STUDY OF A RIBONUCLEASE AND ITS INHIBITOR FROM BACILLUS AMYLOLIQUEFACIENS

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和氨基酸序列以及x- 两者的射线结构以及核磁共振溶液结构都是已知的 巴纳萨斯。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。接近于 每种蛋白质都产生了数百个定向突变。其中一些 这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。的两个半胱氨酸残基 Barstar都可以被ALA取代而不失去活性(体外或 在体内，但随着键的强度有所降低)或收益率将 极大地简化了未来对Barstar折叠的研究。这样的替换 其中一个或两个半胱氨酸残基使巴司塔的稳定性仅降低到 在硫醇或DTT存在下测量的野生型。 已经发展了几种测量相对和绝对的方法 不同组合下Barnase和Barstar之间的结合强度 野生型和突变型蛋白质。对于野生型蛋白质来说 解离系数在10到-13的数量级。 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的可能应用的相当大的兴趣 也是各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 ribonuclease 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 and x- ray structures of both are known as well as the NMR solution structure of barnase. 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. Close to a hundred directed mutations in each protein have been produced. 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. The two Cys residues of barstar can both be replaced by Ala without loss of activity (in vitro or in vivo, but with some reduction in the strength of the bond) 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. Several methods have been developed for measuring the relative and absolute strength of the bond between barnase and barstar for various combinations of wild-type and mutant proteins. For the wild-type proteins the dissociation coefficient is on the order of 10 to the -13. 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 developmental studies and is the key to a variety of anti-viral strategies.