Structure/function analysis will continue on the interaction of proteins with the surface of membranes and with steroids. Both processes figure heavily in the control of intracellular metabolism; the former through the enzymatic release of second mssengers from the cytosolic leaflet of the plasma membrane; the later through steroid activation of genetic control elements. Our principal tool will be high resolution crystallography of the appropriate proteins and of their physiologically important complexes. Phosphlipase A2 (PLA2) offers an ideal model for studying the interaction of proteins with membrane surfaces since it prefers to attack phospholipid head groups if they are in membranes or membrane-like aggregates. Despite several high resolution crystal structures of PLA2 we do not yet understand its action. To resolve this issue we will continue to systematically build a stereochemical picture of the enzyme's functional interactions. The steps are: (1) To complete the 1.7 A refinement of the ligand free form of C. atrox PLA2; (2) To solve the high resolution crystal structure of the calcium complex of C. atrox of PLA2; (3) To solve the high resolution crystal structure of crotoxin, a phosphlipiasic neurotoxin whose action is targeted to the presynaptic membrane; (4) To prepare crystals of PLA2 in complexes with phosphlipid analogues that emulate the interaction between PLA2 and phosphlipids in membrane-like aggregates. This requires the synthesis of stable analogues in which the head groups are constrained to resemble those in an aggregate, but that are small enough to cocrystallize with the enzyme; (5) To explore the preparation of crystalline intracellular phospholipases that mediate internal cell responses. As a first model for protein steroid interaction, we will interpret the 2.5 A map of delta 5-KSI and contrast the structure with that of its isomorphous steroid inhibitor complex. Once refined, these structures should reveal the enzyme's mechanism of specific steroid binding and catalysis. We hope to prepare crystals of the rather plentiful human sex steroid binding globulin and ultimately to crystallize estrogen and progesterone receptors.
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