The long-term objective of this research program is to understand the relationship between the amino acid sequences of proteins and their three-dimensional structures and stabilities. This is one of the central, unsolved problems in molecular biology and is important because protein folding is a prerequisite of most biological processes. In addition, much of the promise of biotechnology for improving human health depends on our eventual ability to manipulate the function of a protein by altering its sequence. We will use P22 Arc repressor, a dimeric protein of known structure, to study the effects of sequence changes produced by directed or combinatorial mutagenesis on the structure, stability, and folding kinetics of the protein. Because its is small and accessible to genetic, biophysical, and structural study, the Arc system is one of the premier models for studying the sequence determinants of protein folding and association. In other studies, we will investigate the conformational determinants of the prion state, using the URE2 protein of yeast as a model system. Prions provide a fascinating but poorly understood phenomena in which an altered state of the protein, which arises rarely but spontaneously, can be stably propagated in a dominant, epigenetic fashion by a mechanism in which altered molecules cause normal molecules to assume the altered prion state. In all studies, thermodynamic stability will be investigated by reversible denaturation; changes in folding and unfolding kinetics will be determined by stopped-flow methods; and conformational properties of selected molecules will be probed by x-ray crystallography or NMR. The data acquired will be important for testing and refining our understanding of the sequence determinants of protein structure and stability and for evaluating algorithms that seek to predict the effects of sequence changes on protein structure and function.
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