TABLE 1.

Stability of wild-type and mutant proteins

Protein	m* (kcal mol−1 M−1)	U1/2† (M)	ΔG‡ (kcal mol−1)	ΔGav§ (kcal mol−1)
WT	2.27 ± 0.06	3.270 ± 0.022	7.43 ± 0.16	7.10 ± 0.05
D96A/N128A	2.25 ± 0.05	3.175 ± 0.001	7.14 ± 0.16	6.89 ± 0.03
D96A	2.15 ± 0.21	3.181 ± 0.003	6.85 ± 0.66	6.90 ± 0.01
N128A	2.02 ± 0.06	3.176 ± 0.010	6.41 ± 0.23	6.90 ± 0.02

Urea denaturation was performed at 25.0°C, in 50 mM MOPS, pH 7.0, with 0.5 M NaCl.

^*Slope of a linear plot of ΔG versus urea concentration. Mean of two determinations ± SE.

^†Urea concentration of mid-denaturation. Mean of two determinations ± SE.

^‡Standard free energy of unfolding calculated for each protein as m_i times U_1/2i Mean of two determinations ± SE.

^§Standard free energy of unfolding calculated for each protein as m_av times U_1/2i, where m_av = 2.17, is the average slope of all determinations. Mean of two determinations ± SE. We consider this data to be more accurate.