CYTOCHROME C MECHANISMS BY RECOMBINANT DNA TECHNIQUES

传统上，朝着全面的 对蛋白质结构的机制的理解 从属于其功能，是实施控制的前提 它的生理活动，来自于研究分析 特定活动中的变化，由以下两种情况之一引起 特定氨基酸化学修饰或来源于氨基酸 由一种基因损伤造成的替换。然而，化学 氨基酸侧链的修饰仅限于一小部分 残留比例，而幸存的基因修改是 随机且必然限于非致命性突变。这些 经典的方法现在正在戏剧性地扩展到 利用重组DNA技术获得细胞色素c 任何所需的氨基酸序列。基因的定点突变 真核细胞色素c基因的克隆及其产物的制备 在异源表达系统中的蛋白质，将被执行以 研究特定的氨基酸替代如何影响蛋白质 结构和稳定性及其生物合成，以及电子 运输和结合亲和力与各种生理功能 反应伙伴，如线粒体细胞色素C氧化酶 细胞色素c还原酶和酵母细胞色素c过氧化物酶。自.以来 我们目前生产突变细胞色素c的技术 要求它们至少部分发挥作用，这是一个重要的 次要目标是制定程序以获得 无功能突变体在酵母中的表达。对.的使用 生物合成中间体--载脂蛋白的突变，开启了 几个生理上相关的门的检查 描述蛋白质生命周期的过程。这些 包括外膜识别脱辅基蛋白。 线粒体膜，它通过膜的运输， 酶催化的血红素修复基团与 载脂蛋白，将完整蛋白释放到 线粒体膜间间隙及其作用机制 线粒体中细胞色素c的水平受到调节。最后，我们的 目前酵母中的系统产生N-末端乙酰化和 非乙酰化大鼠细胞色素c，两者都携带完整的 三甲基化赖氨酸72；它还产生果蝇黑腹果蝇 细胞色素C，其赖氨酸以三、二、一和 非甲基化形式，已用高效液相色谱分离。这些 蛋白质提供了一个到目前为止研究的独特机会 这些众所周知的次生物质的结构和功能效应 细胞色素c的修饰。

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 lesions. 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 structure and stability its biosynthesis, as well as electron transport and binding affinities with various physiological reaction partner, such as the mitochondrial cytochrome c oxidase cytochrome c reductase and yeast cytochrome c peroxidase. Since our present technique for the production of mutant cytochromes c requires them to be at least partially functional an important secondary objective is the development of procedures for obtaining the expression in yeast of functionless mutants. 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. These include the recognition of the apoprotein by the outer mitochondrial membrane, its transport through the membrane, the enzyme-catalyzed 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, our present system in yeast produces both N-terminally acetylated and non-acetylated rat cytochrome c, both carrying a fully trimethylated lysine 72; it also yields Drosophila melanogaster cytochrome c which has that lysine in tri-, di-, mono- and unmethylated forms, which have been separated by HPLC. These proteins provide a so far unique opportunity for studying the structural and the functional effects of these well known secondary modifications of cytochrome c.