Thermal unfolding of variants hFXN90–210 and hFXN90–195.
Unfolding was followed by (A) Sypro-orange fluorescence in 10 mM sodium phosphate, pH 7.0, and by far-UV CD spectroscopy (B, C, and D). For both variants hFXN90–210 and hFXN90–195 (B and D, respectively) unfolding experiments were also conducted in the presence of different salt concentrations (0, 250 and 500 mM NaCl). In (C), the measurements were carried out in 20 mM sodium phosphate, 100 mM NaCl, 0.1 mM EDTA, at pH 7.0. Heating from 4 to 90°C and cooling from 90 to 4°C are shown in filled and open symbols, respectively. The inset in (B) shows for both proteins the dependence of the free energy of unfolding with the temperature for the buffer condition 20 mM sodium phosphate, 100 mM NaCl, 0.1 mM EDTA, pH 7.0. The curves in dotted lines are plotted taking into account the two extremes of the standard deviation of the ?HNU parameter. The inset in (C) shows the CD signals at 220 nm normalized between 0 and 1 to facilitate the comparison between Tm values obtained in unfolding and refolding and reversibility.