Structure Function Studies of Cytochromes

9707680麦克伦登这项研究的目的是研究蛋白质细胞色素C1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酿酒酵母中的细胞色素Cc1。CC1突变将通过三种程序产生：传统的寡核苷酸定向突变，人工合成的寡核苷酸直接转化酵母，以及聚合酶链式反应产生的随机突变。Cc1突变体将通过在不可发酵的介质(NFS+或NFS-)上生长来测试功能，通过光谱分析测试Cc1的水平，并通过显性-负遗传分析来保存NfS-突变体的总体结构。这些改变的形式将被用来研究Cc1的结构-功能关系以及Cc1与细胞色素c(CC)在体内和体外的相互作用。Cc1和CC之间以及细胞色素和Qcr6p之间的相互作用部位将用抑制突变的遗传学方法进行研究。体外反应活性将通过稳态和单周转方法进行研究，包括利用CC的光诱导电子转移进行的高强度(G)反应。本研究的目的是研究细胞色素Cc1蛋白的结构与功能的关系。这种蛋白质在线粒体电子传递中起关键作用，从而帮助控制细胞内的能量产生。本研究将探索影响CC1氧化还原电势的因素，从而影响该体系的热力学和动力学反应活性。这项工作将使用遗传、分子生物学和生物物理程序。***

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. ***