Structural and Dynamic Mechanisms in Classical Protein Allostery

摘要 蛋白质活性的变构调节是一种物理-机械现象，它是协调 整个生物学中的细胞事件。信号转导、新陈代谢和其他基本的细胞过程 完全依赖于构象和动态变化的执行，使变构蛋白能够 在远程站点之间进行通信。为了理解这种生物机制--并由此延伸到 理解如何合理地改变细胞过程，无论是通过药物还是蛋白质工程-是理解 变构调节如何发挥作用这一根本问题。然而，尽管变构调节一直是 几十年来一直被认识到，尽管最近人们意识到动力有助于变构，但我们的 对变构作用机理的认识还处于初级水平。其中一个限制是， 变构的动力学仅来自几个系统，其中大多数缺乏经典的 功能性变构。另一个限制是获得关于功能动力学的准确信息是 在实验上具有挑战性。识别变构的基本工作原理，变构行为的机理 必须在“强烈变构”的蛋白质中观察到，在这些蛋白质中，变构运动和信号 更容易辨认。从长远来看，对变构机制的了解将促进蛋白质在 并对变构药物和变构蛋白的设计产生了巨大的积极影响。这件事的重点是 工作将在变构酶分支酸变位酶(CM)上进行。综上所述，这种酶似乎是 非常适合于对其变构机理的高分辨率解剖研究。CM是一种典型的变构酶，与 有许多特征证明：它是一个对称的二聚体，活性中心被40？隔开；它 经历T-R构象转变；表现出各向同性变构(Hill系数=1.6)；它表现出 具有小分子效应器的异向性变构作用，通过色氨酸(Trp)或酪氨酸(Tyr)调节活性。Cm 60岁 KDA使其易于进行溶液核磁共振研究，并且它具有极高的溶解性和耐用性，并且产量 高质量的核磁共振波谱。CM丰富的变构特性将使经典变构成为 使用核磁共振和其他生化和生物物理方法(包括计算)进行实验检查 以前所未有的细节。在本提案中，目标1和目标2使用核磁共振、计算方法和化学方法 合成以表征溶液中CM的载脂蛋白和配位态的结构和动力学特征。这个 将监测CM对结合效应器和过渡态类似物的反应，所有这些都是为了实现以下目标 各向异性远程通信机制的识别。《目标3》专注于扩展一部小说 同质变构监测机制的标记方法学。“点击”化学将用于 共价且特定地将CM启动子捆绑在一起以稳定用于研究难以捉摸的单独的样品 结扎状态。这种方法对于蛋白质二聚体的核磁共振研究将是有用的。

Abstract Allosteric regulation of protein activity is a physico-mechanical phenomenon that underlies the coordination of cellular events throughout biology. Signal transduction, metabolism, and other essential cellular processes are completely reliant on the executions of conformational and dynamic changes that enable allosteric proteins to communicate between distant sites. To understand such biological mechanisms – and by extension to understand how to rationally alter cellular processes, either with drugs or protein engineering – is to understand this fundamental problem of how allosteric regulation works. Yet, even though allosteric regulation has been recognized for decades and despite the recent realization that dynamics contributes to allostery, our understanding of allosteric mechanism is still at a rudimentary level. One limitation has been that the roles of dynamics in allostery have been drawn from just a few systems, most of which lack the classic indicators of functional allostery. Another limitation is that gaining accurate information on functional dynamics is experimentally challenging. To identify basic working principles of allostery, mechanisms of allosteric behavior must be observed in proteins that are “strongly allosteric”, where allosteric movements and signatures will be more easily identified. In the long term, knowledge of allosteric mechanism will enhance protein research in general and have a huge positive impact on design of allosteric drugs and allosteric proteins. The focus of this work will be on the allosteric enzyme chorismate mutase (CM). By all considerations, this enzyme appears to be ideal for high-resolution dissective studies of its allosteric mechanisms. CM is a canonical allosteric enzyme as evidenced by a number of characteristics: it is a symmetric dimer with active sites separated by 40 Å; it undergoes T-to-R conformational transitions; it exhibits homotropic allostery (Hill coefficient = 1.6); and it exhibits heterotropic allostery with small molecule effectors that modulate activity up (by Trp) or down (by Tyr). CM is 60 kDa which makes it amenable to solution NMR studies, and it is extremely soluble and durable and yields outstanding quality NMR spectra. The rich allosteric characteristics of CM will allow classical allostery to be examined experimentally using NMR and other biochemical and biophysical methods (including computations) in unprecedented detail. In this proposal, Aims 1 and 2 employ NMR, computational methods, and chemical synthesis to characterize the structural and dynamic features of apo and liganded states of CM in solution. The responses of CM to binding effectors and a transition state analog will be monitored, all towards the goal of identification of mechanisms of heterotropic long-range communication. Aim 3 is focused on extending a novel labeling methodology for monitoring mechanisms of homotropic allostery. “Click” chemistry will be used to covalently and specifically tether CM promoters together to stabilize samples used for studying the elusive singly ligated state. This approach will be useful for NMR studies of protein dimers in general.