BIOPHYSICAL STUDIES OF THROMBOMODULIN EGF DOMAINS

血栓调节蛋白 EGF 结构域的生物物理学研究

• 批准号: 2519328
• 负责人: ELIZABETH A. KOMIVES
• 金额: $ 18.07万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-02-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2519328
• 关键词: X ray crystallography; active sites; biophysics; conformation; crystallization; enzyme activity; epidermal growth factor; mutant; nuclear magnetic resonance spectroscopy; protein C; protein purification; protein sequence; protein structure function; prothrombin; site directed mutagenesis; thrombin; thrombomodulin; yeasts

DESCRIPTION: Thrombomodulin (TM) is an essential anticoagulant protein that binds to thrombin and the resulting complex shuts down further production of thrombin. In the complex, the thrombin active site has new substrate specificity toward protein C, which when activated by the thrombin- TM complex shuts down the coagulation cascade. The proposed experiments will analyze both the structure and function of TM in order to obtain an atomic-level understanding of how TM alters the activity of thrombin. Structural information gained from multi-dimensional NMR methods as well as X-ray crystallography will be integrated with functional information gained from site-directed mutagenesis of residues in both TM and thrombin. Particular emphasis will be placed on the nature of the protein-protein interaction and on the conformational changes that occur during the interaction. The experimental plan is as follows: 1) Determine the structure of the smallest active fragment of TM , TMEGF(4-5) using multi-dimensional NMR methods. 2) Determine the function of essential residues within the fourth EGF-like domain of TM. Essential residues that have been shown to coalesce in a clefT on the fourth domain will be conservatively mutated and analyzed for functional changes using clotting, protein C activation, and direct binding assays. 3) Determine the function of essential residues within the fifth EGF-like domain of TM. Essential residues within the C-terminal loop and tail will be conservatively mutated and each mutant will be analyzed for functional changes as in Aim 2. 4) Determine the structural consequences of interesting functionally altered mutants using methods developed in Aim 1. 5) Express the S195A mutant of human prothrombin-2 in large quantities in Pichia pastoris. This mutant thrombin is required for both NMR and X-ray crystallographic studies. 6) Determine the surface of TMEGF(4-5) that interacts with thrombin using a combination of X-ray crystallography and isotope-edited NMR as well as complementary mutagenesis of residues on both TM and thrombin.

描述：血小子瘤蛋白（TM）是一种必不可少的抗凝蛋白 与凝血酶结合，由此产生的复合物关闭了进一步的生产 凝血酶。 在综合大楼中，凝血酶活性位点具有新的底物 蛋白C的特异性，当蛋白C激活时 复合物关闭凝血级联。 提出的实验将 分析TM的结构和功能，以获得 原子水平的理解TM如何改变凝血酶的活性。 从多维NMR方法获得的结构信息以及 X射线晶体学将与获得的功能信息集成 从TM和凝血酶中残基的位置定向诱变。 特别重点将放在蛋白质蛋白质的性质上 相互作用和在构象中发生的变化 相互作用。 实验计划如下：1）确定 TM的最小活性片段的结构，TMEGF（4-5）使用 多维NMR方法。 2）确定必需品的功能 TM的第四个EGF样结构域中的残基。 必不可少的残留物 已显示在第四个领域的裂缝中结合在一起 保守突变并分析了使用凝结的功能变化， 蛋白C激活和直接结合测定。 3）确定功能 TM的第五个EGF样结构域中的基本残基。 基本的 C末端环和尾部内的残留物将保守突变 并且将分析每个突变体的功能变化，如AIM2。4） 确定有趣的功能变化的有趣的结构后果 使用目标1。5）表达S195a突变体的突变体 Pichia Pastoris中大量的人类凝血酶蛋白-2。 这个突变体 NMR和X射线晶体学研究都需要凝血酶。 6） 确定使用A与凝血酶相互作用的TMEGF（4-5）的表面 X射线晶体学和同位素编辑的NMR以及 在TM和凝血酶上残基的互补诱变。