MUTAGENESIS ASSISTED FUNCTIONAL STUDIES OF CYTOCHROME C

细胞色素 C 的诱变辅助功能研究

• 批准号: 2177150
• 负责人: ARTHUR G MAUK
• 金额: $ 10.35万
• 依托单位: UNIVERSITY OF BRITISH COLUMBIA
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-09-20 至 1997-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2177150
• 关键词: acidity /alkalinity; chemical kinetics; circular dichroism; computer simulation; conformation; cytochrome b; cytochrome c; electron transport; flavins; hemoprotein; hemoprotein structure; ionic bond; microorganism culture; molecular dynamics; mutant; nuclear magnetic resonance spectroscopy; oxidation reduction reaction; peroxidases; potentiometry; protein structure function; recombinant proteins; site directed mutagenesis; stop flow technique; synthetic nucleic acid

Three fundamental aspects of cytochrome c function that have general implications for many other proteins will be studied: (1) Regulation of cytochrome c conformational dynamics and midpoint reduction potential by pH. At alkaline pH, the Met80 ligand to the heme iron is replaced by either Lys79 or Lys73 to produce two alkaline conformational states. Electrochemical, stopped-flow, and NMR analysis of these two conformational isomers will be undertaken through analysis of the Lys79Ala and Lys73Ala variants to assess their structural and functional properties. Related studies will consider (a) the conformational dynamics of mutations at Phe82 that have previously been shown to lower the PKa for the conformational equilibrium, (b) the conformational and functional consequences of replacing residues 79 and 73 with alternative "alkaline ligands," and (c) the identity of the conformationally-linked titratable group that deprotonates prior to loss of the Met80 ligand. (2) Interaction and reaction of cytochrome c with other heme proteins. (a) The interaction of cytochrome c with cytochrome b5 will be studied through application of a novel NMR technique for identification of surface Lys residues involved in complex formation, through detailed electrostatics modelling of potentiometric titrations of the cytochrome c-cytochrome b5 complex and by rapid kinetics techniques. (b) The cytochrome c-cytochrome c peroxidase complex will be characterized by potentiometric titrations and rapid kinetics techniques that will include use of critically selected mutants of the peroxidase predicted to disrupt each of the two binding sites for the cytochrome. These results will also be simulated by electrostatics modelling calculations. (c) The complex formed by human erythrocyte cytochrome b5 and human hemoglobin and its subunits will be analyzed by potentiometric titrations. Parallel studies will assess the influence of complex formation on the spectroscopic and ligand binding characteristics of hemoglobin. (3) The role of electron donor structure on kinetics of intramolecular electron transfer kinetics. Synthetic flavocytochromes produced by a combination of site-directed mutagenesis and chemical modification will be used to study the effect of electron donor structure on intramolecular electron transfer kinetics by comparison with published results obtained with analogous cytochrome derivatives in which ruthenium complexes serve as the electron donor.

细胞色素C功能的三个基本方面，具有一般 将研究对许多其他蛋白质的影响：（1）调节 细胞色素C构象动力学和中点减少潜力 ph。在碱性pH时，Met80配体向血红素铁取代 lys79或lys73产生两个碱性构象状态。 这两个的电化学，停止流量和NMR分析 将通过分析来进行构象异构体 lys79ala和lys73ala变体，以评估其结构和功能 特性。相关研究将考虑（a）构象动力学 以前已显示降低PKA的PHE82突变的突变 对于构象平衡，（b）构象和功能 用替代碱性替换残留物79和73的后果 配体，“（c）构象连接的滴定的身份 在丢失Met80配体之前将质子化的组。 （2） 细胞色素c与其他血红素蛋白的相互作用和反应。 （一个） 将研究细胞色素C与细胞色素B5的相互作用 通过应用新颖的NMR技术来识别 通过详细的表面LYS残基参与复杂形成 细胞色素电位滴定的静电模型 c-环形B5复合物和快速动力学技术。 （b） 细胞色素c-环形C型C-过氧化物酶复合物的特征是 电位计量滴定和快速动力学技术包括 使用被预测的过氧化物酶突变体的使用 细胞色素的两个结合位点中的每一个。这些结果也将 通过静电建模计算模拟。 （c）复合物 由人类红细胞细胞色素B5和人类血红蛋白及其形成 亚基将通过电位测量滴定分析。平行研究 将评估复杂形成对光谱的影响和 血红蛋白的配体结合特征。 （3）电子的作用 分子内电子转移动力学动力学的供体结构。 由定位的组合产生的合成黄素色素 诱变和化学修饰将用于研究效果 分子内电子转移动力学上的电子供体结构 通过与类似细胞色素获得的已发表的结果相比 唯一复合物充当电子供体的衍生物。