CYT STRUCTURE-FUNCTION VIA SITE-DIRECTED MUTAGENESIS

通过定点诱变的 CYT 结构-功能

• 批准号: 3283852
• 负责人: ARTHUR G MAUK
• 金额: $ 7.79万
• 依托单位: UNIVERSITY OF BRITISH COLUMBIA
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-09-20 至 1991-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3283852
• 关键词: acidity /alkalinity; chemical binding; chemical structure function; circular dichroism; conformation; cytochrome c; electron spin resonance spectroscopy; enzyme substrate; fungal genetics; genetic manipulation; hemoprotein structure; high performance liquid chromatography; hydrogen bond; iron; isomer; ligands; molecular cloning; mutant; nuclear magnetic resonance spectroscopy; oligonucleotides; oxidation reduction reaction; point mutation; protein sequence; site directed mutagenesis; species difference; stop flow technique; tissue /cell culture; yeasts; zinc

Although cytochrome c has been studied extensively since its rediscovery by Kelin in the 1920s, many uncertainties remain regarding the manner in which its functional properties are determined by its structure. These questions remain largely from the inability of chemical modification techniques to probe many types of amino acid residues and from the structural ambiguities that invariably occur in comparative studies of proteins from different species. Recent advances in molecular genetics now provide a new method of addressing these concerns through the application of oligodexyribonucleotide-directed site-specific mutagenesis. This approach, in principle, permits the replacement of any residue in any position of the protein sequence with any other naturally occurring amino acid residue if a functional clone of the gene coding for the protein is available. Previous work in our laboratories has employed or, in fact, developed all of the genetic and physical techniques relevant to this proposal. This experience will now be directed to preparing and characterizing the functional properties of five types of mutant yeast iso-l-cytochromes c which we have classified on the basis of the functional features of the protein that they are designed to probe. These classes of mutants include axial ligand mutants, heme thioether linkage mutans, hydrogen-bonding mutants, redox partner recognition mutants, and high-pH transition mutants. Initial studies in our laboratories have demonstrated that these techniques can readily generate 15-30 mg of mutant protein from 20 L cultures of yeast and have demonstrated that mutant cytochromes produced by these techniques provide a powerful means for studying the effect of cytochrome structure on the function of the protein. Combined, the initial results reported unambiguously demonstrate the feasibility and potential productivity of the work proposed.

虽然细胞色素c自从被重新发现以来已经被广泛研究， 在20世纪20年代的科林，许多不确定性仍然存在的方式， 其功能特性由其结构决定。 这些问题 主要是由于化学改性技术无法 探测多种类型的氨基酸残基， 在对不同来源的蛋白质进行比较研究时， 物种 分子遗传学的最新进展现在提供了一种新的方法， 解决这些问题， 寡脱氧核糖核苷酸定向的位点特异性诱变。 这种方法， 原则上，允许在该结构的任何位置上的任何残基的置换， 蛋白质序列与任何其他天然存在的氨基酸残基，如果 编码该蛋白质的基因的功能性克隆是可获得的。 我们实验室以前的工作已经使用或实际上开发了所有 与此提案相关的遗传和物理技术。 这 现在将把经验用于准备和表征 5种突变型酵母异-l-细胞色素c的功能特性 我们已经分类的基础上的功能特点， 它们被设计用来探测的蛋白质。 这些突变体包括 轴向配体突变体，血红素硫醚键突变体，氢键 突变体、氧化还原配偶体识别突变体和高pH过渡突变体。 我们实验室的初步研究表明，这些技术 从20 L酵母培养物中可容易地产生15-30 mg突变蛋白 并且已经证明了通过这些技术产生的突变细胞色素 为研究细胞色素结构对 蛋白质的功能。 综合起来，初步结果报告说， 明确地证明了的可行性和潜在的生产力， 工作建议。