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-蛋白质的模型系统 交互. Barnase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.杆菌质粒 已经为这两种蛋白质设计了载体， 以100 mg的量基本上纯。 DNA和氨基酸序列和x- 两者的射线结构是已知的，并且NMR溶液结构为 barnase 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。 靠近一个 在每种蛋白质中已经产生了数百个定向突变。 一些 这些调查针对的是具体问题，但大多数都是对 蛋白质表面被设计来定位它们的相互作用区域和残基 这两个人都被认为是如此参与。 的两个Cys残基 芽孢杆菌RNA酶抑制剂可以被Ala替代而不丧失活性（体外或体外）。 在体内，但在一定程度上降低了债券的强度）或屈服将 极大地简化了未来对Barstar折叠的研究。 这种替换 Cys残基之一或两者仅降低芽孢杆菌RNA酶抑制剂的稳定性， 在巯基乙醇或DTT存在下测量的野生型的活性。 已经开发了几种方法来测量相对和绝对 芽孢杆菌RNA酶和芽孢杆菌RNA酶抑制剂之间的结合强度 野生型和突变型蛋白质。 对于野生型蛋白质， 解离系数在10到-13的量级。 最近在其他地方的工作中，芽孢杆菌RNA酶基因附着在一个 真核生物启动子，以杀死其中启动子 表达（首先是在植物中产生雄性不育）， 引起了人们对它在发育研究中的可能用途的极大兴趣 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 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.