Dynamic Networks and Mechanisms of Allosteric Communication in Proteins

蛋白质变构通讯的动态网络和机制

• 批准号: 7933132
• 负责人: Andrew L Lee
• 金额: $ 9.76万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7933132
• 关键词: Address; Allosteric Regulation; Behavior; Binding; Biological; Biological Process; Biology; Biotechnology; Cell division; Chemicals; Chemotaxis; Communication; Coupling; Data; Databases; Distal; Drug effect disorder; Drug resistance; Escherichia coli; Event; Fluorescence; Foundations; Free Energy; Funding; Goals; Heart; Heterogeneity; Individual; Industry; Lead; Ligand Binding; Ligands; Light; Maps; Measurement; Measures; Mediating; Metabolism; Methodology; Methods; Modeling; Modification; Motion; Motor; Mutation; Pathway interactions; Pattern; Peptides; Pharmaceutical Preparations; Phosphorylation; Play; Population Dynamics; Process; Property; Protein Dynamics; Proteins; Regulation; Relative (related person); Relaxation; Research; Role; Serine Proteinase Inhibitors; Side; Signal Pathway; Signal Transduction; Site; Solutions; Stimulus; Structure; Surface; Technology; Tertiary Protein Structure; Testing; Therapeutic; Therapeutic Intervention; Thermodynamics; Time; base; beryllium fluoride; density; design; drug discovery; engineering design; falls; genetic regulatory protein; globular protein; mutant; protein folding; protein function; public health relevance; research study; response; theories

DESCRIPTION (provided by applicant): How proteins regulate themselves is a fundamental question in biology. Regulation of protein activity drives cell division, metabolism, signal transduction, and therapeutic intervention. The most common and versatile regulatory proteins are allosteric proteins. Allosteric proteins are distinguished by their capacity to respond to ligand binding or chemical modification at one site, and alter ligand binding or activity at a distal site. Although many allosteric proteins have been characterized structurally, and models of allostery exist, most of the details of how allosteric transitions proceed remain unknown. The long-range goal of this research is to experimentally reveal the time-resolved structural processes that constitute allosteric function. Central to the approach to be taken is the idea that proteins are highly dynamic, especially allosteric proteins, and that dynamic motions are an essential component of allosteric conformational change. Classical oligomeric allosteric proteins are too large for in-depth studies of site-specific dynamics by NMR. Therefore, the monomeric, 14 kDa bacterial response regulator CheY protein will be used as a model allosteric domain. CheY is a chemotaxis signal transduction protein that, upon phosphorylation at Asp-57, undergoes a conformational change at a distal surface that modulates binding to the flagellar motor. The ps-ns and ¿s-ms timescale dynamics of CheY will be extensively characterized in the absence and presence of phosphoryl group mimic, BeF3-, using NMR relaxation methods. In separate experiments, long-range thermodynamic couplings will be mapped using high-throughput methodology. Because, recently, long-range communication has been observed in proteins that are not functionally allosteric, mechanisms similar to those in allosteric proteins may exist in non-allosteric proteins, albeit to a lesser extent. The serine protease inhibitor eglin c is a good example of this: conservative mutations in eglin c lead to long-range dynamic effects in the absence of structural change. In the proposed research, four Specific Aims fall into two main thrusts. In the first thrust, patterns of long-range coupling (or "communication") - both dynamic and thermodynamic - will be compared between the non-allosteric eglin c and the allosteric CheY. These comparisons will shed light on any basic differences in coupling networks between allosteric and non-allosteric proteins; they will also provide a test of the role of dynamics in mediating thermodynamic coupling. In the second thrust, the mechanism of intramolecular signal transduction in CheY will be investigated from an NMR dynamics perspective, using CheY's various biological states and mutations that modulate its activity. Overall, by increasing understanding of the biophysical properties and role of dynamics in allostery, this research will help to lay the foundation for the rational design of allosteric proteins and drugs. PUBLIC HEALTH RELEVANCE: Allosteric conformational change in proteins lies at the heart of regulatory processes such as cell division, metabolism, signal transduction, and drug action. This research seeks to understand the dynamic underpinnings of allostery by experimentally contrasting motional dynamics in non-allosteric and allosteric proteins. The small bacterial signal transduction protein CheY will serve as a model allosteric domain. A detailed understanding of allosteric mechanisms will be needed to rationally design proteins and drugs that take advantage of allosteric principles, as well as understand mechanisms of drug resistance.

描述(申请人提供)：蛋白质如何自我调节是生物学中的一个基本问题。蛋白质活性的调节驱动细胞分裂、新陈代谢、信号转导和治疗干预。最常见、用途最广的调节蛋白是变构蛋白。变构蛋白的不同之处在于它们能够对一个位点的配体结合或化学修饰做出反应，并改变远端位点的配体结合或活性。尽管许多变构蛋白已经在结构上被表征，变构模型也存在，但关于变构如何进行的大部分细节仍不清楚。这项研究的长期目标是从实验上揭示构成变构功能的时间分辨结构过程。要采取的方法的中心是这样一个想法，即蛋白质是高度动态的，特别是变构蛋白质，动态运动是变构构象变化的重要组成部分。经典的寡聚体变构蛋白太大，不能用核磁共振深入研究特定部位的动力学。因此，14 kDa细菌反应调节蛋白单体Chey蛋白将被用作模型变构结构域。Chey是一种趋化信号转导蛋白，在Asp-57处发生磷酸化后，在远端表面发生构象变化，调节与鞭毛马达的结合。利用核磁共振弛豫方法，在没有和存在磷酰基模拟物BeF_3-的情况下，将广泛地表征CHEY的PS-ns和S-ms的时间标度动力学。在单独的实验中，将使用高通量方法绘制远程热力学耦合图。因为，最近在非功能变构的蛋白质中观察到了远程通讯，所以在非变构蛋白质中可能存在类似于变构蛋白质的机制，尽管程度较小。丝氨酸蛋白酶抑制剂Eglin c就是一个很好的例子：在没有结构变化的情况下，Eglin c的保守突变会导致长期的动态效应。在这项拟议的研究中，四个具体目标分为两个主要目标。在第一个推力中，我们将比较非变构的Eglin c和变构的Chey之间的长程耦合(或“通信”)模式--包括动力学和热力学。这些比较将阐明变构和非变构蛋白之间耦合网络的任何基本差异；它们还将提供对动力学在调节热力学耦合中的作用的测试。在第二个推力中，将从核磁共振动力学的角度，利用Chey的各种生物学状态和调节其活性的突变来研究Chey的分子内信号转导机制。总之，通过加深对变构的生物物理性质和动力学作用的了解，本研究将有助于为变构蛋白和药物的合理设计奠定基础。公共卫生相关性：蛋白质的变构构象变化是细胞分裂、新陈代谢、信号转导和药物作用等调控过程的核心。这项研究试图通过实验对比非变构和变构蛋白质的运动动力学来理解变构的动态基础。细菌小分子信号转导蛋白Chey将作为模型变构域。为了合理地设计利用变构原理的蛋白质和药物，以及了解耐药性的机制，需要对变构机制有详细的了解。