MUTAGENESIS-ASSISTED FUNCTIONAL STUDIES OF CYTOCHROME C

细胞色素 C 的诱变辅助功能研究

• 批准号: 3283855
• 负责人: ARTHUR G MAUK
• 金额: $ 10.11万
• 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/3283855
• 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.

一个针对细胞色素C的三个主要功能特性的程序是 概述。 （1）调节细胞色素C结构和功能 ph的属性。 （a）细胞色素c还原的pH依赖性 电势高度依赖于物种。 电位计量学，电化学， 突变体的NMR特性，旨在测试可能的起源 将进行该物种变化。 （b）尽管形成 用PK〜8.5-9的细胞色素c的碱性构象是众所周知的，现在 似乎是细胞色素C的生理功能， 这种构象变化和碱性的活跃位点结构 细胞色素C的理解不足。 变化的突变细胞色素 碱构象将由Kinetic，EPR，NIR-MCD和NMR研究 方法，突变细胞色素的功能特性改变了 残留物对于细胞色素c构象的氧化还原连接变化至关重要 将进一步表征。 （2）细胞色素的相互作用和反应 C与其他血红素蛋白。 （a）将使用电位计量和NMR方法 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，部分是细胞色素B5与血红蛋白的结合。 这 电位测量技术提供了以前无法获得的信息 关于质子结合的络合变化和精确的变化 平衡常数用于复杂形成。 （b）经过特殊修改 野生型和突变体细胞色素c衍生物在特定Lys上修饰 残留物允许通过NMR分辨率来识别 涉及蛋白质蛋白识别的残基。 （c）厌氧停止 - 流动动力学将与布朗动力学计算一起研究 双子分子电子在成对的血红素蛋白之间转移，以评估 静电和热力学考虑的相对贡献 观察到的速率。 （d）在细胞色素上修饰的两种蛋白的突变体 C-杂化色素C过氧化物酶蛋白 - 蛋白质界面将通过 各种形式的细胞色素c-（Zncytoochrome c）的动力学分析 过氧化物酶）复合物。 （3）掺入电子的新策略 细胞色素中的供体/受体中心c。 创建第二个方法的两种方法 提出了细胞色素C中的氧化还原活性位点。 这些涉及（a） 黄素通过突变表面Cys的烷基化的共价附着 残留物和（b）重建CYS-102周围环境 酵母细胞色素C创建（I型（蓝色））铜中心。