STUDY OF SERPIN INHIBITION MECHANISM

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。 抑制性丝氨酸是一类结构上具有相同功能的同源蛋白，它们与不能催化底物水解酶的靶蛋白形成长寿命、稳定的十二烷基硫酸钠共价复合体。这些复合体被表示为E*I*，以强调在形成复合体时两种蛋白质中发生的构象变化。对于每个丝氨酸蛋白来说，反应中心环(RCL)是与其目标蛋白酶相互作用的主要部位。这些结构和功能上的相似性导致许多研究人员隐含地假设，对于一个丝氨酸-蛋白酶对所发现的机制结果或多或少可以推广到所有这样的对。然而，随着更多关于丝氨酸-蛋白酶相互作用的细节的出现，这一假设受到了质疑，揭示了越来越多的明显差异。一个这样的差异与E*I*复合体的结构有关。一些证据支持这样一种结构(记为E*I*1)，在这种结构中，连接到P1残基的酶保持在靠近蛇夹顶部的位置，定义为RCL在完整的、有活性的蛇夹中的位置，而另一些证据表明，这种结构(记为E*I*2)中的酶已经跨越整个蛇夹的长度(约70E)移位到丝夹底部。评估表观差异的一个复杂因素是，并不总是清楚差异是否反映了丝氨酸：蛋白酶对之间的真正差异，或者更确切地说，是由于不同实验室使用的实验条件的差异，还是由于对模棱两可的结果的解释的差异。在此背景下，本提案的主要目的是利用一套共同的实验方法和条件，对不同的丝氨酸-蛋白水解酶对E*I*复合体的形成动态机制进行详细的描述，以便能够严格比较差异和相似之处。具体地说，对于三对丝氨酸：蛋白酶复合体、糜蛋白酶：1-抗糜蛋白酶(CHTR：ACT)、胰酶：1-抗酪蛋白和凝血酶：抗凝血酶III(ATIII)(肝素激活)，我们将解决以下问题：P14-P1 RCL二级结构是如何沿着导致E*I*的反应途径变化的？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光谱(FT-IR)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.