Structural and Functional Roles of Modules in Protein Architecture

球状蛋白质由紧凑的模块组成。蛋白质的模块边界与编码蛋白质的基因中的内含子位置密切相关。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，而模块本身就是原始酶。本项目的目的是获得模块作为原始酶的支持证据，并了解自然界中蛋白质设计的原理。本研究中使用的蛋白质有细菌核糖核酸酶Barnase、RNA聚合酶II、原核生物转录抑制物和叶绿体核核蛋白等。Barnase被分解为六个模块(M1-M6)。氢键主要位于Barnase中的模块内。这一事实表明，氢键的形成有助于决定棒石结构中模块的紧密构象。我们用化学方法合成了模块，并对各模块在溶液中的构象及其功能进行了实验研究。有三个模块(M2、M3和M6)具有核糖核酸酶的酶功能。这是有关模块的酶功能的首次报道。我们的结果清楚地表明，这些模块在早期进化中作为原始酶发挥了作用。我们还用2D-核磁共振和距离几何确定了模块M2和M3的二级结构。它们具有与完整的藤壶中发现的相似的二级结构。此外，我们在RNA聚合酶II中发现了一个类似于Barnase的结构域，并预测了该区域的核糖核酸酶催化功能。其他研究小组报道了RNA聚合酶II的核糖核酸酶活性。我们已经提出，如果有的话，这个区域可能参与了校对。通过对转录抑制因子中模块组织的分析，我们发现其中一些转录抑制因子具有两个或三个螺旋-旋转-螺旋模块，并通过模块组合讨论了抑制因子的进化过程。

A globular protein consists of compact modules. Module boundaries of proteins are closely correlated with intron positions in the genes encoding the proteins. This fact implies that exon-shuffling in genes is module shuffling in protein level and modules themselves were primitive enzymes. The purpose of this project is to obtain supporting evidences of modules as primitive enzymes and to understand the principle of protein design in nature. Proteins used in this study were barnase that is a bacterial RNase, RNA polymerase II, transcription repressors of procaryotes and ribonuclear proteins in chloroplast, etc. Barnase was decomposed into six modules(M1-M6). Hydrogen bonds were located predominantly within modules in barnase. This fact shows that hydrogen bond formation contributes to determine the compact conformation of modules in barnase architecture. We synthesized modules chemically and carried out experimental study on conformation of each module in solution and its function. Three modules (M2, M3 and M6) were revealed to have an enzymatic function as RNase. This is the first report on the enzymatic function of modules. Our results clearly imply that the modules carried a role as primitive enzymes in early evolution. We also determined the secondary structures of modules M2 and M3 using 2D-NMR and distance geometry. They had the similar secondary structures to those found in intact barnase. Furthermore, we found a barnase-like domain in RNA polymerase II and predicted RNase catalytic function in this region. By other research groups RNase activity of RNA polymerase II was reported. We have suggested that this region might be involved in proof reading, if any. By analysis of module organization in transcription repressors, we found that some of them have two or three helix-turn-helix modules and discussed evolutionary process of repressors through module combinations.