The major objective of the revised PPG is to investigate the tertiary structure, function and dynamics of lipid-associated exchangeable apolipoproteins and their interactions with other apolipoproteins. Our central hypothesis is that the properties of the different apolipoprotein domains involved in the workings of the HDL:TG-rich lipoprotein nexus---the metabolic network linking HDL to VLDL, chylomicrons and their remnants--- are determined by predictable variations in the amphipathic helix motif between and among the exchangeable apolipoproteins. We propose to use the preliminary maps we have generated of the amphipathic helixes in apo A-I, C-III, C-I and E as working models for a structural molecular biology and molecular biophysics approach to an understanding of the molecular basis for the apolipoprotein-lipid and apolipoprotein-apolipoprotein interactions involved in the HDL:TG-rich lipoprotein nexus. Integral to the growth and evolution of our program, we have incorporated several new research strategies and directions into our PPG: these include computer-aided analysis of the different classes of amphipathic helixes in exchangeable apolipoproteins, site-directed mutagenesis of apolipoproteins, and computer-based molecular dynamic simulations of lipids and lipid-protein interactions. Projects l, 2 and 3 represent three different, but interdependent and complementary, experimental approaches to a common structural/functional problem: the relationship of the amphipathic helix to apolipoprotein function, including lipid interactions. Project 1 proposes to study this problem via synthesis and analysis of peptide analogs of the amphipathic helix. Project 2 will apply site-directed mutagenesis and expression of apolipoprotein recombinants to the study of the structural/function problem in intact apolipoproteins. Project 3 proposes to use computer-based molecular modeling, a well established method for the study of protein and nucleic acid structure and dynamics, to develop tools for the study of the otherwise virtually inaccessible problem of the dynamics of apolipoprotein-lipid interactions. Three core facilities are proposed to provide support for the three proposed projects. These are: an administrative core A, a lipoprotein core B, and a new instrumentation and computer core C.
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