STUDY OF A RIBONUCLEASE AND ITS INHIBITOR FROM BACILLUS AMYLOLIQUEFACIENS

两种蛋白质，芽孢杆菌RNA酶，芽孢杆菌的胞外核糖核酸酶 解淀粉菌及其细胞内抑制剂芽孢杆菌RNA酶抑制剂 作为研究蛋白质折叠和蛋白质-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） 促进野生型和突变蛋白的产生;（2） 检查基因的结构和控制序列;以及（3） 在序列中做出特定的改变来测试折叠理论， 来探测巴恩斯和巴斯塔之间的互动 这两种蛋白质现在都可以从大肠杆菌中的重组基因中获得。杆菌 产率为100 mg/l或更高。 共表达芽孢杆菌RNA酶抑制剂是必要的， 对抗芽孢杆菌RNA酶表达的致死作用。 X射线结构 蛋白质和它们的复合物都是已知的， barnase的结构 一种快速且相对精确的测定方法， 荧光底物允许我们使用barnase-barstar滴定法 曲线和活性和非活性芽孢杆菌RNA酶突变体之间的竞争， barstar来研究络合物形成的动力学和稳定性。 复杂结构的解决方案证实了参与 由蛋白质工程鉴定的几个残基组成。 一个或 直接接触的所有芽孢杆菌RNA酶抑制剂残基的更多突变 复合物中的芽孢杆菌RNA酶，以及几种芽孢杆菌RNA酶， 制备 所有组合的吉布斯自由能的测定 这样的突变体在复杂的将隔离的能量贡献， 接口的不同部分。 芽孢杆菌素A（芽孢杆菌素（Cys 42，80 Ala））， 与芽孢杆菌RNA酶的结合几乎和野生型一样好， 很多这样的工作。 已经设计了一种系统来在体内选择， 与芽孢杆菌RNA酶突变体结合改善的芽孢杆菌RNA酶突变体， 被野生型芽孢杆菌抑制得很差。 几个这样的巴斯塔突变体 已经找到了 最近的工作，其他地方，其中芽孢杆菌RNA酶基因连接到一个 真核启动子，以杀死其中启动子 表达（首先在植物中产生雄性不育） 它在发展研究中的应用引起了相当大的兴趣 是多种抗病毒策略的关键

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 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 to probe the barnase-barstar interaction. Both proteins can now be obtained from recombinant genes in E. coli with yields of 100 mg\l or better. Co-expression of barstar is necessary to counter the lethal effect of barnase expression. X-ray structures of both proteins and their complex are known as well as the NMR solution structure of barnase. A fast and relatively precise assay based on a fluorogenic substrate has allowed us to use barnase-barstar titration curves and competition between active and inactive barnase mutants for barstar to study the kinetics and stability of complex formation. Solution of the structure of the complex has confirmed the involvement of several residues identified as such by protein engineering. One or more mutations of all of the barstar residues in direct contact with barnase in the complex, as well as several such for barnase, have been prepared. Determination of the Gibbs free energy for all combinations of such mutants in complex will isolate the energy contributions of different portions of the interface. Barstar A (barstar(Cys42,80Ala)), Which binds barnase almost as well as the wild type, is being used for much of this work. A system has been devised to select, in vivo, barstar mutants with improved binding to barnase mutants which are poorly inhibited by wild type barstar. Several such barstar mutants have been found. Recent work, elsewhere, in which the barnase gene is attached to a eukaryotic promoter in order to kill the tissue in which the promoter is expressed (in the first instance to produce male sterility in plants) has aroused considerable interest in its use in developmental studies and is the key to a variety of anti-viral strategies.