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Study Of A Ribonuclease And Its Inhibitor From Bacillus

芽孢杆菌核糖核酸酶及其抑制剂的研究

基本信息

批准号：
6983601
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
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

项目摘要

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. The genes for barstar homologs from three other strains of B. amyloliquefaciens plus one, the yrdF gene from B. subtilis 168 and another, Sti from S. erythreus have been cloned and their products found to cross react with barnase. Cloning of the Sti gene required the use of phage display technology. 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 hydrophobic core residues with a multiplicity (the number of independently randomized sequences) on the order of 10exp9. From a library for which a compact clump of eight core residues were randomized ( to L, I, V, M or F) 30 clones producing functional barstars were obtained, 21 of which provided yields comparable to the wild type. At only one position in this group, at F74, was the wild type residue required. From a larger library with all 22 of the core residues similarly randomized, we have found fewer functional barstars but in this larger context no wild type residues are irreplaceable. Is the limited number of basic protein folds found in nature an historical accident of evolution or is it due to some fundamental geometric limitation? We are currently trying to answer this question by using ribosome display to search for a well folded protein produced by a synthetic gene library designed to yield a novel tertiary structure made up of elements of secondary structure taken from barnase and barstar but with a randomized hydrophobic core.

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。在Barstar的帮助下，克隆并在大肠杆菌中表达，其中几个已经得到了很好的鉴定。另外三个菌株的barstar同源物基因已被克隆，其中一个是枯草芽孢杆菌168的yrdF基因，另一个是红苏链霉菌的Sti，它们的产物与Barnase发生交叉反应。STI基因的克隆需要使用噬菌体展示技术。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具有10exp9量级的重复性(独立随机序列的数目)的所选疏水核心残基的随机化来全合成barstar基因的程序。从一个由8个核心残基组成的紧凑型文库中(随机分为L、I、V、M或F)，获得了30个产生功能性Barstar的无性系，其中21个的产量与野生型相当。在这个组中只有一个位置，即F74，是所需的野生型残留物。从一个更大的文库中，所有22个核心残基都是类似随机的，我们发现功能棒星较少，但在这个更大的背景下，没有野生型残基是不可替代的。自然界中发现的有限数量的碱性蛋白质折叠是进化的历史偶然，还是由于一些基本的几何限制？我们目前正试图通过使用核糖体展示来搜索由合成基因库产生的折叠良好的蛋白质，该基因库旨在产生一种新的三级结构，该三级结构由来自barnase和barstar的二级结构元素组成，但具有随机的疏水核心。