Study Of A Ribonuclease And Its Inhibitor From Bacillus

解淀粉芽孢杆菌的胞外核糖核酸酶barnase和胞内抑制剂barstar被用作研究蛋白质折叠和蛋白质-蛋白质相互作用的模型系统。巴纳酶是存在于原核生物和真核生物中的同源核糖核酸酶群之一。重组DNA技术的应用主要有三个目的：(1)促进野生型和突变型蛋白质的生产；（二）检查基因的结构和控制序列；(3)对序列进行特定的改变，以检验折叠理论和探索barnase-barstar相互作用。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，其中barstar的表达可以对抗barnase表达的致命作用。这两种蛋白质及其复合物的结构都是已知的，在1.5埃的分辨率下。通过体内选择技术获得的几种在界面上具有互补突变的barnase-barstar对的晶体结构已经得到解决，为确定键强度的机制提供了见解。Barstar还抑制链霉菌菌株的一组rna酶。这些酶与藤壶酶有较远的亲缘关系，序列同源性只有25%。在这四种酶中，识别度从40%到70%不等。利用barstar在大肠杆菌中克隆和表达，其中一些已被很好地表征。从另外3株解淀粉芽孢杆菌和枯草芽孢杆菌168的yrdF基因以及S. erythreus的Sti基因中克隆出了barstar同源基因，并发现它们的产物能与barnase发生交叉反应。Sti基因的克隆需要使用噬菌体展示技术。一种噬菌体展示系统已经被开发出来，用于选择与藤壶酶或其突变体紧密结合的barstar的品种或同源物。已经开发了barstar基因的全合成程序，随机选择疏水核心残基，其多重性（独立随机序列的数量）为10exp9。从一个由8个核心残基组成的紧凑簇（随机分配到L、I、V、M或F）的文库中，获得了30个产生功能性酒吧星的克隆，其中21个提供了与野生型相当的产量。在这组中只有一个位置，在F74，是需要野生型残基。在一个包含所有22个核心残基的大型文库中，我们发现功能barstars较少，但在这个更大的背景下，没有野生型残基是不可替代的。

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 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 to probe the barnase-barstar interaction. Both proteins can now be obtained from recombinant genes in E. coli where expression of barstar counters the lethal effect of barnase expression. The structures of both proteins and their complex are known, barnase at 1.5 Angstrom resolution. Crystal structures of several barnase-barstar pairs having complementary mutations in the interface, obtained by an in vivo selective technique, have been solved, providing insight into the mechanisms that determine the strength of the bond. Barstar also inhibits a group of RNases from Streptomyces strains. These enzymes are distantly related to barnase with a sequence identity of only 25%. Among the four such enzymes in hand, identities range from 40% to 70%. Cloned and expressed in E.coli with the aid of barstar, several of these have been well characterized. The genes for barstar homologs from three other strains of B. amyloliquefaciens plus one, the yrdF gene from B. subtilis 168 and another, Sti from S. erythreus have been cloned and their products found to cross react with barnase. Cloning of the Sti gene required the use of phage display technology. A phage display system has been developed for selection of varieties or homologs of barstar that bind tightly to barnase or its mutants. Procedures have been developed for total synthesis of the barstar gene with randomization of selected hydrophobic core residues with a multiplicity (the number of independently randomized sequences) on the order of 10exp9. From a library for which a compact clump of eight core residues were randomized ( to L, I, V, M or F) 30 clones producing functional barstars were obtained, 21 of which provided yields comparable to the wild type. At only one position in this group, at F74, was the wild type residue required. From a larger library with all 22 of the core residues similarly randomized, we have found fewer functional barstars but in this larger context no wild type residues are irreplaceable. Is the limited number of basic protein folds found in nature an historical accident of evolution or is it due to some fundamental geometric limitation? We are currently trying to answer this question by using ribosome display to search for a well folded protein produced by a synthetic gene library designed to yield a novel tertiary structure made up of elements of secondary structure taken from barnase and barstar but with a randomized hydrophobic core.