9514280 Krebs The structure of membrane proteins that span the lipid bilayer is thought to be determined by protein-protein interactions in the aqueous environment and by protein-protein and protein-lipid interactions within the bilayer. The nature and strength of interactions that occur in the aqueous environment can be understood from the perspective of water-soluble protein folding and oligomerization, but little is known about those that occur within the lipid bilayer. In the experiments proposed here, intramembrane interactions will be analyzed by studying the effect of amino acid substitutions on the oligomerization of bacteriorhodopsin (BR) in Halobacterium salinarium. In this organism, BR and associated lipids form two-dimensional crystalline lattice known as the purple membrane. This system is attractive because a structural mode of BR in the purple membrane has been derived from diffraction studies, and because genetic methods have been developed to produce BR mutants in H. salinarium. Protein-protein and protein-lipid interactions in the purple membrane will be probed by mutational analysis of lattice stability. BR mutants will be prepared initially by targeted mutagenesis at sites identified from the structural model, and subsequently by random mutagenesis and screening. Amino acid substitutions will be chosen to minimize disruption of protein tertiary structure, which will be analyzed by spectroscopic and diffraction methods. The targeted approach will be used to test predictions about the role of specific amino acid residues in lattice stability, and will provide mutant proteins for developing in vivo and in vitro stability assays. Preliminary studies have identified several substitutions that appear to disrupt lattice stability without affecting the tertiary structure of the protein, and have provided evidence for a model of BR self-assembly that will be used to evaluate the thermodynamic effects of mutations. Random mutagenesis will be used to test additional subst itutions at sites identified by the targeted approach and at potentially interacting residues. BR mutants obtained by this method will be screened with a rapid assay developed to measure lattice accumulation in vivo. The proposed experiments will address the following questions: (i) which amino acid contacts within the lipid bilayer are most important for lattice stability? (ii) do intramembrane protein-lipid interactions play a significant role in lattice stability? and (iii) what is the strength of protein-protein and protein-lipid interactions within the lipid bilayer? These studies will provide fundamental insights into the nature of non-covalent interactions that determine membrane protein tertiary and quaternary structure. ***
