Urea Unfolding Study of E. coli Alanyl-tRNA Synthetase and Its Monomeric Variants Proves the Role of C-Terminal Domain in Stability

Figure 1

Biophysical characterization of mutant alaRSs along with WT protein. (a) Molecular weight determination of WT-alaRS, G674D, N700 alaRS, and N461 alaRS by analytical ultracentrifugation (AUC). Sedimentation equilibrium profiles of WT-alaRS and its mutants at concentrations 5 μM (upper left panel), 10 μM (upper right panel), and 20 μM (lower panel) at 20°C. The buffer was 100 mM Tris-HCl, pH 8.0. Fitting of the data was performed using SEDPHAT software. (b) Far-UV CD and (c) near-UV CD spectra of WT-alaRS, G674D, N700 alaRS, and N461 alaRS. The result has been represented as mean residue ellipticity (MRE) at 25°C. Protein concentrations were kept at 4 μM for far-UV CD and 15 μM for near-UV CD with the same buffer as the analytical ultracentrifugation experiment. Path length of the cuvettes was 1 mm and 10 mm for far-UV CD and near-UV CD, respectively. Scan speed was 50 nm/min and five independent scans were performed and from that average spectra were taken.

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