Table 2.
Equilibrium unfolding of p53tet with natural mutations in the hydrophobic core
| Mutant | Hydrophobic core represented* | m, kcal/(M.mol) | [D]50%, M | ΔGuH2O, kcal/mol) | ΔΔGuH2O, kcal/mol | Expected additive ΔΔGuH2O, kcal/mol |
|---|---|---|---|---|---|---|
| wt tetS | Human p53, mammals | 5.1 ± 0.2 | 2.78 ± 0.01 | 32.5 ± 0.5 | ||
| F328L | 7.5 ± 0.2 | 1.41 ± 0.01 | 29.0 ± 0.3 | 3.5 ± 0.6 | ||
| L330F | 7.4 ± 1.0 | 1.19 ± 0.03 | 27.3 ± 1.2 | 5.2 ± 1.3 | ||
| I332V | 4.2 ± 0.3 | 2.25 ± 0.04 | 27.5 ± 0.9 | 5.0 ± 1.0 | ||
| M340I | 6.9 ± 0.4 | 2.14 ± 0.02 | 33.2 ± 0.9 | −0.7 ± 1.0 | ||
| F341L | 5.4 ± 0.3 | 2.05 ± 0.02 | 29.2 ± 0.6 | 3.3 ± 0.8 | ||
| F341I | Xenopus | 6.0 ± 0.2 | 1.21 ± 0.01 | 25.6 ± 0.2 | 6.9 ± 0.6 | |
| L344F | 5.0 ± 0.2 | 1.60 ± 0.01 | 26.4 ± 0.3 | 6.1 ± 0.6 | ||
| L344I | 5.3 ± 0.1 | 2.42 ± 0.01 | 31.3 ± 0.3 | 1.2 ± 0.6 | ||
| F341L/L344F | Trout | 4.7 ± 0.4 | 1.20 ± 0.02 | 24.1 ± 0.5 | 8.4 ± 0.7 | 9.4 ± 0.7 |
| L332V/F341L | 6.3 ± 0.4 | 1.88 ± 0.02 | 30.3 ± 0.7 | 2.2 ± 0.8 | 8.3 ± 1.1 | |
| L332V/M340I/F341L | Human p73, zebrafish | 6.2 ± 0.1 | 1.76 ± 0.01 | 29.4 ± 0.2 | 3.1 ± 0.5 | 7.6 ± 1.4 |
| F341L/L344I | 4.9 ± 0.0 | 2.62 ± 0.00 | 31.2 ± 0.1 | 1.3 ± 0.5 | 4.5 ± 0.7 | |
| L332V/F341L/L344I | Chicken, clam | 5.5 ± 0.1 | 2.55 ± 0.01 | 32.4 ± 0.3 | 0.1 ± 0.6 | 9.5 ± 1.1 |
| F328L/L332V/F341L/L344I | Human p51, rat KET | 6.9 ± 0.5 | 1.20 ± 0.02 | 26.8 ± 0.5 | 5.7 ± 0.7 | 13.0 ± 1.3 |
m, Variation in the free energy of unfolding, ΔGu, with the GdmCl concentration; [D]50%, concentration of GdmCl at which the transition is half-completed by using 40 μM protein (monomer) concentration. ΔGuH2O, ΔGu extrapolated to absence of denaturant and given for a standard 1 M protein concentration; ΔΔGuH2O difference in ΔGu between wild-type p53tetS and any mutant at zero denaturant concentration. Expected additive ΔΔGuH2O values for the multiple mutants were obtained by addition of the experimental values found for the corresponding single mutants. The standard errors of fitting and the propagated errors are indicated. The m values obtained for some mutants were somewhat different than that obtained for wt tetS, possibly due to the nature of the mutation(s) introduced. For these mutants, ΔΔGu values differed somewhat depending on the denaturant concentration at which they were calculated. ΔΔGu values are thus given at zero denaturant concentration, because this corresponds to more physiological conditions and because the required extrapolation of the experimental data was relatively short, so that the fitting errors were not large. The values obtained for a 2 M GdmCl concentration (data not shown) led to essentially the same conclusions discussed in the text.
For the mutants indicated, all residues at the thermodynamically most critical positions of human p53tet (residues 330, 332, 340, 341, 344, and 348; ref. 30) correspond to those of p53 or p53-like protein specified. These six residues form the central hydrophobic minicores of each primary dimer and the critical, central part of the interdimer interface. See also text, Fig. 1, and Table 1.