Structure and Function of Modules Constituting Proteins

球状蛋白质由紧凑的模块组成，模块边界与分裂基因中接近内含子的位置相对应。这一事实表明，基因中的外显子改组在蛋白质水平上是模块改组，而结节必须是原始酶本身或其部分。本项目的目的是获得支持这一模块假说的证据。我们用化学方法合成了模块，并对每个模块在溶液中的构象及其功能进行了实验研究和计算工作。我们选择了由淀粉解性芽孢杆菌产生的胞外核糖核酸酶Barnase作为适合这一目的的蛋白质。Barnase是一个由110个氨基酸残基组成的小蛋白质，它没有二硫键，分析了它的X射线结构，原子坐标由G.Dodson博士和C.Hill博士提供。Barnase被分解成至少六个模块(M1-M6)，合成并纯化与每个模块相对应的多肽片段。CD测量表明，M1和M2在亲脂溶剂中有稳定的α-螺旋。因此，相互断开的模块的螺旋形成潜力反映了连接的模块中作为天然蛋白质的螺旋含量。M5在水中具有β-折叠结构。利用距离几何方法对M2中的螺旋形成残基进行了核磁共振研究。利用氢键网络的计算方法，分析了使模块在晶格中紧凑的原子力。氢键网络主要存在于每个模块内，模块之间的氢键网络很少。这一事实表明，氢键网络在使球状蛋白质中的模块紧凑方面做出了重要贡献。本研究首次提出了至少有三个模块的棒状酶在溶液中具有稳定的二级结构的证据。这一结果促使我们对模块的结构和功能进行进一步的研究。

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.