STUDY OF SERPIN INHIBITION MECHANISM

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。抑制性蛇形蛋白是一个结构上同源的蛋白家族，具有与靶蛋白酶形成长寿命、sds稳定的共价复合物的整体功能，而靶蛋白酶不能催化蛋白酶底物的水解。这些复合物用E*I*表示，以强调两种蛋白质在复合物形成过程中发生的构象变化。对于每个蛇形蛋白，反应性中心环（RCL）是与其靶蛋白酶相互作用的主要位点。这些结构和功能上的相似性使许多研究者隐含地假设，对一个蛇形蛋白蛋白酶对发现的机制结果或多或少可以推广到所有这样的对。然而，随着蛇形蛋白-蛋白酶相互作用的更多细节的出现，这一假设受到了质疑，揭示了越来越多的显著的明显差异。其中一个差异与E*I*复合物的结构有关。一些证据支持一种结构结构（标记为E*I*1），在这种结构中，附着在P1残基上的酶仍然靠近蛇形蛋白的顶部，定义为完整的活性蛇形蛋白中RCL的位置，而其他证据显示一种结构（标记为E*I*2），在这种结构中，酶已经在整个蛇形蛋白长度（约70 E）上转移到蛇形蛋白的底部。评估明显差异的一个复杂因素是，这种差异是否反映了丝氨酸蛋白酶对之间的真正差异，或者是由于不同实验室使用的实验条件的差异，或者是由于对模糊结果的解释的差异，并不总是很清楚。在此背景下，本研究的主要目的是利用一套通用的实验方法和条件，对不同蛇形蛋白蛋白酶对形成E*I*复合物的动态机制进行详细的描述，从而对差异和相似性进行严格的比较。具体来说，对于三对丝氨酸蛋白酶复合物，凝乳胰蛋白酶：1-抗凝乳胰蛋白酶（Chtr:ACT），胰蛋白酶：1-抗胰蛋白酶和凝血酶：抗凝血酶III (ATIII)（肝素激活），我们将解决以下问题：P14-P1 RCL二级结构如何沿着导致E*I*的反应途径变化？这些变化的时机是什么？这个问题将通过时间分辨傅里叶变换红外（FT-IR）光谱和13C/12C同位素编辑来解决。

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. Inhibitory serpins constitute a family of structurally homologous proteins with the same overall function of forming long-lived, SDS-stable, covalent complexes with target proteinases that are incapable of catalyzing hydrolysis of proteinase substrates. These complexes are denoted E*I* to emphasize the conformational changes that occur in both proteins on complex formation. For each serpin, the reactive center loop (RCL) is the primary site for interaction with its target proteinase. These structural and functional similarities have led many investigators to implicitly assume that mechanistic results found for one serpin-proteinase pair were more or less generalizable to all such pairs. However, this assumption has been called into question as more details of serpin-proteinase interactions have emerged, revealing an increasing number of significant apparent disparities. One such disparity concerns the structure of the E*I* complex. Some evidence favors a structu re (denot ed E*I*1) in which enzyme attached to the P1 residue remains close to the top of the serpin, defined as the position of the RCL in the intact, active serpin, while other evidence shows a structure (denoted E*I*2) in which enzyme has translocated across the entire length of the serpin (some 70 E) to the serpin bottom. A complicating factor in evaluating an apparent disparity is that it is not always clear whether the disparity reflects a real difference between serpin:proteinase pairs, or is rather due either to differences in experimental conditions used in different laboratories or to differences in interpretations of ambiguous results. Against this background, the major aim of this proposal is to obtain a detailed picture of the dynamic mechanism of E*I* complex formation for different serpin-proteinase pairs, using a common set of experimental approaches and conditions that will permit a rigorous comparison of differences and similarities. Specifically, for three pairs of serpin:proteinase complexes, chymotrypsin:1-antichymotrypsin (Chtr:ACT), trypsin:1-antityrpsin and thrombin: antithrombin III (ATIII) (heparin activated) we will address the following question: How does the P14-P1 RCL secondary structure change along the reaction pathway leading to E*I*? What is the timing of such changes? This question will be addressed using time-resolved Fourier Transform Infra-Red (FT-IR) spectroscopy and 13C/12C isotope-editing.