Structure Function Studies of Cytochromes

本研究的目的是研究细胞色素C1蛋白的结构和功能关系。遗传和分子生物学方法将用于系统地改变酵母的细胞色素c1 （Cc1）。Cc1突变体将通过三种方法产生：传统的寡核苷酸定向诱变，用合成的寡核苷酸直接转化酵母，以及pcr生成的随机诱变。Cc1突变体将通过在不可发酵培养基（Nfs+或Nfs-）上的生长来测试其功能，通过光谱分析来测试Cc1水平，并通过显性负遗传分析来测试Nfs-突变体的整体结构。在体内和体外，改变后的形式将用于研究Cc1的结构-功能关系以及Cc1与细胞色素c （Cc）之间的相互作用。Cc1和Cc之间的相互作用位点，以及细胞色素和Qcr6p之间的相互作用位点，将通过使用抑制突变的遗传方法进行研究。体外反应性将通过稳态和单次转换方法进行研究，包括利用Cc的光诱导电子转移进行高ergonic (G)反应。本研究的目的是研究蛋白质细胞色素c1 （Cc1）的结构功能关系。这种蛋白质在线粒体电子传递中起着关键作用，从而有助于控制细胞内的能量产生。本研究将探讨影响Cc1氧化还原电位的因素，从而影响该体系的热力学和动力学反应性。这项工作将采用遗传、分子生物学和生物物理学程序。＊＊＊

9707680 McLendon The aims of this research are to examine structure function relationships in the protein cytochrome C1. Genetic and molecular biological procedures will be used to systematically alter cytochrome c1 (Cc1) in the yeast Saccharomyces cerevisiae. Cc1 mutants will be generated by three procedures: conventional oligonucleotide-directed mutagenesis, transformation of yeast directly with synthetic oligonucleotides, and PCR-generated random mutagenesis. The Cc1 mutants will be tested for functionality by growth on nonfermentable media (Nfs+ or Nfs-) , Cc1 levels by spectral analysis, and preservation of the over-all structure of Nfs- mutants by dominate-negative genetic analysis. The altered forms will be used to investigate structure-function relationships of Cc1 and the interactions between Cc1 and cytochrome c (Cc), both in vivo and in vitro. The sites of interaction between Cc1 and Cc, as well as between the cytochromes and Qcr6p, will be investigated with genetic approaches using suppressor mutations. Reactivity in vitro will be studied by steady state and single turnover methods, including highly exergonic (G( reactions using photoinduced electron transfer from Cc. The objective of this research is to examine structure function relationships in the protein cytochrome c1 (Cc1). This protein plays a critical role in mitochodrial electron transport, and thereby helps control energy production within cells. This research will probe factors which influence the redox potentials of Cc1, and thereby affect both thermodynamic and kinetic reactivity of this system. The work will employ genetic, molecular biological and biophysical procedures. ***