Mutation of Orthogonal Aminoacyl-tRNA Synthetase for Incorporating para-azido-L-phenylalanine and Its Structural Modeling

In this study, we investigated the effect of mutations in orthogonal aminoacyl-tRNA synthetase (aaRS) on the incorporation efficiency of para-azido-L-phenylalanine (AzF), and performed molecular modeling.We targeted three amino acid positions (178, 248, and 256) in the aaRS cloned in the previously reported pEVOL-pAzF-C1-K776 and generated aaRS mutants (M178T, F248L, K256E) using site-directed mutagenesis.Subsequently, we transformed Escherichia coli DH10B (pSEVA631pt-sfGFP204amb) with each of the pEVOL-pAzF-C1 plasmids containing the mutant aaRS and compared the specific fluorescence intensity upon AzF addition.With AzF addition, M178T and F248L mutants exhibited 1.36-and 1.19-fold increases, respectively, in specific fluorescence intensity compared to the control, while K256E mutant showed a 0.46-fold decrease.The selectivity for AzF was 1.987 for M178T, 1.931 for F248L, and 1.619 for K256E, all lower than the control.To interpret these experimental results, modeling and docking using Swiss-Model were performed.The results obtained in this study could be utilized for the development and interpretation of various aaRS for incorporating non-canonical amino acids into proteins.