The long-term goal of this research is to elucidate, in molecular terms, the structure-function relationships in human high density lipoproteins (HDL). This project focuses on the structure and function of apolipoprotein A-I. the major protein component of HDL. The main hypothesis we wish to test is that the three-dimensional structure of apo A-I in lipid-bound states consists of two domains: (l) an N-terminal domain which is compact, relatively stable yet conformationally flexible, which binds to lipid through hydrophobic anchoring residues in addition to the amphipathic (X-helices, and contains the specific LCAT activating region; and (2) a C-terminal domain which mediates oligomerization in solution, initial insertion of apo A-I into lipid aggregates, and retains nonspecific lipid binding and LCAT activating properties. To study the domain structure and functions of apo A-I we will prepare site-directed mutants and deletion mutants, as well as large, chemically produced fragments of plasma apo A-I and recombinant proapo A-I. These purified variants of apo A-I will then be investigated in terms of (a) their structure, stability, and oligomerization in aqueous solution and in 30% n-propanol (which induces a native-like structure in apo A-I); (b) their ability to interact with phospholipids on surfaces, in liposomes, and in reconstituted HDL (rHDL); (c) their structure in homogeneous rHDL; (d) their functions, in the defined rHDL, as activators of lecithin cholesterol acyltransferase and as mediators of binding to cells and of cholesterol uptake from cells and low density lipoproteins; (e) their potential as models for high-resolution structural studies. A variety of methods will be used to accomplish these aims, including recombinant DNA methods, expression in E. coli, protein purification, fluorescence, far and near UV-CD spectroscopy, lipid-binding kinetics and equilibrium approaches, chromatographic isolation of rHDL, electrophoretic analysis of protein fragmentation, cross-linking, and rHDL sizes. The functional studies will include the determination of the kinetics of the LCAT reaction in our laboratory, and a collaborative study of cell binding and cholesterol efflux from cells.
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