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

蛋白质运动对于酶功能和变构作用是重要的。本研究的目的是测试的假设，构象波动是重要的催化和变构的双特异性磷酸酶VHR。溶液核磁共振（NMR）光谱将用于定量测定该酶的快速和缓慢运动，游离和结合磷酸化肽底物。氮（15 N）和2 H侧链NMR为基础的动态测量将收集在整个VHR的网站，并使用“无模型”的形式主义进行分析，以确定网站特定的顺序参数，S2（一个参数，表明限制或动态的程度）。S2的变化将用于绘制由于底物结合引起的动态响应。扰动的残基将被突变以评估它们在催化中的重要性。弛豫补偿CPMG实验将用于识别和表征沿着骨干的缓慢（微米-毫秒）运动。将获得的构象交换速率与已知的催化参数进行比较。最后，将确定导致变构激活的磷酸化VHR（VHR-pY 138）的生物化学、结构和动力学特征。这些实验的结果有望对结构、动力学和磷酸化在酶功能中的作用产生广泛的影响。该项目将增加有关蛋白质运动如何调节催化的知识体系。将产生广义的规则，解释如何快速和缓慢的运动夫妇，以提高酶的活性。此外，关于蛋白质磷酸化如何影响结构和动力学的新信息将被发现。这些研究将为培养酶学和结构生物学的本科生和研究生提供一个工具。

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.