Role of Protein Dynamics in Modulating the Thermodynamic Linkages in Allostery

CAMP受体蛋白(CRP)是转录因子超家族的原型。的一项关键功能 CRP的生物活性一直是本实验室研究的重点，它的基本规律是 通过cAMP序列特异性的DMA结合来控制变构激活。最近，我们建立了 在任何变构体系中，变构参数的能量学第一次是 蛋白质动力学。以蛋白质动力学为焦点，得到的结果具有一定的收敛特性 从结晶学、计算和功能能量学数据。这一初步成功使我们能够 将我们的研究重点放在作为基本物理性质的蛋白质动力学上，以建立定量的 C反应蛋白结构与变构作用机制的联系(S)。在未来几年，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的基本性质之一吗？二 将采用策略来测试这种关系的有效性：a)可通过溶剂访问的循环是 靶向作为改变蛋白质动力学和变构的结构元件；b)渗透压，影响动力学 蛋白质的运动，将被用作溶剂添加剂来扰乱蛋白质的动力学和变构。 2.是否存在受变构调节特别影响的结构元件网络？这个 其扰动会导致蛋白质动力学和变构发生变化的结构元素将是 确定为：a)X射线数据中热B因子因突变而变化；b)H/D交换 与质谱学相结合的测量；以及c)结构连接性的计算证据。 3.是否可以使用在目标1和目标2中获得的知识来帮助确定结构要素 在定义激活效应器的专一性方面发挥长程效应？C反应蛋白是最活跃的 CAMP可以有效地结合其他cNMP，而不被激活。文献结果表明， CAMP结合部位外的多肽，特别是DNA结合域，决定了 C反应蛋白对结合的配体作出反应。基于显示连通性的初步计算结果 在cAMP结合点和DNA结合域之间，将使用突变体来测试连接性 图案。CAMP结合域的X射线结构数据将用于测试DNA是否存在 结合结构域改变cAMP结合结构域中的结构连接模式。

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.