CYT STRUCTURE-FUNCTION VIA SITE-DIRECTED MUTAGENESIS

通过定点诱变的 CYT 结构-功能

• 批准号: 3283849
• 负责人: ARTHUR G MAUK
• 金额: $ 8.5万
• 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/3283849
• 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自重新发现以来已被广泛研究 基林在1920年代，许多不确定性仍然存在 其功能性能取决于其结构。 这些问题 在很大程度上保持化学修饰技术的无能为力 探测许多类型的氨基酸残基和结构歧义 这种情况总是发生在来自不同不同的蛋白质的比较研究中 物种。 现在，分子遗传学的最新进展现在提供了一种新方法 通过应用解决这些问题 寡脱核苷酸导向的位点特异性诱变。 这种方法， 原则上，允许在任何位置的任何位置更换任何残留物 如果A 可以使用该蛋白质的基因编码的功能克隆。 我们的实验室的先前工作已经雇用或实际上已经开发了所有 与该提案相关的遗传和物理技术。 这 现在，经验将用于准备和表征 五种类型的突变酵母ISO-L-环体色素C的功能特性C 我们根据该功能特征对此进行了分类 它们设计用于探测的蛋白质。 这些类别的突变体包括 轴向配体突变体，血红素硫醚连锁突变，氢键 突变体，氧化还原伴侣识别突变体和高ph过渡突变体。 我们实验室的初步研究表明这些技术 可以很容易地从20升酵母培养物中产生15-30 mg突变蛋白 并证明了这些技术产生的突变细胞色素 提供了研究细胞色素结构对的强大手段 蛋白质的功能。 结合了最初的结果 明确地证明了 提议的工作。