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.

抽象的 蛋白质活性的变构调节是一种物理机械现象，是协调 整个生物学中的细胞事件。 完全依赖于构象和动态变化的执行，使变构蛋白能够 理解这样的生物机制——并延伸至远程站点之间的通信。 了解如何通过药物或蛋白质工程合理地改变细胞过程——就是了解 然而，尽管变构调节已经被解决，但变构调节如何发挥作用这一根本问题。 几十年来，尽管最近认识到动力学有助于变构，但我们的 对变构机制的理解仍处于初级水平，一个限制是其作用。 变构动力学仅从少数几个系统中得出，其中大多数缺乏经典的指标 功能变构的另一个限制是获得功能动力学的准确信息。 确定变构的基本工作原理和变构行为机制具有实验挑战性。 必须在“强​​变构”的蛋白质中观察到，其中变构运动和特征将是 从长远来看，变构机制的知识将加强蛋白质研究。 一般并对变构药物和变构蛋白的设计产生巨大的积极影响。 工作将集中在变构酶分支酸变位酶（CM）上。综合考虑，这种酶似乎是。 CM 是一种典型的变构酶，非常适合对其变构机制进行高分辨率剖析研究。 有许多特征可以证明：它是一种对称二聚体，活性位点间隔 40 Å； 经历 T 到 R 构象转变；它表现出同向变构（希尔系数 = 1.6）； 具有上调（通过色氨酸）或下调（通过酪氨酸）活性的小分子效应子的异向变构为 60。 kDa，这使得它适合溶液核磁共振研究，并且它具有极强的可溶性和耐用性，并且产率 CM 丰富的变构特征将使经典变构成为可能。 使用核磁共振和其他生物化学和生物物理方法（包括计算）进行实验检查 在该提案中，目标 1 和 2 采用了 NMR、计算方法和化学方法。 合成来表征溶液中 APO 的结构和动态特征以及 CM 的配体状态。 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： 目标 3 的重点是扩展一种新颖的异向性远程通信机制。 “点击”化学将用于 共价且特异性地将 CM 启动子连接在一起，以稳定用于单独研究难以捉摸的样本 这种方法对于一般蛋白质二聚体的 NMR 研究很有用。