Role of Protein Dynamics in Modulating the Thermodynamic Linkages in Allostery

描述（由申请人提供）：cAMP受体蛋白（CRP）是转录因子超家族的原型。CRP的生物活性的一个关键特征一直是本实验室研究的焦点，这是控制cAMP对其序列特异性DNA结合的变构激活的基本规则。最近，我们建立，在任何变构系统中，第一次，变构参数的能量是蛋白质动力学的线性函数。以蛋白质动力学为焦点，从晶体学、计算和功能能量数据得到的结果之间存在收敛。这一初步的成功使我们能够将我们的研究工作集中在蛋白质动力学作为基本的物理性质，以建立CRP结构和变构机制之间的定量联系。在未来几年，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的基本性质之一吗？将采用两种策略来测试这种关系的有效性：a）将溶剂可接近的环作为改变蛋白质动力学和变构的结构元件; B）将影响蛋白质动力学运动的渗透剂作为溶剂添加剂来扰动B蛋白质动力学和变构。 2.是否存在一个特别受变构调节影响的结构元件网络？结构元素的扰动可能导致蛋白质动力学和变构的变化，将通过以下方式确定：a）由于突变导致的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;以及c）结构连接性的计算证据。 3.能否利用目标1和目标2中获得的知识来帮助定义在定义激活效应物的特异性时发挥长期作用的结构元件？CRP被cAMP最有效地激活，尽管它可以结合其他cNMP而不被激活。文献结果表明，cAMP结合位点外的多肽，特别是DNA结合结构域，决定了CRP对结合配体的反应能力。基于初步计算结果（显示cAMP结合位点和DNA结合结构域之间的连接性），突变体将用于测试连接性模式。将使用cAMP结合结构域的X射线结构数据来测试DNA结合结构域的存在是否改变了cAMP结合结构域中的结构连接模式。

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.