Protein Engineering for New Functional Hemoproteins Based on Module Substitution

本课题的主要研究结果如下：(1)珠蛋白和过氧化物酶的模块替换。对模取代珠蛋白和过氧化物酶的结构和功能特性进行了表征。大多数模块取代的蛋白质表现出高度不稳定的蛋白质结构，可能是由于模块之间的关键相互作用缺失或严重的扰动。不稳定的蛋白质结构也干扰了球蛋白中特定二聚体或四聚体的形成和过氧化物酶的酶活性。因此，可以得出结论，模块之间的相互作用对于设计新的稳定和功能蛋白以及模块替代也是必不可少的。(2) Globins中新的模块结构的识别。基于边界位于常规模块中间的结构单元的氨基酸取代表明，先前提出的模块可以被划分为新的和更小的结构单元。在这个研究项目中，我重点研究了一个新发现的结构单元，它包括铁配体组氨酸和近端约20个氨基酸残基，“子模块m6”。球蛋白中子模块m6的取代极大地影响了血红素的电子状态和铁配体组氨酸的构型，表明子模块m6将是血红素结合结构的“血红素结合模块”。

The primary results in this research project are as followa :(1)Module Substitution in Globins and Peroxidases. The structural and functional properties of the module-substituted globins and peroxidases have been characterized. Most of the module-substituted proteins exhibited highly destabilized protein structure, probably due to the missing or severe perturbation in the key interactions between the modules. The destabilized protein structure also interfered the formation of the specific dimers or tetramers in globins and enzymatic activity in peroxidases. It can be, therefore, concluded that the module-module interactions are also essential to design new stable and functional proteins as well as the module substitution.(2)Identification of New Module Structure in Globins. The amino acid substituteions based on the structural unit of wchic boundaries are located at the middle of the conventional modules have revealed that the module previously proposed can be divided into new and smaller structural units. In this research project, I focused upon one of the newly identified structural units, which includes the iron-liganded histidine and about 20 amino acid residues in the proximal site, "sub-module m6". The substitution of the sub-module m6 in globins drastically affect the electronic state of heme and configuration of the iron-liganded histidine, indicating that the sub-module m6 would be a "heme binding modules" respondible for the heme binding structure.