Study Of A Ribonuclease And Its Inhibitor From Bacillus

以淀粉解冻芽孢杆菌胞外核糖核酸酶barnase和胞内核糖核酸酶抑制物barstar两种蛋白质作为蛋白质折叠和蛋白质相互作用研究的模型体系。Barnase是存在于原核生物和真核生物中的一组同源核糖核酸酶之一。重组DNA技术的应用有三个主要目的：(1)促进野生型和突变型蛋白质的生产；(2)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的表达抵消了barnase的表达的致死作用。这两种蛋白质及其复合体的结构都是已知的，Barnase的分辨率为1.5埃。通过体内选择技术获得的几个在界面上具有互补突变的barnase-barstar对的晶体结构已经被解决，这为决定键强度的机制提供了洞察。Barstar还抑制链霉菌菌株的一组核糖核酸酶。这些酶与Barnase有较远的亲缘关系，序列同源性仅为25%。在手头的四种这样的酶中，同一性从40%到70%不等。两个RNase Sa和St的结构是从非重组材料上得知的，第三个RNase SA2是从我们的重组材料中得知的。还解决了重组核糖核酸酶Sa与Barstar形成的络合物的结构问题。从链霉菌中克隆并表达了SA2的两个Barstar同源物，由于Barstar对红苏链霉菌的RNase ST的抑制作用不够强，其同源抑制物正被积极寻找。枯草杆菌YrdF基因在枯草杆菌168的全序列中被鉴定为与barstar相似的基因，已经被克隆，并经过几次密码子修改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Barnase同源物，但yrdF在体外和体内都是一种有效的Barnase抑制剂。两种SA抑制剂与Barnase络合的解离常数(Kd)分别为3×10~(-11)和3×10~(-10)，而yrdH和Barstar的解离常数分别不超过10~(-12)和6×10~(-14)。已建立了一种噬菌体展示系统，用于筛选与barnase或其突变体紧密结合的barstar的变种或同源物。已经开发了通过选择的残基的随机化和10exp9量级的多样性(独立随机序列的数量)来全合成barstar基因的程序。我们筛选了3个空间紧凑的Barstar文库，其中疏水核心的8、11和11个残基部分是随机的，另一个是核心的全部22个残基的随机化。每个取代位上可能的残基有亮氨酸、异亮氨酸、Val、蛋氨酸或苯丙氨酸。未选择的基因被转移到质粒Barstar载体中，没有显示有功能的Barstar，而所有选择的基因似乎在体内都有功能，允许携带Barnase的相容质粒转化它们的宿主。所有后者在体外也产生可测量的Barstar活性，大多数都处于非常低的水平。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.