CAREER: NMR Studies of Enzyme Structure and Dynamics

职业：酶结构和动力学的核磁共振研究

• 批准号: 0092962
• 负责人: Mark Foster
• 金额: $ 50万
• 依托单位: Ohio State University Research Foundation -DO NOT USE
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0092962&HistoricalAwards=false
• 关键词: CAREER; NMR; Studies; Enzyme; Structure

Foster, Mark P.MCB-0092962The objective of this CAREER project is to reveal how molecular motions affect enzyme function. The limited knowledge of structure/motion/function relationships severely limits our ability to predict molecular behavior. This knowledge is critical both for understanding catalytic mechanisms and for the design of modified enzymes that catalyze novel chemistries or specific high-affinity ligands. In this project, functionally important molecular motions in bacteriophage l-Integrase are characterized with an integrated approach involving nuclear magnetic resonance (NMR), pre-steady state kinetics, thermodynamics, computation and site-directed mutagenesis. Biochemical and biophysical evidence suggests that large amplitude concerted loop motions are essential to the function of this enzyme, which catalyzes site-specific DNA recombination via the formation of a Holliday junction intermediate. To study these motion/function relationships the project includes: (1) Comparison of the equilibrium structures, dynamics and thermodynamics of the free and DNA-bound enzyme in order to identify the nature of the conformational changes required for catalysis. (2) Measurement of the kinetics and thermodynamics of DNA binding and cleavage with NMR, mass spectrometry, fluorescence and calorimetry. (3) Obtaining a physical description of relevant internal motions from molecular dynamics simulations. (4) Probing the dependence of function on internal motions by use of site-directed mutagenesis and alternate DNA substrates.The educational component of this CAREER award involves development of descriptive tools to enable scientists and educators to effectively communicate to students the dynamic nature of macromolecules. Currently most didactic treatments of molecular structure/function are limited to static pictures that provide little insight into the nature of the motional processes that interconvert functional substates.

Foster，Mark P.MCB-0092962该职业项目的目标是揭示分子运动如何影响酶功能。结构/运动/功能关系的有限知识严重限制了我们预测分子行为的能力。这些知识对于理解催化机制和设计催化新化学或特定高亲和力配体的修饰酶都是至关重要的。在这个项目中，功能上重要的分子运动的噬菌体l-整合酶的特点与一个综合的方法，涉及核磁共振（NMR），前稳态动力学，热力学，计算和定点突变。生物化学和生物物理学证据表明，大幅度一致的环运动是必不可少的功能，这种酶，催化位点特异性DNA重组通过形成一个霍利迪交界处中间。为了研究这些运动/功能关系，该项目包括：（1）比较自由和DNA结合的酶的平衡结构，动力学和热力学，以确定催化所需的构象变化的性质。(2)用核磁共振、质谱、荧光和量热法测量DNA结合和裂解的动力学和热力学。(3)从分子动力学模拟中获得相关内部运动的物理描述。(4)通过使用定点突变和替代DNA底物来探索功能对内部运动的依赖性。该CAREER奖的教育部分涉及开发描述性工具，使科学家和教育工作者能够有效地向学生传达大分子的动态性质。目前，大多数教学的分子结构/功能的治疗是有限的静态图片，提供很少的洞察力的运动过程的性质，相互转换功能亚状态。