NMR and Biophysical Characterization of Functional Millisecond Enzyme Motions

分子内运动在生物功能中的作用被广泛接受，但还没有完全被理解。函数取决于时间平均的三维结构以及该结构的随时间变化的涨落。因此，对依赖于时间的蛋白质波动与生物功能，特别是酶催化的机制的不完整图景，限制了酶设计和从头蛋白质工程的科学进展。这项研究项目将通过整合溶液核磁共振弛豫分散实验、X射线结晶学、生化和生物物理实验以及计算化学方法来解决这些不足，为蛋白质运动、结构和功能的相互作用提供一个模型。核磁共振实验将产生有关酶运动的详细的原子分辨率信息，这些信息将与生化实验和定点突变相结合，以解决功能以及运动和功能之间的关系。结构研究将提供酶的三维视图，用来框架动力学和生化结果，并将通过计算研究得到加强，这将允许对更高能量的构象状态和构象运动机制进行建模。除了科学价值，这个项目还将影响其他领域。这项研究的计算方面将涉及与维克托·巴蒂斯塔教授的实验室合作。这一伙伴关系将通过促进计算/理论科学家和实验者之间的互动来加强耶鲁的基础设施。来自美国经济困难和农村地区的本科生通常无法接触到最先进的科学仪器或研究。这个项目将继续在夏季的几个月里让这些学生参加研究活动。首席调查员正在与西弗吉尼亚州的几所大学联系，以确定感兴趣的学生。来自这些地区的学生往往会成为科学教师。拓宽他们的科学背景将对他们未来的教学能力产生积极的影响。此外，首席调查员积极地将高中生纳入他的研究，这与耶鲁大学关于未成年人在实验室里的政策是一致的。除了促进科学发现，本科生和高中生与首席研究员实验室的研究生配对还有另一个好处，因为它使研究生在正常的助教任务之外接触到教学，从而增加了他们的教学和培训机会。这一项目产生的原创研究将及时发表。此外，首席研究员将继续发表这些核磁共振方法和应用的评论文章，面向一般的非核磁共振读者。这些类型的出版物有可能影响其他领域，并允许将新想法纳入到可能没有考虑过的研究计划中。

The role of intramolecular motion in biological function is widely accepted but not fully understood. Function depends on the time-averaged three-dimensional structure and time-dependent fluctuations from that structure. Thus, the incomplete picture of the mechanism by which time-dependent protein fluctuations couple to biological function, in particular enzyme catalysis, has limited the scientific progress in enzyme design and de novo protein engineering. This research project will address these deficiencies by integrating solution NMR relaxation dispersion experiments, X-ray crystallography, biochemical and biophysical experiments, and computational chemistry approaches to provide a model for the interplay of protein motion, structure and function. The NMR experiments will yield detailed, atomic resolution information on the enzyme motions that will be combined with biochemical experiments and site-directed mutagenesis to address function and the relationship between motion and function. The structural studies will provide a three-dimensional view of the enzyme with which to frame the dynamics and biochemical results and will be augmented by computational studies, which will allow modeling of higher energy conformational states and the mechanism of conformational motion. In addition to the scientific merit, this project will impact other areas. The computational aspects of this research will involve collaboration with the laboratory of Professor Victor Batista. This partnership will enhance the infrastructure at Yale by fostering interactions between computational/theoretical scientists and experimentalists. Undergraduates from economically disadvantaged and rural areas of the United States typically do not have access to state-of-art scientific instrumentation or research. This project will continue to include these students in research activities during the summer months. The principal investigator is in contact with several colleges in West Virginia to identify interested students. Often students from these areas become science teachers. Broadening their science background will positively influence their future teaching ability. In addition, the principal investigator actively includes high school students in his research, in accord with Yale policy on 'minors in the laboratory'. In addition to advancing scientific discovery, the pairing of undergraduates and high school students with a graduate student in the principal investigator's lab provides another benefit as it exposes the graduate student to teaching, outside of the normal TA assignment thereby enhancing their teaching and training opportunities. Original research resulting from this project will be published in a timely fashion. Furthermore, the principal investigator will continue to publish review articles of these NMR methods and applications for a general, non-NMR audience. These types of publications have the potential to impact other fields and allow incorporation of new ideas into research programs for which NMR may not have been considered.