MOLECULAR INTERACTIONS AT THE CELL SURFACE

细胞表面的分子相互作用

• 批准号: 2173917
• 负责人: PAUL B SIGLER
• 金额: $ 22.51万
• 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 1995-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2173917
• 关键词: G protein; X ray crystallography; active sites; biological signal transduction; cell membrane; chemical aggregate; electron microscopy; enzyme complex; enzyme inhibitors; enzyme mechanism; inflammation; intermolecular interaction; macromolecule; membrane lipids; neutron diffraction; phosphodiesterases; phospholipase A2; phospholipase C; protein structure function; receptor; site directed mutagenesis; stereochemistry

Our purpose is to understand as exactly as possible the chemical details that characterize the key reactions at the membrane aqueous interface. We will continue to proceed at two levels simultaneously; (1) High resolution crystallographic, biochemical and mutational and studies on systems where diffraction-quality crystals already exist; primarily, phospholipase A2. (2) Development of new crystalline systems to study problems where the stereochemistry of the biological process is poorly understood; for example, the receptor/G protein system. 1. Mechanism of phospholipase A2 (PLA2) action: We will continue to focus on the structure and action of phospholipase A2 (PLA2). As a calcium- requiring enzyme that attacks phospholipids in lamellar and micellar aggregates, PLA2 serves as a paradigm for action at the lipid/water interface. Moreover, the attack on membrane phospholipids stands at the control point of the release of many second messengers in the signal transduction cascade. Having established the canonical stereochemistry of both the catalytic mechanism and productive-mode binding, we will proceed as follows to complete our understanding: (a) We will better define the distribution of bound water and the location of protons involved in catalysis by extending X-ray and neutron crystallography to the 1.5 A diffraction limit of parent enzyme and its transition-state analogue complexes; (b) We will explore biochemically and crystallographically new analogues designed to probe the mechanism of catalysis and specific binding. (c) We will study the biochemistry and crystal structure of site- directed mutational variants designed to test the functional inferences drawn from the crystallographic work. (d) We will establish by low dose EM and electron diffraction the disposition of the PLA2 molecule relative to the face of the lamellar substrate aggregate during productive mode binding. (e) We will intensify our focus on the PLA2 from the inflamed synovial cavity. Our purpose here is to better understand the mechanism of arachidonate release and thereby provide a rational approach to the design of anti-inflammatory agents. 2. Receptor/G proteins. We will initiate crystallographic studies on the mechanism of signal transduction through the receptor/G- protein/phosphodiesterase (or phospholipase C) system. Our main goal is to develop and study crystalline specimens that will reveal the stereochemistry of signal transduction between an activated receptor and its cognate G protein and/or of the inhibitory interaction between a phosphorylated receptor and arrestin.

我们的目的是尽可能准确地了解化学细节 表征膜水界面的关键反应。 我们 将继续在两个层次上同时进行;（1）高分辨率 结晶学、生物化学和突变学，以及对系统的研究， 衍射质量的晶体已经存在;主要是磷脂酶A2。 (2)开发新的晶体系统来研究 生物过程的立体化学知之甚少; 例如，受体/G蛋白系统。 1.磷脂酶A2（PLA 2）的作用机制：我们将继续关注 磷脂酶A2（PLA 2）的结构和作用。 作为一种钙- 需要酶攻击磷脂在层状和胶束 聚集体，PLA 2作为作用于脂质/水的范例 接口. 此外，对膜磷脂的攻击在 信号中许多第二信使释放的控制点 转导级联 建立了的典型立体化学 无论是催化机制和生产模式的约束，我们将继续 （一）我们将更好地理解 结合水的分布和参与质子的位置 通过将X射线和中子晶体学扩展到1.5A的催化 母体酶及其过渡态类似物的衍射极限 复合物;（B）我们将探索生物化学和晶体学新的 类似物设计用于探测催化和特异性的机制 约束力 (c)我们将研究生物化学和晶体结构的网站- 定向突变变体，旨在测试功能推断 从晶体学工作中提取的。 (d)我们将通过低剂量EM 和电子衍射的PLA 2分子的相对于 在生产模式期间层状基底的表面聚集 约束力 (e)我们将加强对PLA 2的关注， 滑膜腔 我们的目的是更好地理解 花生四烯酸释放，从而提供了一个合理的方法， 消炎药的作用 2.受体/G蛋白。 我们将开始晶体学研究 通过受体/G- 蛋白质/磷酸二酯酶（或磷脂酶C）系统。 我们的主要目标是 开发和研究晶体标本， 活化受体和受体之间信号转导的立体化学 其同源G蛋白和/或其同源G蛋白之间的抑制性相互作用。 磷酸化受体和抑制蛋白。