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.
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