Protein Engineering for New Functional Hemoproteins Based on Module Substitution

在本研究项目中，我们对模块取代的珠蛋白的结构和功能特性进行了广泛的表征，以期建立一种基于自然界分子进化的蛋白质设计新策略。人血红蛋白α亚基和肌红蛋白之间的模块替换表明，将血红蛋白α亚基中的模块M4植入肌红蛋白中，产生了相当稳定的二聚体球蛋白MbMbalpha-珠蛋白。基于肌红蛋白的单体珠蛋白中二聚体的形成强烈地表明，特定的模块替换可以成为一种有效的策略来增加蛋白质的新功能。然而，在血红蛋白a亚基和肌红蛋白之间的模块取代的珠蛋白中，除了MbMbalpha-珠蛋白外，它们的结构高度不稳定，没有显示出任何特定的亚基结合。由于蛋白质结构不稳定，部分模块取代的珠蛋白在大肠杆菌中没有表达。这种对珠蛋白结构的破坏性效应表明，稳定的珠蛋白形成所必需的分子内相互作用受到模块替换的严重干扰，需要额外的突变才能产生稳定的功能蛋白质和模块替换。

In this research project, the structural and functional properties of the module-substituted globins have extensively been characterized in order to establish a new strategy for the protein design based on the molecular evolution in nature. The module substitutions between the alpha-subunit of human hemoglobin and myoglobin have revealed that the implantation of the module M4 in the hemoglobin alpha-subunit into myoglobin produced a fairly stable dimeric globin protein, MbMbalpha-globin. The formation of dimers in the monomeric myoglobin-based globin strongly suggest that the specific module substitution can be a potent strategy to add the new function to proteins. In the module-substituted globins between the hemoglobin a-subunit and myoglobin except for MbMbalpha-globin, however, their structure were highly destabilized and did anot show any specific subunit association. Some of the module-substituted globins were not expressed in Escherichia coli due to the unstable protein structure. Such destructive effects on the globin structure imply that the intramolecular interactions essential for the stable globin formation are severely perturbed by the module substitution and additional mutations would be required to produce stable functional proteins as well as the module substitution.