STRUCTURAL STUDIES OF LIPID/PROTEIN SYSTEMS

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DESCRIPTION (Adapted from abstract): The long-term objective is to provide data on the interaction of lipids with transport proteins or enzymes. To do this, the molecular structure of lipid-containing macromolecules will be used to describes lipid-protein interactions. A nearly complete study of lipovitellin has led to similar studies of microsomal triglyceride transfer protein (MTP), which is involved in assembly of the serum lipoproteins. A major aim is to compete the crystal structure of a complex of MTP with protein disulfide isomerase. For lipovitellin, new x-ray measurements have provided preliminary evidence about the conformation of 20 bound acyl chains. The new data will be refined and an effort will be made to reconstruct the entire domain of phospholipids present in lipovitellin. Attempts to crystallize apo A1, the principal protein component of HDL will be continued. In the beta-oxidation spiral, more than one enzyme appears to be associated with each reaction. For the beta-OH to beta-ketoacyl CoA reaction, humans have two different enzymes: one for short chain L-3-hydroxyacyl CoA substrates (SCHAD) and one for long chain metabolites (LCHAD). Preliminary x-ray data have identified the coenzyme site of SCHAD. The important goal now is to characterize the molecular structure that defines the origin of fatty acid chain specificity. This will be done by x-ray analyses of SCHAD associated with appropriate substrates. In a second stage, the LCHAD segment of a multifunctional protein will be expressed in E. coli, crystallized, and the 3D structure determined by diffraction methods. In a continuation of structural studies of intracellular proteins for shuttling fatty acids and retinoids, mutagenesis, titration calorimetry, and x-ray crystallography will be used to explore ligand specificity. One approach will combine experimental determination of proton binding with electrostatic calculations of characteristic pKs.