Thermodynamics of Protein Fluctuations

描述(申请人提供)：利用核磁共振弛豫和氢交换对蛋白质进行的实验研究明确地证明，即使在自然条件下，也存在作为规范高分辨结构的构象漂移的状态。然而，尽管人们知道它们的存在，但对这些状态的性质和能量学知之甚少。理解在自然条件下存在的构象状态的结构和能量细节的重要性怎么强调都不为过。由于观察到的蛋白质的生物活性是系综中组成微态的能量(或玻尔兹曼)加权贡献，因此了解这些微态的结构和热力学特征是从分子水平理解蛋白质功能的先决条件。开发了一种实验策略，利用稳定性和结合亲和力之间的热力学联系。根据这一连锁方案，通过监测ALA到Gly突变对观察到的结合亲和力(使用等温滴定热法)和稳定性(使用氢-氚交换)的影响，可以直接确定：1)波动和“局部展开”之间的相似性；2)波动对结合的定量影响；3)尿素、渗透压、温度和pH对这种行为的影响；以及4)在特定环内的不同位置和两个模型蛋白质中的不同环获得的结果的可变性。该策略被应用于两个模型蛋白质：SEM5的C-SH3结构域和E.Coli的几个环的分析。二氢叶酸还原酶(DHFR)。这里描述的研究代表了用于阐明即使在自然条件下也被填充的正则结构的构象变体的结构和能量的独特策略。

DESCRIPTION (provided by applicant): Experimental studies on proteins using NMR relaxation and hydrogen exchange have unequivocally established the existence of states that are conformational excursions of the canonical high resolution structure, even under native conditions. Despite knowledge of their existence, however, little is known of the nature and energetics of these states. The importance of understanding the structural and energetic details of the conformational states that exist under native conditions cannot be overstated. As the observed biological activity of a protein is the energy (or Boltzmann)-weighted contribution of the component microstates in the ensemble, knowledge of the structural and thermodynamic features of these states is a prerequisite to a molecular-level understanding of protein function. An experimental strategy has been developed that takes advantage of the thermodynamic linkage between stability and binding affinity. According to this linkage scheme, by monitoring the effects of ALA to GLY mutations on the observed binding affinity (using isothermal titration calorimetry) and stability (using hydrogen-deuterium exchange), it is possible to directly determine; 1) the similarity between fluctuations and 'local unfolding;' 2) the quantitative impact of fluctuation on binding; 3) the effect of urea, osmolytes, temperature, and pH on this behavior; and 4) the variability of the results obtained at different sites within a particular loop, and at different loops within the two model proteins. The strategy is applied to the analysis of several loops in two model proteins; the C-SH3 domain of SEM5, and E. Coli. dihydrofolate reductase (DHFR). The studies described herein represent a unique strategy for elucidating the structure and energy of the conformational variants of the canonical structure, which are populated even under native conditions.