STUDY OF SERPIN INHIBITION MECHANISM

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。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。所列机构为中心，不一定是研究者所在机构。抑制性丝氨酸蛋白酶抑制剂构成了一个结构同源蛋白家族，具有与不能催化蛋白酶底物水解的靶蛋白酶形成长效、SDS稳定的共价复合物的相同总体功能。这些复合物表示为E*I*，以强调复合物形成时两种蛋白质中发生的构象变化。对于每个丝氨酸蛋白酶抑制剂，反应中心环（RCL）是与其靶蛋白酶相互作用的主要位点。这些结构和功能的相似性，导致许多研究人员含蓄地假设，发现一个丝氨酸蛋白酶抑制剂蛋白酶对的机械结果或多或少概括到所有这样的对。然而，随着丝氨酸蛋白酶抑制剂-蛋白酶相互作用的更多细节的出现，这一假设受到了质疑，揭示了越来越多的明显差异。其中一个差异涉及E*I* 复合体的结构。一些证据支持这样一种结构（表示为艾德E*I*1），即附着在P1残基上的酶仍然靠近丝氨酸蛋白酶抑制剂的顶部，定义为RCL在完整的活性丝氨酸蛋白酶抑制剂中的位置，而另一些证据则表明这样一种结构（表示为E*I*2），即酶已经穿过丝氨酸蛋白酶抑制剂的整个长度（约70 E）移位到丝氨酸蛋白酶抑制剂的底部。在评估明显差异时的一个复杂因素是，并不总是清楚差异是否反映了丝氨酸蛋白酶抑制剂：蛋白酶对之间的真实的差异，或者是由于不同实验室使用的实验条件的差异或对模糊结果的解释的差异。在此背景下，本建议的主要目的是获得一个详细的图片的动态机制E*I* 复合物形成不同的丝氨酸蛋白酶抑制剂-蛋白酶对，使用一组共同的实验方法和条件，将允许严格比较的差异和相似之处。具体而言，对于三对丝氨酸蛋白酶抑制剂：蛋白酶复合物，胰凝乳蛋白酶：1-抗胰凝乳蛋白酶（Chtr：ACT），胰蛋白酶：1-抗胰蛋白酶和凝血酶：抗凝血酶III（ATIII）（肝素活化），我们将解决以下问题：P14-P1 RCL二级结构如何改变沿着导致E*I* 的反应途径？这种变化的时机是什么？这个问题将使用时间分辨傅里叶变换红外（FT-IR）光谱和13 C/12 C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.