CYTOCHROME C MECHANISMS BY RECOMBINANT DNA TECHNIQUES

通过重组 DNA 技术研究细胞色素 C 机制

• 批准号: 3276425
• 负责人: EMANUEL MARGOLIASH
• 金额: $ 15.76万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 1994-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3276425
• 关键词: DNA; Drosophilidae; X ray crystallography; acetylation; apoenzymes; covalent bond; cytochrome c; cytochrome oxidase; electron transport; enzyme mechanism; enzyme structure; gene expression; genetic manipulation; hemoprotein structure; high performance liquid chromatography; immunochemistry; laboratory mouse; laboratory rabbit; laboratory rat; methylation; mitochondrial membrane; molecular cloning; mutant; nuclear magnetic resonance spectroscopy; nucleic acid hybridization; nucleic acid sequence; protein engineering; protein folding; protein sequence; protein structure function; site directed mutagenesis; spectrometry; yeasts

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.

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。

项目成果

Developmental expression of nuclear genes that encode mitochondrial proteins: insect cytochromes c.

• DOI: 10.1073/pnas.82.7.1964
• 发表时间: 1985-04
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: M. Swanson;S. M. Zieminn;D. Miller;E. Garber;E. Margoliash

Role of phospholipid in the low affinity reactions between cytochrome c and cytochrome oxidase.

磷脂在细胞色素 c 和细胞色素氧化酶之间的低亲和力反应中的作用。

• DOI: 10.1016/0014-5793(83)80321-4
• 发表时间: 1983
• 期刊: FEBS letters
• 影响因子: 3.5
• 作者: Speck,SH;Neu,CA;Swanson,MS;Margoliash,E
• 通讯作者: Margoliash,E

Changing the invariant proline-30 of rat and Drosophila melanogaster cytochromes c to alanine or valine destabilizes the heme crevice more than the overall conformation.

将大鼠和果蝇细胞色素 c 的不变脯氨酸 30 更改为丙氨酸或缬氨酸会比整体构象更不稳定地破坏血红素缝隙。

• DOI: 10.1073/pnas.87.22.8697
• 发表时间: 1990
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Koshy,TI;Luntz,TL;Schejter,A;Margoliash,E
• 通讯作者: Margoliash,E

The interaction of cytochrome c with cytochrome oxidase.

细胞色素c与细胞色素氧化酶的相互作用。

• 发表时间: 1988
• 期刊: Progress in clinical and biological research
• 作者: Garber,EA;Luntz,TL;Margoliash,E
• 通讯作者: Margoliash,E

Single catalytic site model for the oxidation of ferrocytochrome c by mitochondrial cytochrome c oxidase.

• DOI: 10.1073/pnas.81.2.347
• 发表时间: 1984
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: S. Speck;D. Dye;E. Margoliash