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: 7188518
• 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/7188518
• 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; 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

DESCRIPTION (provided by applicant): 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 DNA 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.

描述（申请人提供）：cAMP受体蛋白（CRP）是转录因子超家族的原型。 CRP 生物活性的一个关键特征是控制 cAMP 对其序列特异性 DNA 结合进行变构激活的基本规则，也是该实验室研究的重点。最近，我们在任何变构系统中首次确定，变构参数的能量学是蛋白质动力学的线性函数。以蛋白质动力学为重点，晶体学、计算和功能能量数据得出的结果之间存在收敛。这一初步成功使我们能够将研究重点放在蛋白质动力学上，作为建立 CRP 结构和变构机制之间定量联系的基本物理特性。未来几年我们将解决这些问题： 1. 蛋白质动力学是调节变构的蛋白质基本特性之一吗？将采用两种策略来测试这种关系的有效性：a）将可溶剂进入的环作为结构元件来改变蛋白质动力学和变构； b) 影响蛋白质动态运动的渗透剂将被用作溶剂添加剂来扰乱蛋白质动态和变构。 2. 是否存在特别受变构调节影响的结构元件网络？其扰动可导致蛋白质动力学和变构变化的结构元件将通过以下方式识别： a) 由于突变导致 X 射线数据中热 B 因子的变化； b) H/D 交换测量与质谱联用； c) 结构连通性的计算证据。 3. 人们能否利用在目标 1 和 2 中获得的知识来帮助定义在定义激活效应器的特异性时发挥长程效应的结构元件？ CRP 被 cAMP 最有效地激活，尽管它可以结合其他 cNMP 而不会被激活。文献结果表明，cAMP 结合位点之外的多肽，特别是 DNA 结合结构域，决定了 CRP 响应结合配体的能力。基于显示 cAMP 结合位点和 DNA 结合结构域之间连接性的初步计算结果，将使用突变体来测试连接模式。 cAMP 结合域的 X 射线结构数据将用于测试 DNA 结合域的存在是否会改变 cAMP 结合域中的结构连接模式。