STUDY OF A RIBONUCLEASE AND ITS INHIBITOR FROM BACILLUS AMYLOLIQUEFACIENS

解淀粉芽孢杆菌核糖核酸酶及其抑制剂的研究

• 批准号: 6432055
• 负责人: ROBERT W HARTLEY
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6432055
• 关键词: Bacillus; bacterial proteins; chemical stability; enzyme activity; enzyme biosynthesis; enzyme inhibitors; enzyme mechanism; enzyme structure; esterase inhibitor; exoribonucleases; gene expression; gene mutation; genetic regulatory element; intermolecular interaction; microorganism metabolism; molecular cloning; nucleic acid sequence; protein engineering; protein folding; protein structure function; recombinant DNA

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.

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基因在枯草芽孢杆菌168的全序列中被鉴定为与barstar相似的基因，经过几次密码子修改后，已在大肠杆菌中表达。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。我们已经筛选了一个空间紧凑的疏水核心的八个残基部分随机分布的合成Barstar文库。每个取代位上可能的残基是亮氨酸、异亮氨酸、Val、蛋氨酸或苯丙氨酸。200个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120个被选择的基因似乎都在体内具有功能，允许携带Barnase的相容质粒转化它们的宿主。后者中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。已经对30个产生体外活性的基因进行了测序。没有两个是一样的。Barstar第74位的随机密码子，野生型中的Phe，在所有选择的基因序列中都是Phe，这表明了该残基的结构重要性。有一种产品，在6个位置有非天然残基，产量高，已被暂时选择用于结构研究。我们目前正在筛选一个Barstar基因库，其中亲水性核心的所有22个残基都已被随机排列。