MUTAGENESIS-ASSISTED FUNCTIONAL STUDIES OF CYTOCHROME C

细胞色素 C 的诱变辅助功能研究

• 批准号: 3283850
• 负责人: ARTHUR G MAUK
• 金额: $ 9.7万
• 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 1994-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3283850
• 关键词: Saccharomyces cerevisiae; acidity /alkalinity; active sites; circular dichroism; conformation; cytochrome b; cytochrome c; electron spin resonance spectroscopy; flavins; fungal genetics; hemoglobin; hemoprotein structure; high performance liquid chromatography; ligands; mutant; nuclear magnetic resonance spectroscopy; oxidation reduction reaction; peroxidases; point mutation; potentiometry; protein sequence; protein structure function; site directed mutagenesis; stop flow technique; temperature sensitive mutant; tissue /cell culture; yeasts; zinc

A program addressing three major functional properties of cytochrome c is outlined. (1) Regulation of cytochrome c structure and functional properties by pH. (a) The pH-dependence of cytochrome c reduction potentials is highly species dependent. Potentiometric, electrochemical, and NMR characteristics of mutants designed to test possible origins of this species variation will be undertaken. (b) Although formation of an alkaline conformation of cytochrome c with a pK~8.5-9 is well known and now appears to be a physiological function of cytochrome c, the mechanism of this conformational change and the active site structure of alkaline cytochrome c are poorly understood. Mutant cytochromes with altered alkaline conformations will be studied by kinetic, EPR, NIR-MCD, and NMR methods, and the functional properties of mutant cytochromes altered at residues critical to the redox-linked change in cytochrome c conformation will be further characterized. (2) Interaction and reaction of cytochrome c with other heme proteins. (a) Potentiometric and NMR methods will be used to study binding of cytochrome c to cytochrome b5 and cytochrome c peroxidase and, in part, binding of cytochrome b5 to hemoglobin. The potentiometric technique provides previously unavailable information concerning complexation-linked changes in proton binding and precise equilibrium constants for complex formation. (b) Specifically modified wild-type and mutant cytochrome c derivatives modified at specific Lys residues to permit their resolution by NMR will be used to identify residues involved in protein-protein recognition. (c) Anaerobic stopped- flow kinetics will be used with Brownian dynamics calculations to study the bimolecular electron transfer between pairs of heme proteins to assess the relative contributions of electrostatic and thermodynamic considerations to the observed rates. (d) Mutants of both proteins modified at the cytochrome c-cytochrome c peroxidase protein-protein interface will be studied by kinetic analysis of various forms of the cytochrome c-(Zncytochrome c peroxidase) complex. (3) New strategies for incorporation of electron donor/acceptor centers in cytochrome c. Two methods for creating a second redox-active sites in cytochrome c are proposed. These involve (a) covalent attachment of flavins through alkylation of mutant surface Cys residues and (b) reconstruction of the environment surrounding Cys-102 in yeast cytochrome c to create a (type I(blue)) copper center.

解决细胞色素c的三个主要功能特性的程序是 概述。(1)细胞色素c结构和功能的调节 （a）细胞色素c还原的pH依赖性 潜力是高度依赖于物种的。 电位，电化学， 和核磁共振特征的突变体，旨在测试可能的起源， 将进行这种物种变异。 (b)虽然形成了 细胞色素c的碱性构象，pK约为8.5-9是众所周知的， 似乎是细胞色素c的生理功能， 这种构象变化和碱的活性中心结构 细胞色素C的了解很少。 具有改变的突变细胞色素 碱性构象将通过动力学、EPR、NIR-MCD和NMR进行研究 方法，和突变细胞色素的功能特性改变， 对细胞色素c构象的氧化还原连接变化至关重要的残基 将得到进一步表征。 (2)细胞色素的相互作用和反应 C与其他血红素蛋白质。(a)将使用电位法和核磁共振法 研究细胞色素c与细胞色素b5和细胞色素c的结合 过氧化物酶和部分细胞色素b5与血红蛋白的结合。 的 电位技术提供了以前无法获得的信息 关于质子结合中的络合相关变化和精确 络合物形成的平衡常数。 (b)特异性修饰 在特异性Lys处修饰的野生型和突变型细胞色素c衍生物 允许通过NMR进行拆分的残基将用于鉴定 参与蛋白质识别的残基。(c)厌氧停止- 流动动力学将与布朗动力学计算一起使用， 血红素蛋白对之间的双分子电子转移，以评估 静电和热力学考虑的相对贡献 观察到的比率。(d)两种蛋白质的突变体在细胞色素 c-细胞色素c过氧化物酶蛋白质-蛋白质界面将通过 不同形式的细胞色素c-（Zn-cytochrome c）的动力学分析 过氧化物酶）复合物。 (3)引入电子的新策略 细胞色素c的供体/受体中心。 创建第二个的两种方法 提出了细胞色素C中的氧化还原活性位点。 其中包括：（a） 通过突变体表面Cys的烷基化共价连接黄素 （B）Cys-102周围环境的重建 酵母细胞色素c产生一个（I型（蓝色））铜中心。