LIPOPROTEIN STRUCTURE AND APOPROTEIN CONFORMATION

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Our long-term objectives are to elucidate in molecular detail the structure, stability and dynamic properties of the plasma lipoproteins and apolipoproteins that are vital to understanding of lipid interactions, apoprotein exchange, lipoprotein cell surface interactions, receptor- mediated lipoprotein uptake, and lipoprotein inter-conversions. The stabilization and their functional roles as cofactors for enzymes, ligands for receptors, or mediators of reverse cholesterol transport. A detailed understanding of apoprotein structural stability and adaptability is vital to further progress in understanding lipoprotein structure and function. The precise molecular mechanisms of this unique structural adaptability remain unclear, and will be the focus of the proposed research. Continuing our previous work, we will focus on structural investigations of lipoproteins (HDL and LDL) and apolipoproteins to highest possible resolution, using state-of-the-art methods of molecular biophysics and structural biology. The structure and stabilizing interactions of synthetic of expressed peptides that model important structural and functional units in the sequences of the exchangeable peptides that model important structural and functional units in the sequences of the exchangeable apoproteins (primarily apoA-I) will be determined. These peptides will be designed on the basis of the 11/22 residue helical segments comprising native apoA-I, and on an "idealized" consensus sequence for the fundamental 11/22-mer tandem repeat in the sequence of the exchangeable apoproteins. Structural and themodynamic studies of mutant forms of apoA-I encompassing point and deletion mutants will concentrate on the role of specific regions of the apoA-I molecule in its conformation and stability. The three-dimensional structure of intact LDL, with emphasis on the topology and the molecular conformation of the apo-B100 at the lipoprotein surface will be determined by cryo- electron microscopy and 3D-image reconstruction. The focus will be on the analysis of the organization of apo-B and the localization of structural and functional domains of the LDL particle, using a combination of site- specific immuno-nanogold labeling and direct visualization of the bound LDL receptor.