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

研究启动资助：蛋白质动力学在 VHR 催化和变构中的作用

• 批准号: 0624451
• 负责人: Ernesto Fuentes
• 金额: $ 5万
• 依托单位: University of Iowa
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-15 至 2009-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0624451&HistoricalAwards=false
• 关键词: Reseach; Starter; Grant; Role; Protein

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.

蛋白质运动对酶的功能和变构是重要的。本研究的目的是验证双特异性磷酸酶VHR的构象波动对催化和变构的重要假设。溶液核磁共振（NMR）光谱将用于定量确定这种酶的快速和缓慢运动，游离和结合到磷酸化的肽底物上。基于氮（15N）和2H侧链核磁共振的动力学测量将在整个VHR站点收集，并使用“无模型”形式进行分析，以确定特定站点的顺序参数S2（指示限制或动力学程度的参数）。S2的变化将用于映射由于底物结合而产生的动态响应。扰动残基将发生突变，以评估其在催化中的重要性。弛豫补偿CPMG实验将用于识别和表征沿骨干的慢（微米-毫秒）运动。得到的构象交换速率将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- py138）的生化、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具有广泛的意义。更广泛的影响。这个项目将增加关于蛋白质运动如何调节催化的知识体系。将产生广义的规则来解释快速和慢速运动是如何结合起来增强酶活性的。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。