Relatively little is known about folding mechanisms of integral membrane proteins, embedded in lipid bilayers. The overall goal of this proposal is to answer three specific questions about integral membrane protein folding. 1. How does surface roughness affect membrane protein folding? The lipid-exposed surfaces of some integral membrane proteins appear to be rougher than the helix=helix contact surfaces buried in the protein core. This suggests the hypothesis that surface roughness contributes to the thermodynamic stability of membrane proteins. To test this hypothesis, smoother surfaces will be engineered on bacteriorhodopsin, a small integral membrane protein that can spontaneously fold into an active structure. The smooth surfaces will be introduced by recombinant genetic modification and the ability of the altered proteins to fold properly will be measured. The hypothesis predicts that individual helices will form in the smooth-surface bacteriorhodopsin, but the smooth helices will not coalesce to form an active conformation. Surface roughness could contribute to thermodynamic stability of a membrane protein either through greater disordering effects of the rough surface on the lipid acyl chains or through stronger protein-lipid interactions. These two mechanisms will be tested in synthetic peptides, by measurement of lipid order parameters and peptide transfer enthalpies. 2. How do prosthetic groups affect the folding and assembly of integral membrane proteins? Many integral membrane proteins contain prosthetic groups. Do these groups influence the pathway of folding? Is the apo-protein in a compact, nearly native state, or is it an expanded structure or a heterogeneous mixture of conformations? The role of the prosthetic group retinal will be examined in the folding pathway of bacteriorhodopsin. Patterns of chemical crosslinking will be compared between apo-protein, refolded, and native structures. Site- directed photoaffinity crosslinking agents would have a narrow range of crosslinking products in the native or nearly native structure. By contrast, a heterogeneous mixture of partially folded structures would crosslink randomly, and an unfolded structure would show little crosslinking. 3. How are multi-subunit integral membrane proteins assembled? A multi- subunit integral membrane protein must have exterior surfaces with dual functions. First, the individual subunits must fold into tertiary structures in the lipid bilayer, and then subunits must associated into the active quaternary structure. Do the individual subunits separately attain final active conformations and then associate together, or do they assist each other in folding and form tertiary and quaternary structures simultaneously? Are assembly catalysts such as chaperonins necessary? The refolding if cytochrome c oxidase from Paracoccus denidrificans will be examined. Refolding will be attempted under conditions that permit refolding of single subunit integral membrane proteins. Prosthetic group binding, subunit assembly, and oxidase activity will be measured as tests of refolding. Health significance: The process of membrane protein assembly is a fundamental biochemical mechanism, found in all living cells. The results of this research will provide information that may be used to study and treat disease states involving assembly of membrane proteins. Transmembrane helix association is a key step in signaling by some hormone receptors and these pathways are often involved in development of cancer. Defective assembly of a membrane protein is thought to be involved in the prion diseases, such as Creutzfeldt-Jakob disease, and spongiform encephalopathies. Some diseases involved diminished levels of cytochrome c oxidase activity, as Alzheimer's, and some of these diseases have been attributed to defective protein assembly in the membrane.
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