MUTAGENESIS ASSISTED FUNCTIONAL STUDIES OF CYTOCHROME C

细胞色素 C 的诱变辅助功能研究

• 批准号: 2177149
• 负责人: ARTHUR G MAUK
• 金额: $ 10.4万
• 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/2177149
• 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性 将研究对许多其他蛋白质的影响：（1） 细胞色素 c 构象动力学和中点还原电位 酸碱度。在碱性 pH 值下，血红素铁的 Met80 配体被替换为 Lys79 或 Lys73 产生两种碱性构象状态。 这两种物质的电化学、停流和核磁共振分析 构象异构体将通过分析进行 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 Phe82 的突变先前已被证明可降低 PKa 对于构象平衡，（b）构象和功能 用替代品“碱性”替换残基 79 和 73 的后果 配体”，以及（c）构象连接的可滴定分子的身份 在 Met80 配体丢失之前去质子化的基团。 (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 通过应用新型核磁共振技术来识别 表面赖氨酸残基参与复杂的形成，通过详细 细胞色素电位滴定的静电建模 c-细胞色素b5复合物并通过快速动力学技术。 （二） 细胞色素c-细胞色素c过氧化物酶复合物的特征为 电位滴定和快速动力学技术，包括 使用经过严格选择的过氧化物酶突变体预计会破坏 细胞色素的两个结合位点中的每一个。这些结果也将 通过静电建模计算进行模拟。 (c) 综合体 由人红细胞的细胞色素b5和人血红蛋白组成及其 亚基将通过电位滴定进行分析。平行研究 将评估复合物形成对光谱和 血红蛋白的配体结合特征。 (3)电子的作用 供体结构对分子内电子转移动力学的影响。 通过定点组合产生的合成黄细胞色素 将使用诱变和化学修饰来研究效果 电子供体结构对分子内电子转移动力学的影响 与用类似细胞色素获得的已发表结果进行比较 钌络合物作为电子供体的衍生物。