The broad, long-term objectives of this research proposal are to develop an improved understanding of protein-protein recognition phenomena by examining in detail the structure-function relationships and interfacial dynamics of Kunitz-type protease inhibitors complexed with serine proteases. Serine proteases, and the Kunitz protease inhibitors that regulate them, play an increasingly well-recognized role in human physiology and in the pathology of nearly all important illnesses, including cardiovascular, neoplastic, inflammatory and neurodegenerative diseases.
The specific aims of this grant application are: 1. To apply heteronuclear NMR to compare 1H, 13C, and 15N chemical shifts, amide 1H/2H exchange rates, structures, dynamics, and hydration of wild-type BPTI free in solution and bound to the bovine proteolytic enzymes trypsin and chymotrypsin. 2. To characterize effects of alanine substitutions on dynamics, binding kinetics and thermodynamics of BPTI-trypsin complexes using NMR, continuous flow NMR, and titration calorimetry. 3. To determine extensive 15N, 13C, and 1H resonance assignments, high resolution solution structure, and internal dynamics in the Alzheimer's beta amyloid precursor protein Kunitz inhibitor (APPKI). 4. To apply heteronuclear NMR and titration calorimetry to compare the structures, dynamics, and hydration of APPKI free in solution and bound to the proteolytic enzyme trypsin. 5. To apply heteronuclear NMR to determine the structure, dynamics, and hydration of APPKI bound to the serine protease domain of human Factor IXa. Structural and dynamic information on molecular recognition by Kunitz inhibitor-serine protease complexes will be obtained from heteronuclear NMR experiments using 15N, 13C, and/or 2H-enriched recombinant proteins. Dynamic information will also be obtained from amide 1H/2H exchange and 15N relaxation measurements on free and protease-bound inhibitor molecules. Comparisons of NMR data with results from X-ray crystallography, alanine scanning mutagenesis, and titration calorimetry will be used to provide a comprehensive view of interfacial phenomena. The characteristics of kinetic intermediates in the BPTI-trypsin association reaction will also be studied by continuous flow NMR. Work will proceed in a methodical fashion from extremely well-understood model systems to targets of biomedical importance.
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