Table I. Changes in the free energies of urea-induced unfolding of p53 DNA-binding domain mutants.
| p53 variant | m (kcal/mol/M)a | [Urea]50% (M) | ΔGH2OD–N (kcal/mol) | ΔΔGH2OD–N (kcal/mol) | ΣΔΔGH2OD–N (kcal/mol)b |
|---|---|---|---|---|---|
| WT | 3.25 ± 0.12 | 3.13 ± 0.01 | 9.77 ± 0.16 | ||
| T123A | 3.76 ± 0.19 | 3.09 ± 0.01 | 9.64 ± 0.16 | 0.13 ± 0.22 | |
| V143A | 3.20 ± 0.21 | 2.06 ± 0.01 | 6.42 ± 0.13 | 3.34 ± 0.20 | |
| H168R | 2.40 ± 0.11 | 2.25 ± 0.01 | 7.02 ± 0.13 | 2.75 ± 0.21 | |
| G245S | 3.15 ± 0.12 | 2.74 ± 0.01 | 8.55 ± 0.15 | 1.22 ± 0.22 | |
| R249S | 3.13 ± 0.13 | 2.59 ± 0.01 | 8.08 ± 0.14 | 1.69 ± 0.21 | |
| N239Y | 3.01 ± 0.11 | 3.57 ± 0.01 | 11.14 ± 0.17 | –1.37 ± 0.23 | |
| N268D | 3.27 ± 0.10 | 3.52 ± 0.01 | 10.98 ± 0.17 | –1.21 ± 0.23 | |
| V143A/N268D | 3.35 ± 0.13 | 2.41 ± 0.01 | 7.52 ± 0.14 | 2.25 ± 0.21 | 2.13 |
| G245S/N239Y | 3.07 ± 0.06 | 3.13 ± 0.01 | 9.77 ± 0.16 | 0.00 ± 0.22 | –0.14 |
| R249S/T123A | 2.92 ± 0.08 | 2.46 ± 0.07 | 7.68 ± 0.19 | 2.09 ± 0.24 | 1.82 |
| R249S/H168R | 3.05 ± 0.07 | 2.58 ± 0.01 | 8.05 ± 0.14 | 1.72 ± 0.21 | 4.46 |
| T123A/H168R | 2.65 ± 0.07 | 2.13 ± 0.01 | 6.65 ± 0.13 | 3.12 ± 0.20 | 2.88 |
| R249S/T123A/H168R | 3.44 ± 0.10 | 2.52 ± 0.01 | 7.86 ± 0.14 | 1.91 ± 0.21 | 4.57 |
Data were fitted to the equation ΔGDD–N = ΔGH2OD–N – m [D], where ΔGDD–N is the free energy of unfolding in the presence of denaturant and ΔG is the free energy of unfolding in water, using the transformation in equation (1). ΔGH2OD–N = m [D]50% and ΔΔGH2OD–N = <m>Δ[D]50%. The standard errors of fitting for ΔGH2OD–N and ΔΔGH2OD–N are indicated.
aThe mean m value (<m>) of 14 denaturation curves (at 10°C in 50 mM sodium phosphate/5 mM DTT at pH 7.2) is 3.12 ± 0.088 kcal/mol and was used to calculate ΔGH2OD–N and ΔΔGH2OD–N in the table.
bSums of ΔΔGH2OD–N for individual mutations in the double and triple mutants.