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: 8168987
• 负责人: ELIZABETH A. KOMIVES
• 金额: $ 0.53万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8168987
• 关键词: Active Sites; Affect; Anticoagulants; Binding; Complex; Computer Retrieval of Information on Scientific Projects Database; Development; Disseminated Intravascular Coagulation; Enzymes; Escherichia coli; Funding; Grant; Institution; Label; Measures; Protein C; Relaxation; Research; Research Personnel; Resources; Source; Structure; Thrombin; Thrombomodulin; United States National Institutes of Health; Vertebral column; research study

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 我们已经收集了强有力的证据表明，凝血酶调节蛋白（TM）在凝血酶上的外位点1的结合改变了蛋白C活化的酶的活性位点。 我们的假设是TM通过“动态变构”改变凝血酶。 这一发现对开发更好的抗凝剂和了解TM片段在治疗弥散性血管内凝血中的潜力具有重要意义。 为了获得关于凝血酶-TM相互作用中的动态变构的信息，我们将通过NMR测量主链动力学。由于凝血酶的表达只是最近才在E.大肠杆菌中，我们是最早生产15 N，13 C，2 H标记凝血酶的实验室之一，我们首先需要获得主链归属。 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，我们将确定凝血酶的骨架动力学如何受到TM结合的影响。 将进行NMR弛豫实验，以测量存在和不存在TM片段时凝血酶的骨架动力学。 这些实验将揭示TM结合时凝血酶内发生的骨架动力学变化，这些变化从晶体结构中不可见。