Effect of a Single Amino Acid Substitution on the Near-Ultraviolet CD Spectra of Tryptophan Synthase α-Subunit

Publisher Website
Google Scholar
Abstract
The near-ultraviolet CD spectra of the wild-type tryptophan synthase α-subunit from Esche-richia coli and four mutant proteins, trpA88(Glu49→Tyr), trpA3(Glu49→Val), trpA33(Glu49→Met), and trpA11(Glu49→Gin), were examined at pH 7.0 and room temperature (20°C), in order to estimate the CD contribution of the tyrosine residue in the wavelength range above 270 nm. The CD spectrum of the trpA88 protein showed more intense positive bands than those of the other proteins examined, reflecting the contribution of one additional tyrosine residue. The CD spectra of the other mutant proteins, trpA3, trpA33, and trpA11, were considerably different from that of the wild-type protein, even though the substituted amino acid residues at position 49 were not aromatic. The difference CD spectra of the mutant proteins versus the wild-type protein were analyzed in terms of vibrational transition of a tyrosine residue. The difference CD spectrum of the trpA88 protein versus the wild-type protein had well-resolved bands at 286, 280, 270, and 263 nm and one shoulder at 275 nm. These band positions corresponded to those of the CD spectrum for L-tyrosine derivatives in a non-aqueous solvent at 77°K, suggesting that Tyr 49 of the trpA88 protein was buried in the hydrophobic interior of the molecule. The difference CD spectra of the trpA3 and the trpA33 proteins (substituted by more hydrophobic residues at position 49) versus the wild-type protein were also similar to the CD spectrum for L-tyrosine derivatives in a non-aqueous solvent, especially as regards the positions of CD bands. These results suggest that one or more of the seven tyrosyl residues in the protein is(are) near position 49 and interacts with the residue at position 49, and that the state of the tyrosine residue(s) is strongly affected by the hydrophobicity of the residue at position 49.