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完全了解化学细节 这表征了膜水接口处的关键反应。 我们 将同时继续在两个层次上继续进行； （1）高分辨率 晶体学，生化和突变以及对系统的研究 衍射质量晶体已经存在；主要是磷脂酶A2。 （2）开发新的结晶系统以研究问题 生物过程的立体化学知之甚少。为了 例如，受体/G蛋白系统。 1。磷脂酶A2（PLA2）作用的机制：我们将继续集中精力 关于磷脂酶A2（PLA2）的结构和作用。 作为钙 - 需要攻击层状和胶束中磷脂的酶 聚集体，PLA2充当脂质/水的作用范式 界面。 此外，对膜磷脂的攻击位于 信号中许多第二个使者释放的控制点 转导级联。 建立了规范的立体化学 催化机制和生产模式结合，我们将继续 如下所示：（a）我们将更好地定义 绑定水的分布和涉及的质子的位置 通过将X射线和中子晶体学扩展到1.5 A来催化 母酶及其过渡状态类似物的衍射极限 复合物； （b）我们将在生化和晶体上探索新的 旨在探测催化和特定机制的类似物 结合。 （c）我们将研究部位的生物化学和晶体结构 旨在测试功能推断的定向突变变体 从晶体学工作中得出。 （d）我们将通过低剂量建立 和电子衍射相对于PLA2分子的处置 生产模式下层状底物骨料的面孔 结合。 （e）我们将加强对发炎的PLA2的关注 滑腔。 我们的目的是更好地了解 蛛网释放，从而为设计提供了合理的方法 抗炎剂。 2。受体/G蛋白。 我们将启动有关晶体学研究 通过受体/g-信号转导的机理 蛋白/磷酸二酯酶（或磷脂酶C）系统。 我们的主要目标是 开发和研究将揭示的结晶标本 活化受体和信号转导的立体化学 它的同性G蛋白和/或 磷酸化的受体和抑制蛋白。