NMR and Biophysical Characterization of Functional Millisecond Enzyme Motions

功能性毫秒酶运动的核磁共振和生物物理表征

• 批准号: 0744161
• 负责人: joseph loria
• 金额: $ 60万
• 依托单位: Yale University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0744161&HistoricalAwards=false
• 关键词: NMR; Biophysical; Characterization; Functional; Millisecond

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.

分子内运动在生物功能中的作用已被广泛接受，但尚未完全理解。 函数依赖于时间平均的三维结构和来自该结构的随时间变化的涨落。因此，时间依赖性蛋白质波动耦合到生物功能，特别是酶催化的机制的不完整的图片，限制了酶设计和从头蛋白质工程的科学进展。该研究项目将通过整合溶液NMR弛豫分散实验，X射线晶体学，生物化学和生物物理实验以及计算化学方法来解决这些缺陷，为蛋白质运动，结构和功能的相互作用提供模型。核磁共振实验将产生详细的，原子分辨率的酶运动，将与生化实验和定点诱变相结合，以解决功能和运动与功能之间的关系的信息。结构研究将提供一个三维视图的酶与框架的动力学和生物化学的结果，并将通过计算研究，这将允许更高的能量构象状态和构象运动的机制建模增强。除了科学价值外，该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。这种伙伴关系将通过促进计算/理论科学家和实验学家之间的互动来加强耶鲁大学的基础设施。来自美国经济落后地区和农村地区的本科生通常无法获得最先进的科学仪器或研究。该项目将继续在夏季的几个月里让这些学生参加研究活动。首席研究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。这些地区的学生往往成为科学教师。拓宽他们的科学背景将积极影响他们未来的教学能力。此外，主要研究者积极地将高中生纳入他的研究中，这与耶鲁大学关于"实验室未成年人"的政策是雅阁的。除了推进科学发现之外，本科生和高中生与主要研究者实验室的研究生配对提供了另一个好处，因为它使研究生在正常的TA任务之外接受教学，从而增加了他们的教学和培训机会。该项目的原创研究成果将及时发表。此外，首席研究员将继续发表这些NMR方法和应用的一般，非NMR观众的评论文章。这些类型的出版物有可能影响其他领域，并允许将新的想法纳入NMR可能没有考虑的研究计划。