Work

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

Year: 2024

Type: article

Abstract: 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.

Source: KSBB Journal

DOI: 10.7841/ksbbj.2024.39.3.78

Authors: Euncheol Jung, Yu-bin Lim, Jong-il Choi

Institution: Chonnam National University

Language: English

Topic: RNA and protein synthesis mechanisms

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: bronze

Funders: National Research Foundation, Ministry of Education, National Research Foundation of Korea, Ministry of Education, India

Awards: G2237787981, G68318506, G6953522574