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: 3875541
• 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/3875541
• 关键词: Bacillus; DNA binding protein; Escherichia coli; X ray crystallography; bacterial genetics; bacterial proteins; conformation; enzyme complex; enzyme inhibitors; enzyme mechanism; enzyme model; enzyme structure; gel electrophoresis; gene expression; genetic manipulation; genetic recombination; molecular cloning; mutant; natural gene amplification; nuclear magnetic resonance spectroscopy; nucleic acid probes; nucleic acid sequence; pancreatic ribonuclease; protein folding; protein metabolism; protein sequence; structural genes

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 proteinprotein interactions. Barnase is one of a homologous group of ribonucleases occurring 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 Angstrums. The structures of both proteins in solution are being studied by 2-D NMR. 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. Some 50 variations in the sequence of each protein 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. Our most important finding with these mutants is that the two Cys residues of barstar can both be replaced by Ala without loss of activity or yield, greatly simplifying future studies of barstar folding.

两种蛋白质，芽孢杆菌RNA酶，芽孢杆菌的胞外核糖核酸酶 解淀粉杆菌及其细胞内抑制剂芽孢杆菌素被用作 研究蛋白质折叠和蛋白质的模型系统 交互.芽孢杆菌RNA酶是核糖核酸酶的同源群之一 在原核生物和真核生物中都存在。 重组DNA技术的应用有三个主要目的：（1） 促进野生型和突变体蛋白的产生;（2）检查 基因的结构和控制序列;以及（3）使特异性 改变序列来测试折叠理论和探测芽孢杆菌RNA酶， barstar互动 克隆的野生型芽孢杆菌RNA酶基因的致死作用可以通过以下抑制： 在同一质粒上表达芽孢杆菌RNA酶抑制剂基因。E.大肠杆菌质粒载体 已经为这两种蛋白质设计了一种方法， 以100 mg的量基本上纯。DNA和氨基酸序列是 这两种酶都是已知的，芽孢杆菌RNA酶的X射线结构已被精确到2.0 愤怒。这两种蛋白质在溶液中的结构正在研究， 二维核磁共振。一种合成的荧光底物已被用于研究 水解动力学，并着眼于动力学和稳定性的 天然和突变蛋白质的芽孢杆菌RNA酶-芽孢杆菌RNA酶抑制剂相互作用。大约50 每种蛋白质序列的变异已经通过 抗坏血酸定向诱变。其中一些是针对具体的 问题，但大多数是对蛋白质表面设计的调查的一部分 来定位他们的互动区域我们最重要的发现 突变体的一个重要特征是芽孢杆菌RNA酶抑制剂的两个Cys残基都可以被Ala取代 而不损失活性或产率，大大简化了未来的研究， 巴斯塔尔折叠。