METALLOPROTEIN STABILITY AND REDOX CHEMISTRY

金属蛋白稳定性和氧化还原化学

• 批准号: 2188767
• 负责人: DONALD M. KURTZ
• 金额: $ 20.12万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 1998-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2188767
• 关键词: Archaea; Clostridium; bacterial pigment; bacterial proteins; calorimetry; chemical kinetics; computer simulation; gene mutation; hydrogen bond; hydropathy; intermolecular interaction; ionic bond; molecular dynamics; oxidation reduction reaction; peptide chemical synthesis; polymerase chain reaction; protein denaturation; protein folding; protein sequence; rubredoxins; temperature; thermodynamics; thermophilic organism; thermostability; ultraviolet spectrometry

We propose to develop the small Fe-S protein rubredoxin into a paradigm for understanding how a metalloprotein's molecular architecture controls its stability and redox behavior. Comparisons will be made between a mesophilic (Clostridium pasteurianum, optimum growth temperature of 37 degrees C) and a hyperthermophilic (Pyrococcus furiosus, optimum growth temperature of 95 degrees C) rubredoxin to dissect the specific protein determinants that confer the enhanced thermostability of the latter. Mutational analyses and detailed spectroscopic and calorimetric characterization of the mutated proteins will be used to partition the contributions (charge interactions, hydrogen bonding, metal-ligand bonding, etc.) to this stability. Molecular dynamics simulations will be used to predict effects on thermostability of specific mutations; we will test the ability of such simulations to confirm experimentally observed changes in stability. The contribution of the protein in modulating the redox potential of the Fe(S-Cys)4 site will also be explored by mutational analyses. An electrostatic model will be used to predict computationally the effect on redox potential of specific charge mutations and detailed electrochemical measurements (vs. ionic strength and temperature) on mutated proteins will test these predictions. This is expected to allow determination of the temperature dependence of an effective internal protein dielectric constant. Intramolecular electron-transfer kinetics studies will explore the possibility that P. junosus rubredoxin gains its increased stability from stronger or more extensive internal hydrogen bonding. Electronic coupling between the rubredoxin Fe(S-Cys)4 site and attached electron-donor complexes is expected to be sensitive to differences in internal protein architecture and temperature-dependent electron-transfer rate measurements will probe these differences between the P. furiosus and C. pasteurianum proteins. The combination of (a) the comparative nature of this study between two structurally homologous proteins with very different thermostabilities, and (b) the availability of the feedback loop between computational tools and experimental measurements, gives us a unique opportunity to fully characterize the protein determinants of thermostability and the protein tuning of redox potentials and electron-transfer rates. The methods developed in this project will be generally applicable to other metalloprotein systems.

我们建议将小的Fe-S蛋白鲁recredoxin开发到范式中 为了了解金属蛋白的分子结构如何控制 它的稳定性和氧化还原行为。将进行比较 中嗜（梭状芽胞杆菌的梭菌，最佳生长温度为37 学位c）和高疗（富洛氏菌，最佳生长） 温度为95度c）rubredoxin以剖析特定蛋白 赋予后者增强热稳定性的决定因素。 突变分析以及详细的光谱和量热法 突变蛋白的表征将用于划分 贡献（电荷相互作用，氢键，金属配体 结合等）达到这种稳定性。分子动力学模拟将是 用于预测对特定突变的热稳定性的影响；我们将 测试此类模拟确认实验观察到的能力 稳定性的变化。蛋白质在调节 Fe（S-Cys）4位点的氧化还原电位也将通过突变探索 分析。静电模型将用于预测计算 对特定电荷突变的氧化还原潜力的影响和详细的影响 电化学测量（与离子强度和温度） 突变的蛋白质将测试这些预测。这预计将允许 确定有效内部的温度依赖性 蛋白质介电常数。分子内电子转移动力学 研究将探讨junosus rubredoxin获得其获得的可能性 从更强或更广泛的内部氢中提高稳定性 结合。 Rubredoxin Fe（S-Cys）4位点和 预计附着的电子供应复合物对 内部蛋白质结构和温度依赖性的差异 电子转移速率测量将探究这些差异 Furiosus和C. basteurianum蛋白。 （a）的组合 这项研究的比较性质，两个结构同源 具有恒温截然不同的蛋白质，以及（b）可用性 计算工具与实验之间的反馈回路 测量，为我们提供了一个独特的机会，可以完全表征 蛋白质的蛋白质决定因素和氧化还原的蛋白质调整 电势和电子转移率。在此开发的方法 项目通常适用于其他金属蛋白系统。