MOLECULAR INTERACTIONS AT THE CELL SURFACE

细胞表面的分子相互作用

• 批准号: 3271099
• 负责人: PAUL B SIGLER
• 金额: $ 18.81万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-01-01 至 1990-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3271099
• 关键词: atomic absorption spectrometry; calcium binding protein; computer graphics /printing; enzyme complex; enzyme inhibitors; enzyme substrate analog; hormone binding protein; hormone receptor; membrane activity; membrane lipids; membrane proteins; phospholipase A2; phospholipids; stereochemistry; steroid delta isomerase; steroid hormone metabolism

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.

结构/功能分析将继续对蛋白质的相互作用 与细胞膜表面和类固醇结合。 这两个过程都显示 在很大程度上控制细胞内代谢;前者通过 第二信使核糖核酸从细胞质小叶的酶促释放 质膜;后者通过类固醇激活遗传控制 元素 我们的主要工具将是高分辨率晶体学， 合适的蛋白质及其生理上重要的复合物。 磷脂酶A2（PLA 2）是研究蛋白质与蛋白质相互作用的理想模型 因为它更喜欢攻击磷脂 头基，如果它们在膜或膜样聚集体中。 尽管 我们还不了解PLA 2的几种高分辨率晶体结构 它的行动。 为了解决这个问题，我们将继续系统地建立 酶功能相互作用的立体化学图。 的 步骤是：（1）完成1.7 A的配体游离形式的精制， C. atrox PLA 2的高分辨晶体结构 C.钙络合物（3）解决了PLA 2的高分辨率问题 crotoxin的晶体结构，一种磷酸化神经毒素，其作用是 （4）制备PLA_2的晶体， 与磷脂类似物的复合物， 膜样聚集体中的PLA 2和磷脂。 这需要 合成稳定的类似物，其中头部基团被限制为 类似于聚集体中的那些，但它们足够小以共结晶 （5）探索制备结晶性的 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 三角洲5-KSI图，并与其构造对比 同晶类固醇抑制剂复合物。 一旦被提炼，这些结构 应该揭示酶的特殊类固醇结合机制， 催化作用 我们希望准备大量的人类性别的晶体 类固醇结合球蛋白，并最终结晶雌激素， 孕酮受体