Functional Dynamics during Induced-fit Enzyme Turnover

描述(申请人提供)：运动是许多蛋白质作用的关键。人们对动态的理解很少，尤其是在往往限制成交量的微秒级和较慢的制度下。它们超出了大多数实验和计算方法的范围。我们的长期目标是从根本上理解构象动力学是如何支撑机制的。这将通过从结构和动力学上表征单个氨基酸的运动以及它们与诱导适合酶的周转周期中功能的关系来实现。最近发展起来的核磁共振弛豫色散分析方法将应用于一种模型酶来确定构象交换率。这些将结合高分辨率的晶体结构和核磁共振剩余偶极耦合的更快的动力学表征。精氨酸激酶展示了底物诱导的结构域和环运动以及固有的协调运动的例子。对于这种大小(42 KDa)的代表性酶来说，精氨酸激酶的核磁共振谱和X射线衍射都具有异常高的质量。这为阐明运动的结构-动力学-功能关系提供了一个独特的机会，这些运动代表了那些对许多蛋白质具有重要功能的运动。重点将放在绘制每个残基的主干构象交换率图上，特别是在微秒/毫秒范围内。核磁共振弛豫交换率将在各种平衡底物/产物浓度下的酶周转过程中进行测量，以确定哪些运动与结合、解离或反应步骤有关。稳定的酶络合物的晶体结构将被确定为随机顺序的bi-bi机制，而核磁共振弛豫滴定将被用于确定与结合或解离相关的构象变化的交换率。因此，将通过周转周期的步骤来剖析每种氨基酸的结构变化和运动时间常数。运动的功能作用将通过对温度、突变和酶的其他扰动后的动力学和动力学的联合分析来确定。与健康相关：对分子运动的基本了解对于了解人类蛋白质如何实现其正常功能以及基因突变导致疾病的机制至关重要。

DESCRIPTION (provided by applicant): Motions are key to the action of many proteins. Dynamics are poorly understood, especially, in the micro- second and slower regimes that often limit turnover. They lie beyond most experimental and computational methods. Our long term objective is a fundamental understanding of how conformational dynamics underpin mechanism. This will be achieved by characterizing structurally and kinetically the motions of individual amino acids and their relation to function in the turnover cycle of an induced-fit enzyme. Recently developed methods of NMR relaxation dispersion analysis will be applied to a model enzyme to determine conformational exchange rates. These will be integrated with high resolution crystal structures and NMR residual dipolar coupling characterization of faster dynamics. Arginine kinase presents examples of domain and loop movements that are substrate-induced, as well as inherent concerted motions. Both the NMR spectra and x-ray diffraction of arginine kinase are of unusually high quality for a representative enzyme of this size (42 kDa). This presents a unique opportunity to elucidate the structure-dynamics- function correlates of motions representative of those that are functionally important for many proteins. The emphasis will be on mapping the backbone conformational exchange rates for each residue, particularly in the micro-/milli-second regime. NMR relaxation exchange rates will be measured during enzyme turnover at various equilibrium substrate/product concentrations to determine which motions are associated with binding, dissociation or reaction steps. Crystal structures will be determined for the stable enzyme complexes within the random sequential bi-bi mechanism, and NMR relaxation titration will be used to determine the exchange rates for conformational changes associated with binding or dissociation. Thus, the structural changes and time constants of motion will be dissected for each amino acid through the steps of the turnover cycle. The functional role of the motions will be established through joint analysis of kinetics and dynamics following temperature, mutational and other perturbations of the enzyme. Health Relevance: A fundamental understanding of molecular motion is essential to understanding how human proteins achieve their normal function and the mechanisms through which disease may result from genetic mutations.