Study Of A Ribonuclease And Its Inhibitor From Bacillus

两种蛋白质，barnase，解淀粉芽孢杆菌的胞外核糖核酸酶，和barstar，其胞内抑制剂，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。芽孢杆菌RNA酶是原核生物和真核生物中都存在的一类同源核糖核酸酶。重组DNA技术的应用有三个主要目的：（1）促进野生型和突变蛋白的生产;（2）检测基因的结构和控制序列;（3）对序列进行特异性改变，以检验折叠理论并探测芽孢杆菌RNA酶-芽孢杆菌RNA酶的相互作用。这两种蛋白质现在都可以从大肠杆菌中的重组基因获得。其中芽孢杆菌RNA酶抑制剂的表达抵消芽孢杆菌RNA酶表达的致死效应。这两种蛋白质及其复合物的结构是已知的，芽孢杆菌RNA酶的分辨率为1.5埃。通过体内选择性技术获得的在界面中具有互补突变的几个barnase-barstar对的晶体结构已经得到解决，提供了对确定键强度的机制的深入了解。Barstar还抑制来自链霉菌菌株的一组RNA酶。这些酶与芽孢杆菌RNA酶的亲缘关系较远，序列同一性仅为25%。在手头的四种这样的酶中，同一性范围从40%到70%。两种RNA酶Sa和St的结构是从非重组材料的工作中已知的，第三种RNA酶Sa 2来自我们的重组材料。与芽孢杆菌RNA酶抑制剂复合的重组RNA酶Sa的结构也已得到解决。两个芽孢杆菌RNA酶抑制剂同源物Sa 2已从链球菌中克隆并表达，自S.红曲霉不能被芽孢杆菌RNA酶抑制剂充分抑制，其同源抑制剂正在被积极地寻找。在B的全序列中鉴定了yrdF基因。subtilis 168克隆了一个与芽孢杆菌属芽孢杆菌相似的基因，并在经过几个密码子修饰后，在E.杆菌虽然在B中没有可识别的芽孢杆菌RNA酶同系物。在枯草芽孢杆菌168中，yrdF是芽孢杆菌RNA酶的有效抑制剂，在体外和体内。与芽孢杆菌RNA酶复合的两种Sa抑制剂的解离常数（Kd）分别为3 × 10~（exp-11）和3 × 10~（exp-10），相比之下，yrdH的解离常数不超过10~（exp-12），芽孢杆菌RNA酶的解离常数不超过6 × 10~（exp-14）。噬菌体展示系统已被开发用于选择与芽孢杆菌RNA酶或其突变体紧密结合的芽孢杆菌RNA酶的变种或同源物。已经开发了用于全合成芽孢杆菌RNA酶抑制剂基因的程序，其中所选残基随机化并且多重性（独立随机化序列的数目）为10exp9的量级。我们筛选了3个合成的芽孢杆菌RNA酶抑制剂文库，其中疏水核心的空间紧凑的8、11和11个残基部分被随机化，另一个核心的所有22个残基被随机化。在每个取代位置中可能的残基是Leu、Ile、瓦尔、Met或Phe。转移到质粒芽孢杆菌RNA酶抑制剂载体的未选择的基因没有显示出功能性芽孢杆菌RNA酶抑制剂的迹象，而所有选择的基因似乎在体内是功能性的，允许通过携带芽孢杆菌RNA酶的相容质粒转化其宿主。所有后者在体外也产生可测量的芽孢杆菌RNA酶抑制剂活性，大多数处于非常低的水平。然而，每一组中的一些确实产生了足够的物理研究。已经开始对大量这些基因进行测序，并将尽可能多地选择用于物理、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.