Structure and Function of Modules Constituting Proteins

构成蛋白质的模块的结构和功能

基本信息

批准号：
63480515
负责人：
GO Mitiko
金额：
$ 4.16万
依托单位：
Nagoya University (1989)Kyushu University (1988)
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1989
项目状态：
已结题

项目摘要

A globular protein consists of compact modules and module boundaries correspond close to intron positions in split genes. This fact implies that exon shuffling in genes is module shuffling in protein level and niodules must be primitive enzymes themselves or parts of them. The purpose of this project is to obtain supporting evidences for this module hypothesis. We synthesized modules chemically and carried out experimental study as well as computational work on conformation of each module in solution and its function if any. We chose barnase, an extracellular ribonuclease produced by Bacillus amyloliquefaciens, as a suitable protein for this purpose.Barnase is a small protein consisting of 110 amino acid residues, it has no disulfide bonds, its X-ray structure was analyzed and the atomic coordinates were kindly supplied by Drs. G. Dodson and C. Hill. Barnase was decomposed into at least six modules (Ml-M6), the polypeptide segments corresponding to each of them was synthesized and purified. CD measurement shows that Ml and M2 have stable alpha-helices in lipophilic solvent. Thus, the helix forming potential of modules disconnected from each other reflects helical content in connected modules as native protein. M5 has a beta-sheet structure in water. The helix making residues in M2 were identified by NMR study using distance geometry method. The atomic forces to make the modules compact in barnase were analyzed by computational method focusing on the hydrogen-bond network. Hydrogen-bond network was found predominantly within each module and quite few between modules. This fact shows the important contribution of hydrogen-bond network in making modules compact in a globular protein. This research project brought out an evidence for the first time that at least three modules of barnase took stable secondary structures in solution. This result prompts us to carry out a further study on the structure and function of modules.

球状蛋白由紧凑的模块和模块边界组成，相对应靠近分裂基因中的内含子位置。这一事实意味着，基因的外显子改组是模块在蛋白质水平上改组，而niodules必须是原始酶本身或部分酶。该项目的目的是获得该模块假设的支持证据。我们通过化学合成模块，并进行了实验研究以及有关溶液中每个模块构象及其功能的计算工作。我们选择了barnase，这是由阿氨基杆菌法族人产生的细胞外核糖酶作为用于此目的的合适蛋白质。barnase是一种由110个氨基酸残基组成的小蛋白质，它没有二硫键键，其X射线结构是由X射线结构进行分析的，并且由原子均匀的均匀供应。 G. Dodson和C. Hill。将barnase分解为至少六个模块（ML-M6），对与每个模块相对应的多肽片段被合成并纯化。 CD测量表明，ML和M2在亲脂性溶剂中具有稳定的α-螺旋。因此，彼此断开模块的螺旋构成潜力反映了连接模块中作为天然蛋白的螺旋含量。 M5在水中具有Beta片结构。使用距离几何方法通过NMR研究鉴定了M2中的螺旋残留物。通过关注氢键网络的计算方法分析了使barnase中的模块紧凑的原子力。在每个模块中主要发现氢键网络，在模块之间很少发现。这一事实表明了氢键网络在使模块在球状蛋白中紧凑的重要贡献。该研究项目首次提出了一个证据，表明至少三个barnase模块在溶液中采用了稳定的二级结构。该结果促使我们对模块的结构和功能进行进一步研究。