Determinants of Membrane Protein Topology

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DESCRIPTION (provided by applicant): E. coli lactose permease is a twelve-transmembrane domain protein that displays variable topology dependent on the lipid composition of the membrane. The topology is dynamic, changing posttranslationally and post-insertionally with changes in the lipid environment. Transmembrane helix number VII seems to be a hinge point for these transitions, likely because of its relatively hydrophilic nature, and the noncanonical extramembrane loops that precede it. In this proposal, we outline work that will identify specific residues in lactose permease that contribute to topological flexibility. Amino acid changes that lead to either lipid-independent topologies or new lipid-dependent topology profiles will be identified by both site directed mutagenesis and genetic screens. Lipid interactions with these newly identified protein mutants will be assessed. From these studies, we hope to identify lipid-related factors important in determining initial topology and topological flexibility. The general principles governing the folding of lactose permease will apply to other membrane proteins in both E. coli and eukaryotes, and thereby aid in the search for effective therapies of such membrane protein disorders as Alzheimer's Disease and cystic fibrosis.