Protein Engineering for New Functional Hemoproteins Based on Module Substitution

基于模块替换的新型功能性血红素蛋白的蛋白质工程

• 批准号: 06808058
• 负责人: ISHIMORI Koichiro
• 金额: $ 1.22万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06808058/
• 关键词: Module; Exon Shaffling; Globin; モジュール置換; 多量体化蛋白質; キメラグロビン; 会合特性; ミオグロビン; ヘモグロビン

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.

在该研究项目中，为了建立基于自然界分子进化的蛋白质设计的新策略，对模块取代的球蛋白的结构和功能特性进行了广泛的特征。人血红蛋白和肌红蛋白的α-亚基之间的模块取代表明，模块M4在血红蛋白α-亚基中植入肌红蛋白中产生了一种相当稳定的二聚体全球蛋白蛋白，MBMBALBALPHA蛋白。单体肌红蛋白球蛋白中二聚体的形成强烈表明，特定的模块取代可能是将新功能添加到蛋白质中的有效策略。在模块化的全球素中，血红蛋白A-亚基和肌球蛋白之间的全球蛋白除MBMBalpha-珠蛋白外，它们的结构高度不稳定，并且对ANOT表现出了任何特定的亚基关联。由于不稳定的蛋白质结构，某些模块取代的球蛋白未在大肠杆菌中表达。这种对球蛋白结构的破坏性作用暗示着对稳定球蛋白形成必不可少的分子内相互作用受到模块取代的严重扰动，并且需要其他突变来产生稳定的功能蛋白以及模块取代。

项目成果

Unno,M.: "High-Pressure Flash Photolysis Study of Hemoprotein:Effect of Substrate Analogues on Recombination of Carbon Monoxide" Biochemistry. 34. 9762-9768 (1994)

Unno，M.：“血红素蛋白的高压闪光光解研究：底物类似物对一氧化碳重组的影响”生物化学。

Wakasugi,K.: ""Module"Substitution in Hemoglobin Subunits.Preparation and Characterization of a "Chimera βα-subunit"" J.Biol.Chem. 269. 18750-18756 (1994)

Wakasugi, K.：血红蛋白亚基中的“模块”替换。“嵌合体 βα-亚基”的制备和表征”J.Biol.Chem. 269. 18750-18756 (1994)

Ishimori, K: "NMR Studies on Recombinant Cytochrome P450cam Mutants" Biochimie. (印刷中). (1996)

Ishimori, K：“重组细胞色素 P450cam 突变体的核磁共振研究”Biochimie（出版中）。

Wakasugi, K.Ishimori, K.Morishima, I: "NMR Studies on Recombinant Cytochrome P450cam Mutants" Biochimie. 78. 763-770 (1996)

Wakasugi、K.Ishimori、K.Morishima、I：“重组细胞色素 P450cam 突变体的 NMR 研究”Biochimie。

Wakasugi, K.Ishimori, K.Morishima, I: "“Module"-substituted Globins:Artificial Exon Shuffling Among Myoglobin,Hemoglobin α-and β-subunits" Biophys.Chem.68. 265-273 (1997)

Wakasugi、K.Ishimori、K.Morishima、I：““模块”取代的球蛋白：肌红蛋白、血红蛋白 α 和 β 亚基之间的人工外显子改组”Biophys.Chem.68（1997）。