RIBONUCLEASE AND ITS INHIBITOR FROM BACILLUS AMYLOLIQUEFACIENS

解淀粉芽孢杆菌的核糖核酸酶及其抑制剂

• 批准号: 3854534
• 负责人: R W HARTLEY
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3854534
• 关键词: Bacillus; Escherichia coli; X ray crystallography; bacterial genetics; bacterial proteins; chemical kinetics; chemical stability; conformation; enzyme complex; enzyme inhibitors; enzyme mechanism; enzyme model; enzyme structure; enzyme substrate analog; gene expression; hydrolysis; intermolecular interaction; lethal genes; molecular cloning; mutant; nuclear magnetic resonance spectroscopy; nucleic acid probes; pancreatic ribonuclease; protein engineering; protein folding; protein sequence; recombinant DNA; site directed mutagenesis

Two proteins, barnase, the extracellular ribonuclease of Bacillus amyloliquefaciens, and barstar, its intracellular inhibitor, are used as a model system for the study of protein folding and protein-protein interactions. Barnase is one of an homologous group of ribonucleases occuring in both prokaryotes and eukaryotes. Recombinant DNA techniques are being applied with three major aims: (1) to facilitate production of wild type and mutant proteins; (2) to examine the structural and control sequences of the genes; and (3) to make specific changes in the sequences to test theories of folding and probe the barnase- barstar interaction. The lethal effect of the cloned wild type barnase gene can be repressed by expression of the barstar gene on the same plasmid. E. coli plasmid vectors have been devised for both proteins and both can now be obtained essentially pure in 100 mg quantities. DNA and amino acid sequences are known for both and the x-ray structure of barnase has been refined to 2.0 A. The structures of both proteins in solution are being studied by 2-D NMR and x-ray work on both barstar and the barnase-barstar complex are under way. A synthetic fluorescent substrate has been used to study hydrolysis kinetics and to look at the kinetics and stability of the barnase-barstar interaction for native and mutant proteins. A number of variations in the sequence of each protein (80 in barnase, 60 in barstar) have been obtained by oligonucleotide-directed mutagenesis. Some of these were aimed at specific questions but most are part of a survey of the protein surfaces designed to locate their areas of interaction and residues on both have been identified as being so involved. Our finding that the two Cys residues of barstar can both be replaced by Ala without loss of activity or yield will greatly simplify future studies of barstar folding. Such replacement of either or both of the Cys residues reduces the stability of barstar only to that of the wild-type measured in the presence of mercaptoethanol or DTT. A method has been developed for measuring the relative strength of the bond between barnase and barstar for various combinations of wild-type and mutant proteins. Recent work, elsewhere, in which the barnase gene was attached to a eukaryotic promoter in order to kill the tissue in which that promoter is expressed (in the first instance to produce male sterility in plants) has aroused considerable interest in its possible use in development studies and as the key to a variety of anti-viral strategies.

芽孢杆菌的胞外核糖核酸酶Barnase Amyloliquefaciens及其胞内抑制物Barstar被用作 蛋白质折叠和蛋白质-蛋白质研究的模型系统 互动。Barnase是核糖核酸酶的同源基团之一 存在于原核生物和真核生物中。 重组DNA技术的应用主要有三个目标：(1) 促进野生型和突变蛋白的生产；(2)检测 基因的结构和控制序列；以及(3)使特定的 序列的变化，以测试折叠理论和探索藤本酶- 巴斯塔尔互动。 克隆的野生型barnase基因的致死作用可被 Barstar基因在同一质粒上的表达。大肠杆菌质粒 已经为这两种蛋白质设计了载体，现在两种蛋白质都可以获得 基本上纯净量为100毫克。DNA和氨基酸序列是 已知的和，Barnase的X-射线结构已细化到2.0 答：这两种蛋白质在溶液中的结构正在用二维方法研究 对Barstar和Barnase-Barstar络合物的核磁共振和X射线研究都是 正在进行中。一种合成的荧光基质已被用于研究 并考察其动力学和稳定性。 天然蛋白和突变蛋白的Barnase-Barstar相互作用。一批 每种蛋白质的序列变化(Barnase中80个，Barstar中60个) 都是通过寡核苷酸定向诱变获得的。其中一些 都是针对特定问题的，但大多数都是对 蛋白质表面设计用于定位其相互作用区域和残基 这两个人都被确认参与其中。 我们发现Barstar的两个半胱氨酸残基都可以用 不损失活性或产率的ALA将极大地简化未来的研究 巴斯塔尔折叠术。这种半胱氨酸残基之一或两个残基的替换 将Barstar的稳定性降低到仅为在 硫醇或DTT的存在。 已开发出一种测量键的相对强度的方法 在Barnase和Barstar之间，用于野生型和 突变蛋白质。 最近的研究，在其他地方，在这些工作中，barnase基因连接到一个 以杀死该启动子所在的组织 表达(首先在植物中产生雄性不育) 引起了人们对它在发展研究中可能使用的相当大的兴趣 并作为各种抗病毒策略的关键。