Reseach Starter Grant : The Role of Protein Dynamics in VHR Catalysis and Allostery

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Protein motions are important for enzyme function and allostery. The goal of this research is to test the hypothesis that conformational fluctuations are important for catalysis and allostery in the dual-specific phosphatase VHR. Solution nuclear magnetic resonance (NMR) spectroscopy will be used to quantitatively determine the fast and slow motions of this enzyme, free and bound to a phosphorylated peptide-substrate. Nitrogen (15N) and 2H side-chain NMR-based dynamics measurements will be collected at sites throughout VHR and analyzed using the "model-free" formalism to determine site-specific order parameters, S2 (a parameter that indicates the degree of restriction or dynamics). Changes in S2 will be used to map the dynamic response due to substrate binding. Perturbed residues will be mutated to assess their importance in catalysis. Relaxation compensated CPMG experiments will be used to identify and characterize slow (microns-ms) motions along the backbone. The obtained rates for conformational exchange will be compared to known catalytic parameters. Finally, the biochemical, structural, and dynamic features of phosphorylated VHR (VHR-pY138) that lead to allosteric activation will be determined. The results from these experiments are expected to have broad implications for the role of structure, dynamics and phosphorylation in enzyme function.Broader Impact. This project will increase the body of knowledge concerning how protein motions regulate catalysis. Generalized rules will be generated that explain how fast and slow motions couple to enhance enzyme activity. Furthermore, new information on how protein phosphorylation influences structure and dynamics will be discerned. These studies will provide a vehicle for training undergraduate and graduate students in enzymology and structural biology.