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性 对许多其他蛋白质的影响将被研究：(1)调节 细胞色素c构象动力学和中点还原电位 PH值。在碱性pH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 Lys79Ala和Lys73Ala变体以评估其结构和功能 属性。相关研究将考虑(A)构象动力学 之前已被证明降低PKA的Phe82位突变 对于构象平衡，(B)构象和功能 用“碱性”替代第79和73号残留物的后果 配体“和(C)构象连接的可滴定物质的身份 在失去Met80配体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。(A) 细胞色素c与细胞色素b5的相互作用将被研究。 通过应用一种新的核磁共振技术来鉴定 表面赖氨酸残基参与复杂的形成，通过详细的 细胞色素电位滴定的静电学模拟 C-细胞色素b5复合体和快速动力学技术。(B) 细胞色素c-细胞色素c过氧化物酶复合体的特征是 电位滴定和快速动力学技术将包括 使用经过严格挑选的过氧化物酶突变体预测将扰乱 细胞色素两个结合部位中的每一个。这些结果也将 通过静电学模型计算进行模拟。(C)综合大楼 人红细胞细胞色素b5与人血红蛋白的结合 亚基将通过电位滴定进行分析。平行研究 将评估络合物的形成对光谱和 血红蛋白的配体结合特性。(3)电子的作用 施主结构对分子内电子转移动力学的影响。 由定点组合生产的合成黄色素 将使用诱变和化学修饰来研究其效果 电子给体结构对分子内电子转移动力学的影响 通过与发表的类似细胞色素的结果进行比较 以Ru络合物为电子给体的衍生物。