STUDY OF A RIBONUCLEASE AND ITS INHIBITOR FROM BACILLUS AMYLOLIQUEFACIENS

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被barstar抑制不足，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，该基因已被克隆并经过多次密码子修饰，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。我们筛选了一个具有空间紧凑的八个疏水核心随机残基部分的合成barstar库。在每个替代位置上可能的残基是Leu、Ile、Val、Met或Phe。200个未选择的基因转移到质粒barstar载体上，没有显示出功能性barstar的迹象，而120个选择的基因在体内似乎都是功能性的，允许它们的宿主被携带barnase的相容质粒转化。后者约有一半在体外产生可测量的barstar活性，其中一半的产量与野生型基因相当。已对30个产生体外活性的基因进行了测序。没有两个人是一样的。在barstar位置74的随机密码子，即野生型的Phe，在所有选择的基因测序中都是Phe，表明该残基在结构上的重要性。一种具有6个位置的非天然残留物且产量高的产品已被暂时选择用于结构研究。我们目前正在筛选barstar基因文库，其中所有22个亲水性核心残基已随机化。

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 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 a synthetic barstar library with a spatially compact eight residue portion of the hydrophobic core randomized. The possible residues in each substituted position being Leu, Ile, Val, Met or Phe. 200 unselected genes transferred to a plasmid barstar vector showed no indication of functional barstar, while 120 selected genes all appear to be functional in vivo, allowing transfomation of their host by a compatible plasmid carrying barnase. About half of the latter produce measurable barstar activity in vitro and half of these yield amounts comparable to the wild type gene. Thirty of the genes producing in vitro activity have been sequenced. No two are alike. The randomized codon at barstar position 74, Phe in the wild-type, is Phe in all the selected genes sequenced, indicating the structural importance of this residue. One product, with non-native residues in 6 positions and produced in high yield, has been provisionally chosen for structural studies. We are currently screening a barstar gene library in which all 22 residues of the hydrophilic core have been randomized.