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

Table 1

The thermodynamic parameters derived from the urea induced denaturation of WT-alaRS, G674D, N700, and N461 alaRS using the data of fluorescence emission maximum. A three-state equation was used to fit the data (equation (2)) as described in Section 2 using the graph plotting software KyPlot (version 2.0 beta 15 (32 bits), Koichi Yoshioka, 1997–2001). The excitation wavelength was kept at 295 nm and emission spectra were recorded from 310 to 450 nm. The emission maximum value was calculated by taking the first derivative of the spectra. Bandpasses for both excitation and emission were kept at 5 nm.

Fluorescence	(Kcal/mol)	(Kcal/mol)	(Kcal/mol/M)	(Kcal/mol/M)
WT	6.9 ± 0.2	11.7 ± 0.3	1.3 ± 0.1	1.8 ± 0.1
G674D	4.7 ± 0.1	8.9 ± 1.1	0.9 ± 0.1	1.2 ± 0.2
N700	5.9 ± 0.1	8.7 ± 0.2	1.1 ± 0.1	1.3 ± 0.2
N461	3.5 ± 0.2	13.2 ± 0.3	0.8 ± 0.1	1.6 ± 0.1

Values are the means of at least three independent experiments with standard errors indicated.