MOLECULAR INTERACTIONS AT THE CELL SURFACE

将继续对蛋白质之间的相互作用进行结构/功能分析 膜的表面和类固醇。这两个流程都显示 在很大程度上控制细胞内的新陈代谢；前者通过 黄曲霉胞质小叶第二信使的酶促释放 质膜；后者通过类固醇的激活进行遗传控制 元素。我们的主要工具将是高分辨率结晶学 适当的蛋白质及其生理上重要的复合体。 磷脂酶A2(PLA2)为研究相互作用提供了理想的模型 有膜表面的蛋白质，因为它更喜欢攻击磷脂 头部群，如果它们是在膜或膜状聚集体中。尽管 PLA2的几种高分辨晶体结构我们尚不了解 它的行动。为了解决这个问题，我们将继续系统地建立 这种酶的功能相互作用的立体化学图景。这个 步骤是：(1)完成1.7A的无配体形式的精制 (2)解决了赤霉菌PLA2的高分辨晶体结构 PLA2的阿曲霉钙络合物；(3)解决高分辨率 巴毒素的晶体结构，一种磷脂性神经毒素，其作用是 靶向突触前膜；(4)制备PLA2晶体 具有模拟相互作用的磷脂类似物的络合物 膜状聚集体中的PLA2和磷脂。这需要 头基受限制的稳定类似物的合成 类似于聚集体中的那些，但足够小，可以共结晶 用酶制剂；(5)探索结晶的制备方法 介导内部细胞反应的细胞内磷脂酶。 作为蛋白质类固醇相互作用的第一个模型，我们将解释2.5 一张三角洲5-KSI的地图，并将其结构与其 类固醇抑制剂复合体。一旦改进，这些结构 应该揭示酶与特定类固醇结合的机制和 催化作用。我们希望制备出相当丰富的人类性别的水晶 类固醇结合球蛋白，最终结晶雌激素和 黄体酮受体。

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.