CAREER: NMR Studies of Enzyme Structure and Dynamics

这个CAREER项目的目标是揭示分子运动如何影响酶的功能。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至关重要。在这个项目中，噬菌体l-整合酶的功能重要分子运动是通过核磁共振（NMR）、预稳态动力学、热力学、计算和位点定向诱变的综合方法来表征的。生物化学和生物物理证据表明，该酶通过形成Holliday连接中间体催化位点特异性DNA重组，从而实现了大振幅的协同环运动。为了研究这些运动/功能关系，该项目包括：(1)比较自由酶和dna结合酶的平衡结构、动力学和热力学，以确定催化所需的构象变化的性质。(2)利用核磁共振、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)利用位点定向诱变和替代DNA底物探索功能对内部运动的依赖。该职业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与学生交流大分子的动态特性。目前，大多数关于分子结构/功能的教学方法都局限于静态图像，无法深入了解功能亚态相互转换的运动过程的本质。

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.