HIGH DENSITY LIPOPROTEIN STRUCTURE FUNCTION CORRELATIONS

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The overall objective of this research is to elucidate, in molecular terms, the composition-structure-function relationship in human high density lipoproteins (HDL). Using reconstituted HDL (rHDL) containing normal apolipoprotein A-I (apo A-I), apo A-I fragments, or a mutant apo A-I, in conjunction with pure lipids, we propose to investigate the three- dimensional folding of this apolipoprotein, and its role in defining the subclasses of discoidal and spherical rHDL, and their reactivity with lecithin cholesterol acyltransferase. Furthermore, we plan to isolate a homogeneous fraction of native HDL3 containing only apo A-I, and to prepare rHDL from apo A-I isolated by detergent solubilization methods, with the objective of comparing the structure and function of "native" apo A-I with its properties in the conventionally isolated form (by organic solvent extraction). The HDL particles and their apo A-I and lipid components will be characterized by a variety of biochemical and biophysical approaches including isolation of the particles by ultracentrifugal and FPLC methods, analysis by non-denaturing gradient gel electrophoresis, investigation of the protein structure by fluorescence, CD, and FT-TR spectroscopy, monoclonal antibody binding, and limited proteolysis and polypeptide sequencing. The order and dynamics of the lipid constituents of the HDL will be studied by fluorescence and FT-TR methods, and the biological function of all the HDL particles will be assessed by their ability to activate the lecithin cholesterol acyltransferase reaction, using radiolabeled lipids in the enzymatic assays.