Role of Protein Dynamics in Modulating the Thermodynamic Linkages in Allostery

蛋白质动力学在调节变构热力学联系中的作用

• 批准号: 7367841
• 负责人: JAMES C LEE
• 金额: $ 28.59万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7367841
• 关键词: Address; Affect; Algorithms; Allosteric Regulation; Amino Acid Sequence; Be++ element; Beryllium; Binding; Binding Sites; Biological; Complement Factor B; Coupled; Cyclic AMP; Cyclic AMP Receptor Protein; Cyclic GMP; Cyclic Nucleotides; DNA Binding Domain; Data; Elements; Entropy; Family; Helix (Snails); Induced Mutation; Knowledge; Laboratory Research; Lead; Ligand Binding; Ligands; Link; Literature; Maps; Mass Spectrum Analysis; Measurement; Modeling; Motion; Mutagenesis; Mutation; Pattern; Peptide Sequence Determination; Play; Progress Reports; Property; Protein Dynamics; Proteins; Range; Research; Roentgen Rays; Role; Site; Solvents; Specificity; Structure; Sucrose; System; Testing; Thermodynamics; Time; Urea; base; ear helix; mutant; physical property; polypeptide; protein structure; prototype; success; transcription factor

cAMP receptor protein (CRP) is the prototype of a super-family of transcription factors. One key feature of CRP's biological activity that has been the focus of this laboratory's research is the fundamental rules that govern the allosteric activation by cAMP of its sequence-specific DMA binding. Recently we established, for the first time in any allosteric system, that the energetics of allosteric parameters are linear functions of protein dynamics. Using protein dynamics as the focus, there is a convergence among the results derived from crystallographic, computational and functional energetic data. This preliminary success enables us to focus our research effort on protein dynamics as the fundamental physical property to establish quantitative linkages between CRP structure and mechanism(s) of allostery. For coming years we will address these issues: 1. Is protein dynamics one of the fundamental properties of protein that modulate allostery? Two strategies will be employed to test the validity of this relationship: a) loops which are solvent accessible are targeted as structural elements to alter protein dynamics and allostery; b) Osmolytes, which affect dynamic motions of proteins, will be employed as solvent additives to perturb protein dynamics and allostery. 2. Is there a network of structural elements that is particularly affected by allosteric regulation? The structural elements the perturbation of which can lead to changes in protein dynamics and allostery will be identified by: a) changes in the thermal B-factors in X-ray data due to mutation; b) H/D exchange measurements coupled with mass spectrometry; and c) computation evidence for structural connectivity. 3. Can one use the knowledge gained in Aims 1 and 2 to assist in defining the structural elements that exert long range effects in defining specificity of the activating effectors? CRP is activated most efficiently by cAMP, although it can bind other cNMP without being activated. Literature results imply that polypeptide outside of the cAMP binding site, specifically the DNA binding domain, dictates the ability of CRP to respond to the bound ligand. Based on the preliminary computation results which show connectivity between the cAMP binding site and the DNA binding domain, mutants will be used to test the connectivity pattern. X-ray structural data of the cAMP binding domain will be used to test if the presence of the DNA binding domain alters the structural connectivity pattern in the cAMP binding domain.

cAMP 受体蛋白 (CRP) 是转录因子超家族的原型。的一个关键特征 CRP的生物活性一直是本实验室研究的重点，其基本规律是 通过 cAMP 控制其序列特异性 DMA 结合的变构激活。最近我们成立了，为了 在任何变构系统中，变构参数的能量学是第一次 蛋白质动力学。以蛋白质动力学为重点，得出的结果之间存在收敛性 来自晶体学、计算和功能能量数据。这一初步成功使我们能够 我们的研究重点是蛋白质动力学，作为建立定量分析的基本物理性质 CRP 结构和变构机制之间的联系。未来几年我们将解决这些问题 问题： 1. 蛋白质动力学是调节变构的蛋白质基本特性之一吗？二 将采用策略来测试这种关系的有效性：a）可溶剂访问的循环是 作为改变蛋白质动力学和变构的结构元件； b) 渗透调节剂，影响动态 蛋白质的运动，将被用作溶剂添加剂来扰乱蛋白质动力学和变构。 2. 是否存在特别受变构调节影响的结构元件网络？这 结构元件的扰动会导致蛋白质动力学和变构的变化 通过以下方式识别： a) 由于突变导致 X 射线数据中热 B 因子的变化； b) H/D 交换 与质谱联用的测量； c) 结构连通性的计算证据。 3. 能否利用目标 1 和 2 中获得的知识来协助定义结构要素 在定义激活效应器的特异性方面发挥长期作用？ CRP被激活最多 尽管它可以与其他 cNMP 结合而不被激活，但它可以通过 cAMP 有效地结合。文献结果表明 cAMP 结合位点之外的多肽，特别是 DNA 结合域，决定了 CRP 对结合的配体作出反应。基于显示连通性的初步计算结果 cAMP结合位点和DNA结合域之间，突变体将用于测试连接性 图案。 cAMP 结合域的 X 射线结构数据将用于测试 DNA 是否存在 结合域改变 cAMP 结合域的结构连接模式。