Site-Directed Mutagenesis of Tryptophan Residues in the Structure of the Catalytic Module of Tyrosyl-tRNA Synthetase from Bos taurus

Isolation and characterization of the mutant form of N-terminal catalytically module of Bos taurus tyrosyl-tRNA synthetase with the replacement of Trp 40 and Trp 283 by alanine

“…A considerable amount of recombinant protein is required to investigate the properties of the enzyme by experimental methods. Since the final yield of the target recombinant protein in bacterial systems strongly depends on the culture conditions, the experimentally determined optimal parameters for the expression of mini BtTyrRS in E. coli are required [9,10].…”

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“…Based on site-directed mutagenesis, the recombinant plasmid pET-30a (+)-39KYRS was used to create the substitutions of tryptophan residues at positions 40 and 283 by alanine in the cDNA of the synthetase catalytic module [10]. The resulting plasmid pET-30a-39KYRSW87 having only one tryptophan codon in the cloned cDNA was used in this work to obtain one-tryptophan protein for further fluorescence studies of conformational features and intramolecular interactions in protein structure [21,22].…”

“…Previously, we have cloned the cDNA of the tyrosyl-tRNA synthetase catalytic module in the expression plasmid pET32a and investigated its expression [9]. Subsequently, Trp40 and Trp283 codons were replaced with alanine codons by site-directed mutagenesis in cloned cDNA and only one tryptophan codon was left at the site of dimerization of mini BtTyrRS monomers [10].…”

Intrinsic fluorescence of single-tryptophan form of tyrosyl-tRNA synthetase catalytic module with the replacements of Trp 87 and Trp 283 by alanine

“…The different arrangements of Trp40, Trp87 and Trp283 residues in mini TyrRS make promising the monitoring of the local conformational changes, provided only 1 Trp fluorophore at each of these functional sites. Using site-directed mutagenesis, we have replaced the Trp87 and Trp283 codons with alanine codons in the mini BtTyrRS cDNA cloned in plasmid pET30a-39KYRS, leaving only one Trp40 near the active site of the enzyme [17].…”