Guanidine hydrochloride (GdmCl) denaturation study

We calculated the free energy of unfolding using Trp fluorescence. Chemical denaturation of proteins was performed by adding GdmCl solution (Pierce, Thermo Scientific) to each of WT p53 core and mutant protein. To do that, p53 proteins in MOPS buffer, pH 7.4 was mixed with increasing concentrations (0, 0.5, 1.0, 1.25, 1.5, 1.75, 2, 3, 4 and 6 M) of GdmCl such that final protein concentration was 10 μM. The protein mixtures were then incubated overnight for equilibrium unfolding at room temperature. After incubation, Trp fluorescence of these samples was measured with excitation at 290 nm and emission in the range of 300–500 nm, keeping the both excitation and emission slit width at 5 nm. The Trp fluorescence intensity at 350 nm was plotted against various concentration of GdmCl and used for free energy calculation (see Supplementary Text).