Structure and Function of Modules Constituting Proteins

球状蛋白由紧凑的模块组成，模块边界与分裂基因中的内含子位置接近。这一事实表明，基因中的外显子改组是蛋白质水平上的模块改组，而纽蛋白本身就是原始酶或其一部分。本项目的目的是为这一模块假设获得支持性证据。我们通过化学方法合成了这些模块，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。我们选择解淀粉芽孢杆菌（Bacillus amyloliquefaciens）产生的一种胞外核糖核酸酶Barnase作为合适的蛋白质，Barnase是一种由110个氨基酸残基组成的小蛋白质，不含二硫键。Dodson和C.之丘将芽孢杆菌RNA酶分解为至少6个模块（M1-M6），合成并纯化了与每个模块对应的多肽片段。CD测量显示Ml和M2在亲脂性溶剂中具有稳定的α-螺旋。因此，彼此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。采用距离几何方法通过NMR研究鉴定了M2中的螺旋形成残基。以氢键网络为中心，通过计算方法分析了使芽孢杆菌RNA酶中各模块紧密结合的原子力。氢键网络主要存在于每个模块内，而模块之间很少。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的重要贡献。本研究首次证明芽孢杆菌RNA酶至少有三个模块在溶液中具有稳定的二级结构。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.