The long term goal of this project is to elucidate the role of protein flexibility in determining the energetic and functional aspects of protein-protein interactions, particularly in the context of serine proteases and their natural protein inhibitors. The serine proteases play numerous metabolic and regulatory roles in humans and other organisms, and an important mechanism of their own regulation involves proteins that bind to their active sites with very high affinities, apparently mimicking a substrate, but without undergoing proteolysis at a significant rate. This study will focus on one member of this large class of inhibitors, bovine pancreatic trypsin inhibitor (BPTI). Although aspects of this protein have been extensively studied by biophysical and mutational methods, it is not yet known why it (or other members of the class) are so dramatically resistant to hydrolysis. Various destabilizing chemical and mutational modifications can increase the rate of cleavage, however, suggesting that the stability or rigidity of the structure when bound to the enzyme somehow prevents motions that are necessary for catalysis. The rigidity of the structure, which undergoes very little change upon binding to trypsin, is also likely to contribute to the very high stability of the enzyme-inhibitor complex. To test these hypotheses, the dynamics of several BPTI variants, both free and when bound to trypsin, will be studied by NMR relaxation methods. In addition, the thermodynamics of the interactions will be studied by calorimetric and fluorescence methods, and x-ray crystal structures will be determined for the BPTI-trypsin complexes. The combination of data from these three approaches will lead to an improved understanding of how protein flexibility, or the lack thereof, influences the regulation of a medically important class of enzymatic reactions. More generally, this work will contribute to the fundamental knowledge necessary to predict and understand the interactions among biological macromolecules, an area of rapidly increasing biomedical importance.
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