ΔGD (80°C)

Stability/Folding

Gibbs Free Energy of Folding/Unfolding (ΔG)

kJ/mol

Circular Dichroism (CD)

0.1 M sodium cacodylate–HCl (pH 7.0)

80 C

None

4 μM

222.6 nm

The thermodynamic parameters were derived from thermal unfolding transitions as shown in Figure 1 (see details under Tm measurement). The accuracy of the ΔGD (80°C) is correlated with Tm. For variants with Tm close to 80°C, the accuracy is ±0.3 kJ/mol; for variants with Tm < 75°C or > 85°C, the accuracy is ±0.5 kJ/mol.
Tm pH7.0 details: Thermal unfolding transitions of 4 μM protein in 0.1 M sodium cacodylate–HCl (pH 7.0) and different concentra- tions of NaCl were followed by circular dichroism at 222.6 nm with a 1 nm bandwidth in 10 mm cells using a Jasco J-600A spectropolarimeter equipped with a PTC-348 WI Peltier device. The data were analyzed according to a two-state model using non-linear regression and the program Grafit (Erithacus Software, Staines, UK). The heat capacity change ΔCp was held constant at 4000 J mol− 1 K− 1. The reversibility of thermal unfolding was examined by heating protein samples for 5 min at a temperature that was 10 °C above its Tm value, followed by rapid cooling to 20 °C. The subsequent thermal unfolding transitions were identical with those obtained for a sample without pre-heating.