modelling studies of $\alpha$-helices associating with alkane chains reveal the possibility of packing voids at the lipid-protein interface. Since $\alpha$-helices are capable of packing well with one another, as are alkane chains with each other, a side-by-side association of $\alpha$-helices in the bilayer would result in the elimination of packing voids. The increase in packing efficiency is postulated to provide the energy driving formation of tertiary and quaternary structure in the membrane embedded regions of proteins.

Experiments were conducted to test this hypothesis by examining the association of $\alpha$-helices in two model systems, bacteriorhodopsin and the transmembrane region of glycophorin A, in the presence of small alkanes, which are expected to pack around the helices at the protein-lipid interface and thereby eliminate packing voids. Bacteriorhodopsin (bR) is a retinal containing protein which consists of seven membrane-spanning $\alpha$-helices. bR was refolded from two proteolytic fragments in the presence of hexanes and pentanes. The small alkanes were found to abolish retinal binding by the combined fragments, indicating disruption of tertiary structure, but were not found to abolish secondary structure. Fragment association was further assayed with fluorescence resonance energy transfer. From this assay it was determined that the fragments were associating in the presence of the small alkanes, indicating that the effect of the alkanes is to block or disrupt the retinal binding site of the protein.

The transmembrane region of glycophorin A (GpA-TM) consists of a single membrane-spanning $\alpha$-helix and had been known to form dimers in ionic detergents. A fluorescence resonance energy transfer assay was used to monitor GpA-TM associations in lipid bilayers. It was found the GpA-TM forms an association in bilayers which is not disrupted by additional lipid and is specific for the precise GpA-TM sequence. Addition of hexanes to this system did not disrupt the observed association.