Structure/function analysis will continue on the molecular details of the interactions of proteins with components of the membrane matrix such as phospholipids and other lipid-like compounds such as steroids. Presently the core of the research centers around the high resolution crystal structure analysis of two proteins; the dimeric phospholipase A2 (PLPA2) from the venom of C. atrox which attacks phopholipids only when they are in membranes and membrane-like aggregates; and the steroid metabolizing enzyme, Delta5-3-ketosteroid isomerase (KSI). The PLPA2 from C. atrox has been solved to 2.5 A resolution by multiple isomorphous replacement augmented with real-space direct methods (MIR/DM). We intend to refine the current model and extend the resolution initially to 1.7 Angstroms and ultimately to 1.5 Angstroms. The 1.7 Angstroms refined model will serve as a basis for comparative studies with the bovine monomeric PLPA2 which prefers more loosely aggregated substrates. The sterochemistry of substrate interaction and the role of Ca2+ (and its competitors) in this process will be examined. Attempts to crystallize other PLPA2 species will be undertaken to extend and strengthen our understanding of the molecular basis of protein-membrane interactions. The KSI sturcture has been solved to 6 Angstroms resolution by MIR/DM. Significant technical difficulties imposed by a 504 Angstroms c-axis and the presence of 4 protomers in each of the 12 asymmetric units have been overcome at the 6 Angstroms level. The structure analysis of the nascent enzyme and steroid complexes will be extended to 2.5 Angstroms resolution to visualize the molecular basis fo steroid recognition in productive-mode binding. Data for the parent and one heavy-atom derivative have been recorded photographically to 2.5 Angstroms using the synchrotron light source in Orsay. Crystallization of estrogen receptor proteins will be undertaken in collaboration with Dr. Elwood Jensen's group.
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