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.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 我们收集了有力的证据表明，在凝血酶上的外志1上的血小板结合蛋白（TM）结合将酶的活性位点改变蛋白C激活。 我们的假设是TM通过“动态变构”来改变凝血酶。 这一发现对更好的抗凝剂的发展具有重要意义，并了解TM片段在治疗传播血管内凝血中的潜力。 为了获得有关凝血酶TM相互作用中动态变构的信息，我们将通过NMR测量骨干动力学。由于凝血酶的表达直到最近才在大肠杆菌中完成，因此我们是最早产生15N，13C，2H标记的凝血酶的实验室之一，我们首先需要获得骨干分配。 释放凝血酶的动力学将被比较活跃的位点抑制凝血酶。 最后，我们将去除了TM结合的影响凝血酶的骨干动力学如何。 将进行NMR弛豫实验，以测量存在和不存在TM片段的凝血酶的主链动力学。 这些实验将揭示在TM结合后凝血酶内发生的骨干动力学变化，而TM结合在晶体结构中不可见。