STRUCTURAL BASIS OF BACTERIORHODOPSIN BIOGENESIS

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DESCRIPTION (provided by applicant): The long-term goal of the proposed research is understand the structural basis of membrane protein biogenesis. The focus is on the polytopic (multispanning) membrane protein, bacteriorhodopsin, an integral membrane protein produced by the archaeon Halobacteriurn salinarum. This protein folds to form a bundle of seven transmembrane alpha-helices, binds retinal and assembles in a two-dimensional crystalline lattice known as the purple membrane. Bacteriorhodopsin is a model for understanding membrane protein insertion, folding and assembly because its three-dimensional structure is known at high resolution in the native membrane environment. The objective in this proposal is to test in vivo whether bacteriorhodopsin fits a two-stage model in which interactions between transmembrane segments are the primary determinant of folding and assembly. The specific aims are (1) to introduce mutations throughout BR by a saturation mutagenesis approach implemented in preliminary studies; (II) to identify those residues at which substitutions can be tolerated without affecting the formation of BR from BO and retinal; and (III) to determine residues at which substitutions can be tolerated without affecting the assembly of the BR lattice. The results from this analysis will be compared with the three-dimensional structure of BR to test whether the predictions of the two-stage model are confirmed. These studies are expected to yield important new information on the structural basis of integral membrane protein folding and assembly in the cell.