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。其中两个RNase Sa和St的结构是在非重组材料中发现的，而第三个RNase Sa2的结构是在重组材料中发现的。重组rna酶Sa与barstar配合物的结构也得到了解析。从链霉菌中克隆并表达了2个针对Sa2的barstar同源物，由于S. erythreus的RNase St未被barstar充分抑制，其同源抑制剂正在积极寻找中。在枯草芽孢杆菌168的完整序列中鉴定出与barstar相似的基因yrdF，并经多次密码子修饰后在大肠杆菌中表达。虽然在枯草芽孢杆菌168中没有可识别的麻秆酶同源物，但yrdF是一种有效的体外和体内麻秆酶抑制剂。两种Sa抑制剂与barnase的解离常数（Kd）分别为3x10exp-11和3x10exp-10，而yrdH的解离常数不超过10exp-12， barstar的解离常数不超过6x10exp-14。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，选择残基随机化，多重性（独立随机序列的数量）为10exp9。我们筛选了3个合成barstar文库，其中疏水核心的8、11和11个残基部分空间紧凑，随机化，另一个文库随机化了核心的所有22个残基。每个取代位上可能的残基分别是Leu、Ile、Val、Met或Phe。未选择的基因转移到质粒barstar载体上，没有显示出功能barstar的迹象，而所有选择的基因在体内似乎都具有功能，允许它们的宿主被携带barnase的相容质粒转化。后者在体外也能产生可测量的barstar活性，但大多数水平很低。然而，每组中的一些确实产生了足够的物理研究。大量这些基因的测序工作已经开始，并将选择尽可能多的基因进行物理、功能和结构研究。

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%. The structures of two, RNases Sa and St, are known from work on nonrecombinant material and a third, RNase Sa2, from our recombinant material. The structure of recombinant RNase Sa in complex with barstar has also been solved. Two barstar homologs from streptomyces, for Sa2, have been cloned and expressed and since RNase St of S. erythreus is insufficiently inhibited by barstar, its homologous inhibitor is being actively pursued. The gene yrdF, identified in the complete sequence of B. subtilis 168 as similar to that of barstar, has been cloned and, after several codon modifications, expressed in E. coli. Although there is no recognizable homolog of barnase in B. subtilis 168, yrdF is an effective inhibitor, in vitro and in vivo, of barnase. The dissociation constants (Kd) of the two Sa inhibitors complexed with barnase are 3x10exp-11 and 3x10exp-10, compared to no more than 10exp-12 for yrdH and 6x10exp-14 for barstar. 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 residues and a multiplicity (the number of independently randomized sequences) on the order of 10exp9. We have screened 3 synthetic barstar libraries with spatially compact 8, 11 and 11 residue portions of the hydrophobic core randomized and another with randomization of all 22 residues of the core. The possible residues in each substituted position were Leu, Ile, Val, Met or Phe. Unselected genes transferred to a plasmid barstar vector showed no indication of functional barstar, while all selected genes appear to be functional in vivo, allowing transformation of their host by a compatible plasmid carrying barnase. All of the latter also produce measurable barstar activity in vitro, most at a very low level. Some of each set, however, do produce enough for physical studies. Sequencing of a large number of these genes has commenced and as many as practicable will be chosen for physical, functional and structural studies.