INSERTION/FOLDING OF POLYTOPIC INTEGRAL MEMBRANE PROTEIN

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DESCRIPTION: The overall goal of this project is to investigate lipid-protein-protein interactions that determine the formation of protein structure in lipid bilayers. Understanding the factors that govern the three-dimensional structure of membrane proteins is important because it is still difficult to obtain high resolution structures of membrane proteins by X-ray or electron crystallography or solid-state NMR. An alternate possibility is to predict polytopic membrane structures from their amino acid sequences, but predictive methods depend critically on the parameters describing the interactions that occur in the membrane. In addition, under-standing membrane protein folding is of practical importance because it will guide researchers in their efforts to refold recombinant proteins for structural and other studies. Protein A (Omp A) from the outer membrane of E. coli is chosen as a model protein for these studies. This protein is thought to form an 8-stranded antiparallel beta-barrel in membranes and will serve as a paradigm for the folding of membrane proteins that contain beta-structure (e.g., porins or acetylcholine receptors). The long-range goal of the proposed research is to formulate general rules for the folding and insertion of membrane proteins. There are six specific aims: (1) to study the kinetics of refolding of urea-denatured OmpA into lipid model membranes with various lipids and at various temperatures; (2) to determine the rate constants and the mechanism of OmpA interconversion from the adsorbed to the native inserted state by temperature-dependent kinetic experiments using wild-type and specifically designed mutant OmpAs; (3) to further characterize the structure of OmpA folding intermediates in fluid phase lipid at low temperature by fluorescence quenching, near UV CD, and polarized ATR-FTIR spectroscopy; (4) to chemically synthesize and fluorescently label all eight membrane-spanning beta-strands and the four antiparallel beta-loops to determine their individual structures in bilayers and the thermodynamics and kinetics of their insertion from buffer into bilayers; (5) to co-reconstitute two or more of these peptides in the adsorbed and/or inserted forms to study their kinetics of self-assembly and their structures in bilayers at different temperatures; and (6) to formulate general rules of membrane protein folding and insertion based on the results of aims 1 through 5.