Structural and Dynamic Mechanisms in Classical Protein Allostery

经典蛋白质变构的结构和动力学机制

• 批准号: 10021672
• 负责人: Andrew L Lee
• 金额: $ 34.83万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-20 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10021672
• 关键词: Active Sites; Address; Affect; Affinity; Allosteric Regulation; Aspartate; Back; Behavior; Binding; Biochemical; Biological; Biology; Carbamoyl Transferases; Cell physiology; Cells; Characteristics; Chemicals; Chemistry; Chorismate Mutase; Communication; Computing Methodologies; Crystallization; Data; Dissection; Distal; Distant; Drug Design; Enzymes; Event; Exhibits; Feedback; Goals; Hemoglobin; Knowledge; Label; Life; Ligand Binding; Ligands; Link; Measures; Mechanics; Metabolism; Methodology; Methods; Molecular Conformation; Monitor; Movement; NMR Spectroscopy; Nature; Pathway interactions; Pharmaceutical Preparations; Process; Protein Engineering; Proteins; Protomer; Regulation; Relaxation; Research; Resolution; Role; Sampling; Signal Transduction; Site; Structural Models; Structure; System; Thermodynamics; Tryptophan; Tyrosine; Work; Yeasts; analog; biophysical techniques; chemical synthesis; computer studies; conformational conversion; crosslink; design; dimer; drug development; drug discovery; enzyme activity; enzyme structure; human disease; inhibitor/antagonist; molecular dynamics; novel; programs; promoter; protein function; protein structure; response; small molecule; tool; tryptophyltyrosine; unnatural amino acids

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.

摘要 蛋白质活性的变构调节是一种物理机械现象，其是蛋白质活性的协调的基础。 细胞事件贯穿生物学。信号转导、代谢和其他基本的细胞过程， 完全依赖于执行构象和动态变化，使变构蛋白质， 远程站点之间的通信。为了理解这种生物学机制， 了解如何合理地改变细胞过程，无论是药物还是蛋白质工程-就是了解 这个关于变构调节如何起作用的基本问题。然而，尽管变构调节已经被 几十年来，尽管最近认识到动力学有助于变构， 对变构机制的了解仍处于初级水平。一个限制是， 变构动力学仅来自少数系统，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， 实验挑战明确变构的基本工作原理，变构行为的机制 必须在“强变构”的蛋白质中观察到，其中变构运动和特征将被 更容易识别。从长远来看，对变构机制的了解将促进蛋白质研究， 这是普遍的，并对变构药物和变构蛋白的设计产生了巨大的积极影响。的重点 工作将在变构酶分支酸酯（CM）。从各方面考虑，这种酶似乎是 理想的高分辨率解剖研究其变构机制。CM是一种典型的变构酶， 由许多特征证明：它是一个对称的二聚体，活性位点相隔40 nm;它 经历T-to-R构象转变;其表现出同向变构（Hill系数= 1.6）;并且其表现出 具有调节活性上调（通过Trp）或下调（通过Tyr）的小分子效应物的异向性变构。CM 60 kDa，这使得它适合于溶液NMR研究，并且它是极其可溶的和持久的， 高质量的核磁共振谱。CM丰富的变构特性将允许经典的变构被 使用NMR和其他生物化学和生物物理方法（包括计算）进行实验检查 前所未有的细节。在该提案中，目标1和2采用NMR、计算方法和化学方法。 合成以表征溶液中CM的载脂蛋白和配体状态的结构和动力学特征。的 将监测CM对结合效应物和过渡态类似物的反应，所有这些都是为了实现以下目标： 异向远距离通讯机制的确定。目标3的重点是扩展一个新的 用于监测同向性变构机制的标记方法学。“点击”化学将被用来 共价和特异性地将CM启动子拴在一起，以稳定用于研究难以捉摸的单个 连接状态。这种方法将是有用的NMR研究蛋白质二聚体一般。