BACKBONE DYNAMICS OF THROMBIN AND THROMBIN-THROMBOMODULIN COMPLEXES

凝血酶和凝血酶-血栓调节蛋白复合物的骨架动力学

• 批准号: 8361179
• 负责人: ELIZABETH A. KOMIVES
• 金额: $ 0.63万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361179
• 关键词: Active Sites; Affect; Anticoagulants; Binding; Complex; Development; Disseminated Intravascular Coagulation; Enzymes; Escherichia coli; Funding; Grant; Label; Measures; National Center for Research Resources; Principal Investigator; Protein C; Relaxation; Research; Research Infrastructure; Resources; Source; Structure; Thrombin; Thrombomodulin; United States National Institutes of Health; Vertebral column; cost; research study

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We have gathered strong evidence that binding of thrombomodulin (TM) at exosite 1 on thrombin alters the active site of the enzyme towards protein C activation. Our hypothesis is that TM alters thrombin by "dynamic allostery". This discovery has important implications for the development of better anticoagulants and for understanding the potential of TM fragments in the treatment of disseminated intravascular coagulation. To gain information on the dynamic allostery in the thrombin-TM interaction, we will measure backbone dynamics by NMR. Since the expression of thrombin has only recently been accomplished in E. coli, we are one of the first labs to produce 15N, 13C, 2H-labeled thrombin, we first need to obtain the backbone assignments. The dynamics of free thrombin will be compared active site-inhibited thrombin. Finally, we will deermine how the backbone dynamics of thrombin are affected by TM binding. NMR relaxation experiments will be performed to measure the backbone dynamics of thrombin in the presence and absence of TM fragments. These experiments will reveal backbone dynamics changes that occur within thrombin upon TM binding that are not visible from the crystal structure.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 我们收集到强有力的证据表明，血栓调节蛋白 (TM) 在凝血酶外位点 1 处的结合会改变该酶的活性位点，从而激活蛋白 C。 我们的假设是 TM 通过“动态变构”改变凝血酶。 这一发现对于开发更好的抗凝剂以及了解 TM 片段在治疗弥散性血管内凝血中的潜力具有重要意义。 为了获得凝血酶-TM 相互作用中动态变构的信息，我们将通过 NMR 测量主链动力学。由于凝血酶的表达最近才在大肠杆菌中完成，我们是最早生产 15N、13C、2H 标记凝血酶的实验室之一，因此我们首先需要获得主干分配。 游离凝血酶的动力学将与活性位点抑制的凝血酶进行比较。 最后，我们将确定 TM 结合如何影响凝血酶的主链动力学。 将进行 NMR 弛豫实验来测量在 TM 片段存在和不存在的情况下凝血酶的主链动力学。 这些实验将揭示 TM 结合时凝血酶内发生的主链动力学变化，这些变化从晶体结构中是不可见的。