Serine proteinase inhibitors (serpins) play a central role in a wide variety of physiologic processes involved in aging. This is especially evident in diseases that increase as a function of age, including certain cancers, cardiac and lung diseases and degenerative diseases of the central nervous system and neuromuscular system. These processes critically depend on the balance of proteinases and proteinase inhibitors, a balance that is ultimately determined by the molecular properties of the reaction between the two proteins. Inhibitory serpins form a family of structurally homologous proteins with the same overall function of forming long-lived, SDS stable acyl enzyme complexes with target proteinases (denoted E*I*) incapable of catalyzing hydrolysis of proteinase substrates. These structural and functional similarities have led to the assumption 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 that indicate substantial disparities. Complicating this picture is that it is not always clear whether such disparities reflect real differences between serpin:proteinase pairs, or are rather due to differences in experimental conditions or in interpretations of data. 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 the three pairs of serpin:proteinase complexes Chtr:ACT, trypsin:antitrypsin and thrombin: antithrombin the principal investigator will endeavor to answer questions regarding: 1) the number of identifiable intermediates in the transition from the encounter complex E.I to the metastable covalent complex E*I*; 2) the structure of the E*I* complex; 3) the timing of changes in P14-Pl RCL secondary structure change along the reaction pathway leading to E*I*; 4) the timing of the opening up of the 'shutter region' believed to be important for E*I* formation; 5) the reversibility of E*I* formation; and 6) which aspects of the structural differences of the serpin:proteinase pairs are most significant for the mechanistic differences observed in answering questions 1) - 5). To address these questions the principal investigator will employ native, variant, chemically modified and/or isotopically-labeled forms of serpins and proteinases in making four time-resolved measurements: fluorescence resonance energy transfer, fourier-transform infrared spectroscopy, monoclonal antibody binding, and E*I* formation.
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