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%。在芽孢杆菌RNA酶抑制剂的帮助下在大肠杆菌中克隆和表达，其中几个已经被很好地表征，沿着三个芽孢杆菌RNA酶抑制剂同源物，它们都抑制芽孢杆菌RNA酶。最近，我们利用噬菌体展示技术发现并克隆了Sti基因，Sti是芽孢杆菌RNA酶St的天然抑制剂。红血丝该基因的有效表达需要对其序列进行大量的沉默突变操作。在E.但仍不足以发挥作用。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10 exp 9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。每个部分随机化组中的一些产生足够的物理研究。一个更大的完全随机化的核心barstar基因库正在准备中。具有全部22个随机化的核心残基的功能性杆状病毒的序列表明没有特定的氨基酸是必不可少的，尽管在某些位置野生型残基是明显优选的。由核心侧链占据的体积，如由非氢原子的数量表示的，比预期的更可变，特别是在低侧，这表明二级结构框架可以相当大地塌陷而不破坏barnase结合位点。连续流电穿孔技术的发展已经允许直接筛选相当大的E。大肠杆菌文库中的芽孢杆菌RNA酶表达的抗性，使我们能够估计产量的活性芽孢杆菌与完全随机化的核心在约一个在10 exp 4或5。

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, along with three barstar homologs, all of which inhibit barnase. Recently, we have used phage display to find and clone the gene for Sti, a barstar homolog and natural inhibitor of RNase St from S. erythreus. Useful expression of this gene required considerable manipulation of its sequence with silent mutations. It allowed some increase in production of RNase St in E. coli but still not enough to be useful. 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 of the partially randomized sets produce enough for physical studies. A much larger library of genes for completely randomized core barstars is being prepared. Sequences of functional barstars with all 22 core residues randomized indicate that no particular amino acid is indispensable, although in some positions the wild type residue is clearly preferred. The volume occupied by the core sidechains, as represented by the number of non-hydrogen atoms, is much more variable than expected, especially on the low side, suggesting that the secondary structure framework can collapse considerably without destroying the barnase-binding site. Development of a continuous flow electroporation technique has allowed direct screening of fairly large E. coli libraries for resistance to barnase expression, allowing us to estimate the yield of active barstars with fully randomized cores at about one in 10exp4 or 5.