Thermodynamics of Protein Fluctuations

蛋白质波动的热力学

• 批准号: 7269537
• 负责人: VINCENT J. HILSER
• 金额: $ 27.45万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7269537
• 关键词: Affect; Affinity; Behavior; Binding; Biological; C-terminal; Calorimetry; Chemicals; Condition; Contracts; Deuterium; Development; Dihydrofolate Reductase; Escherichia coli; Goals; Hydrogen; Knowledge; Ligands; Measures; Modeling; Molecular; Molecular Conformation; Monitor; Mutation; Nature; Proline; Protein C; Proteins; Relaxation; Research Personnel; Resolution; Role; SH3 Domains; Sarcosine; Scheme; Site; Structure; Surface; Temperature; Testing; Thermodynamics; Titrations; Urea; Variant; Weight; alanylglycine; base; innovation; molecular recognition; mutant; protein function; research study; trimethyloxamine

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.

描述（由申请人提供）：使用 NMR 弛豫和氢交换对蛋白质进行的实验研究已明确证实，即使在天然条件下，也存在规范高分辨率结构的构象偏移状态。然而，尽管知道它们的存在，但人们对这些状态的性质和能量知之甚少。了解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也不为过。由于观察到的蛋白质生物活性是整体中组分微观状态的能量（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。已经开发出一种利用稳定性和结合亲和力之间的热力学联系的实验策略。根据该连接方案，通过监测ALA至GLY突变对观察到的结合亲和力（使用等温滴定量热法）和稳定性（使用氢-氘交换）的影响，可以直接确定； 1）波动与“局部展开”之间的相似性； 2）波动对结合的定量影响； 3) 尿素、渗透剂、温度和 pH 值对此行为的影响； 4) 在特定环内的不同位点以及在两个模型蛋白内的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中多个环的分析； SEM5 的 C-SH3 结构域和大肠杆菌。二氢叶酸还原酶（DHFR）。本文描述的研究代表了一种独特的策略，用于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天然条件下也存在。