Protein Engineering for New Functional Hemoproteins Based on Module Substitution

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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.