CAREER: The Role of Dynamics in Enzyme Function by NMR, Chemical Synthesis, and Biochemistry

这个职业项目解决了关于分子内蛋白质动力学在酶催化中的作用的基本问题。新型配体的化学合成和酶/配体复合物的生物物理表征将与溶液核磁共振自旋弛豫实验相结合，为蛋白质柔韧性和蛋白质功能之间的协调提供更丰富的理解。这些研究将与核糖核酸酶a一起进行。核磁共振实验将在与生物功能相关的不同时间尺度上探测蛋白质的运动。表征ps - ns运动将允许解剖位点特定的热力学贡献参与酶复合体的形成。质谱-质谱体系中的运动与催化过程所必需的化学和物理过程具有相似的时间尺度。它们的表征将提供参与催化活性酶运动的原子分辨率。这些实验将提供对酶功能的物理化学力的理解水平，这对于设计生物催化剂和优化蛋白质/配体相互作用至关重要。为了影响科学家和非科学家，研究和教育必须结合起来。实现这一目标的努力将侧重于研究指导、教学和促进研究小组之间的互动。这三项活动将包括对本科生和研究生以及来自地理代表性不足地区的学生进行培训和教学，以努力同时促进发现和学习。首先，教学计划的主要组成部分包括教授两门课程，“生化速率和机制”和“核磁共振波谱学”。这些课程的目标是开始研究生和高级本科生。这两门课程都将理论与实验应用相结合，并依靠解决问题和学生展示来提供一个积极的学习环境。其次，将主要努力作为研究生和本科生的研究导师。除了耶鲁大学的学生外，每年夏天还将邀请一名来自西弗吉尼亚州农村地区的学生进行研究。最后，为了促进来自不同研究小组的学生之间的互动，每月将组织研讨会，并鼓励学生参加和参与讨论。

This CAREER project addresses fundamental questions regarding the role of intramolecular protein dynamics in enzyme catalysis. Chemical synthesis of novel ligands and biophysical characterization of enzyme/ligand complexes will be combined with solution NMR spin-relaxation experiments to provide a richer understanding of the coordination between protein flexibility and protein function. These studies will be performed with the enzyme Ribonuclease A. NMR experiments will probe protein motion on distinct timescales relevant to those of biological function. Characterization of ps - ns motions will allow dissection of site-specific thermodynamic contributions involved in enzyme complex formation. Motions in the ms - ms regime are of similar timescale to chemical and physical processes essential for catalysis. Their characterization will provide atomic resolution of motions involved in a catalytically active enzyme. These experiments will provide the level of understanding of physico-chemical forces in enzyme function essential for the design of biocatalysts and optimization of protein/ligand interactions. To impact scientists and non-scientists alike, research and education must be integrated. Efforts to achieve this will focus on research mentoring, teaching, and the promotion of interaction among research groups. Incorporated within these three activities will be training and teaching of students at the undergraduate and graduate levels and from geographically underrepresented regions in an effort to simultaneously advance discovery and learning. First, the principal component of the educational plan consists of teaching two courses, 'Biochemical rates and mechanisms' and 'NMR spectroscopy.' These courses are targeted to beginning graduate students and advanced undergraduates. Both courses combine theory with experimental applications and rely on problem solving and student presentation to provide an active learning environment. Second, a major effort will be made as research mentor to graduate and undergraduate students. In addition to students from Yale, a student from rural West Virginia will be invited to perform research each summer. Finally, to promote interactions among students from diverse research groups, monthly seminars will be organized and students will be encouraged to attend and participate in discussions.