Thermodynamics of Protein Fluctuations

描述（由申请人提供）：使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确立了规范高分辨率结构构象漂移态的存在，即使在自然条件下也是如此。然而，尽管知道它们的存在，但对这些状态的性质和能量学知之甚少。了解天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也不过分。由于观察到的蛋白质的生物活性是集合中组成微态的能量（或玻尔兹曼）加权贡献，因此了解这些状态的结构和热力学特征是分子水平理解蛋白质功能的先决条件。利用稳定性和结合亲和之间的热力学联系，开发了一种实验策略。根据该连锁方案，通过监测ALA对GLY突变对观察到的结合亲和力（采用等温滴定量热法）和稳定性（采用氢-氘交换法）的影响，可以直接确定；1)波动与局部展开的相似性；’2)波动对绑定的定量影响；3)尿素、渗透物、温度和pH值对这一行为的影响；4)在特定环内不同位点和两种模型蛋白内不同环处获得的结果的可变性。该策略应用于两种模型蛋白的几个环的分析；SEM5的C-SH3结构域和大肠杆菌。二氢叶酸还原酶（DHFR）本文所描述的研究代表了一种独特的策略来阐明规范结构的构象变体的结构和能量，这些构象变体甚至在自然条件下也存在。

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.