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: 2175357
• 负责人: EMANUEL MARGOLIASH
• 金额: $ 16.59万
• 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 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2175357
• 关键词: DNA; Drosophilidae; X ray crystallography; acetylation; apoenzymes; biological products; 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; recombinant DNA; 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，在实现全面 对蛋白质结构的机制的理解 这是实际控制的先决条件 它的生理活动，来自于研究， 特定活动的变化，由以下原因引起： 特定氨基酸化学修饰或来自氨基酸 由基因损伤造成的替换。 然而化学 氨基酸侧链的修饰仅限于小的 残留物的比例，而幸存的遗传修饰是 随机的，并且必然限于非致命的突变。 这些 传统的方法现在正在通过 使用重组DNA技术获得细胞色素c， 任何所需的氨基酸序列。 的定点诱变 真核细胞色素c基因的克隆及其生产 蛋白质在异源表达系统中的表达， 研究特定氨基酸取代如何影响蛋白质 结构和稳定性，其生物合成，以及电子 运输和结合亲和力与各种生理 反应伴侣，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。 以来 我们目前生产突变细胞色素C的技术 要求它们至少部分地起作用， 第二个目标是开发程序， 无功能突变体在酵母中的表达。 使用 脱辅基蛋白的突变体，生物合成的中间体，打开 门的几个生理相关的检查 表征蛋白质生命周期的过程。 这些 包括外膜蛋白对脱辅基蛋白的识别， 线粒体膜，其运输通过膜， 血红素辅基的酶催化共价结合 脱辅基蛋白，全蛋白释放到 线粒体膜间隙和机制， 线粒体中细胞色素c的水平受到调节。 最后我们 酵母中的现有系统产生N-末端乙酰化和 非乙酰化大鼠细胞色素c，均携带完全 三甲基赖氨酸72;它也产生果蝇 细胞色素C，其具有三-、二-、单-和 未甲基化的形式，其已通过HPLC分离。 这些 蛋白质提供了一个迄今为止独特的机会，研究 结构和功能的影响，这些众所周知的次要 细胞色素C的修饰。