TABLE 5.

Thermodynamic parameters for the unfolding transition of VIM-2 and its W87A and W87F mutantsa

Type of unfoldingParameterValue (mean ± SD) for:
VIM-2W87AW87F
GdmCl inducedΔG°(H2O) (kJ/mol)31 ± 422 ± 2NDb
m (kJ/mol·M)−18 ± 2−17 ± 1ND
Cm (M)1.7 ± 0.41.3 ± 0.20.9/1.8c
ThermaldTm (°C)54 ± 0.250 ± 0.144 ± 0.2
  • a The thermodynamic parameters of GdmCl- and temperature-induced unfolding were measured at pH 7.5 and at a temperature of 25°C and computed from the analysis of the equilibrium transitions. The ΔG°, m, and Cm values are the means of those obtained by far-UV CD and fluorescence measurements and were computed as described previously (5, 12). m is a measure of the dependency of the free energy on the denaturant concentration, and Cm is the transition midpoint, i.e., the denaturant concentration at which [N] = [u]. The apparent Tm is the temperature at the midpoint of the denaturation curve.

  • b ND, not determined.

  • c Approximate values retrieved from the transition curves for the first and second transitions, respectively.

  • d The reversibility of thermal unfolding was estimated as the recovery of the CD signal after cooling of the sample as described in Materials and Methods and was <5% in all cases.