Thermodynamic parameters for urea-induced unfolding equilibrium of Pim-1 wild-type and mutants.

Urea-induced unfolding equilibrium data were obtained at 10°C in 20 mM Tris/HCl, pH 7.5, containing 0.2 M NaCl and 200 µM DTT by measuring the relative fluorescence intensity at 345 nm. The free energy of unfolding from the native state to the intermediate 1 (ΔG1), from the intermediate 1 to the intermediate 2 (ΔG2) and to the unfolded state (ΔG3) were calculated from Eqn. 5. [Urea]0.5 and m which are the midpoint and m value for the transition between native, intermediate and unfolded states, respectively, were calculated from Eqn.5. ΔGtot is obtained as the sum of ΔG1, ΔG2, and ΔG3. Data are reported ± SE of the fit.