CYTOCHROME C MECHANISMS BY RECOMBINANT DNA TECHNIQUES

传统上，在全面了解 蛋白质的结构使其功能发挥作用的机制， 实际控制其生理活动的先决条件， 来自于分析特定活动变化的研究， 由特定的氨基酸化学修饰或由 由基因损伤造成的氨基酸替换。 然而，化学 氨基酸侧链的修饰仅限于小部分 而幸存的遗传修饰是随机的， 仅限于非致命突变。 这些经典的方法 现在通过重组DNA的使用， 获得具有任何所需氨基酸序列的细胞色素C的技术。 克隆的真核细胞色素c基因的定点诱变，以及 在异源表达系统中生产蛋白质，将是 研究特定氨基酸取代如何影响蛋白质 稳定性、生物合成以及电子传递和结合 与线粒体的还原酶和氧化酶复合物的亲和力 呼吸链 第二个目标是继续改善 用于在酵母中获得突变体全细胞色素c的方法，以及 开发更有效的技术来生产大量的 大肠杆菌中的野生型和突变型脱细胞色素c。 使用 脱辅基蛋白的突变体，生物合成的中间体，打开了大门， 检查几个生理相关的过程， 描述蛋白质的生命周期。 其中包括承认 脱辅基蛋白的线粒体外膜，其运输 通过膜，血红素的酶催化共价结合通过膜，血红素的酶催化共价结合 辅基的载脂蛋白，全蛋白的释放到 线粒体膜间隙，以及水平的机制， 线粒体中的细胞色素c受到调节。 最后，核苷酸 克隆的真核细胞色素c基因的序列将用于计算 统计系统发育树，定义发生在 所考虑的不同的进化血统。 这将允许 通过表达， 通过定点突变人工改变基因， 追溯过去进化转型的功能伴随物。

Traditionally, significant progress towards a comprehensive understanding of the mechanisms by which a protein's structure subserves its function, a prerequisite for the practical control of its physiological activities, has come from studies which assay the changes in a particular activity, resulting either from specific amino acid chemical modifications or from amino acid substitutions created by a genetic lesion. However, chemical modifications of amino acid side chains are limited to a small proportion of residues, while surviving genetic modifications are random and necessarily limited to non-lethal mutations. These classical approaches are now being dramatically extended through the use of recombinant DNA techniques to obtain cytochromes c with any desired amino acid sequence. Site directed mutagenesis of cloned eukaryotic cytochrome c genes, and the production of the protein in heterologous expression systems, will be performed to study how particular amino acid substitutions affect protein stability, biosynthesis, as well as electron transport and binding affinities with the reductase and oxidase complexes of the mitochondrial respiratory chain. Secondary objectives are the continued improvement of th methodologies employed to obtain mutant holocytochromes c in yeast, and the development of more efficient techniques to produce large quantities of wild-type and mutant apocytochromes c in Escherichia coli. The use of mutants of the apoprotein, the biosynthetic intermediate, opens the door to the examination of several physiologically-relevant processes that characterize the life cycle of the protein. Thes include the recognition of the apoprotein by the outer mitochondrial membrane, its transport through the membrane, the enzymecatalyzed covalent binding of the heme prosthetic group to the apoprotein, the release of the holoprotein into the mitochondrial intermembrane space, and the mechanism by which the level of cytochrome c in mitochondria is regulated. Finally, the nucleotide sequences of cloned eukaryotic cytochrome c genes will be used to calculate statistical phylogenetic trees that define the mutations which occurred in the different lines of evolutionary descent considered. This will allow the production of extinct evolutionary forms of the protein by expressing genes artificially changed through site-directed mutagenesis, and thus retrace the functional concomitants of past evolutionary transitions.