BACKBONE DYNAMICS OF THROMBIN AND THROMBIN-THROMBOMODULIN COMPLEXES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们已经收集了强有力的证据表明，血栓调节蛋白(TM)在外切酶1与凝血酶的结合改变了酶对蛋白C激活的活性部位。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血剂和了解TM片段在治疗弥漫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，我们将通过核磁共振测量骨架动力学。由于凝血酶在大肠杆菌中的表达是最近才完成的，我们是最早生产15N，13C，2H标记凝血酶的实验室之一，我们首先需要获得骨架的指认。游离型凝血酶的动力学将与活性部位抑制凝血酶进行比较。最后，我们将确定TM结合如何影响凝血酶的骨架动力学。核磁共振弛豫实验将被用来测量在存在和不存在TM片段的情况下凝血酶的骨架动力学。这些实验将揭示凝血酶与TM结合时发生的主干动力学变化，这些变化从晶体结构中看不到。

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.