Thermodynamic Analysis of the Structural Stability of the Tetrameric Oligomerization Domain of p53 Tumor Suppressor

The structural stability of an amino acid fragment containing the oligomerization domain (residues 303-366) of the tumor suppressor p53 has been studied using high-precision differential scanning calorimetry (DSC) and circular dichroism spectroscopy (CD). Previous NMR solution structural determinations have revealed that the fragment forms a symmetric 29.8 kDa tetramer composed of a dimer of dimers (p53tet) [Lee, W., Harvey, T. S., Yin, Y., Yau, P., Litchfield, D & Arrowsmith, C. H. (1994) Nature Struct. Biol. 1, 877-890]. Thermal unfolding of the tetramer is reversible and can be described as a two-state transition in which the folded tetramer is converted directly to unfolded monomers (N₄ 4U). According to the DSC and CD data, the population of intermediate species consisting of folded monomers or dimers is insignificant, indicating that isolated dimeric or monomelic structures have a much lower stability than the dimer and do not become populated during thermal denaturation under the conditions studied. The transition temperature of unfolding is found to be highly dependent on protein concentration and to follow the expected behavior for a tetramer that dissociates upon unfolding. Experiments conducted at pH 4.0 in 25 mM sodium acetate at a tetramer concentration of 145.8 M have a transition temperature (T_m) of 75.3 °C while at 0.5 μM the value drops to 39.2 °C. The enthalpy change of unfolding at 60 °C is 26 kcal (mol of monomer)^-1 with a heat capacity change of 387 cai (K-mol of monomer)-1. The stability of p53tet is dependent on pH and salt concentration. Decreasing the pH from 7.0 to 3.0 lowered the stability of the tetramer significantly (T_m's of 84.5 and 34.3 °C, respectively) while higher salt concentrations increased the stability, especially at low pH values. The results of these studies indicate that the tetramer is stabilized primarily by intersubunit interactions rather than intrasubunit interactions. In fact, more than 58% of the total area buried from the solvent in the folded tetramer corresponds to the intersubunit interfaces, and 70% of this area is hydrophobic. These results emphasize the role of quaternary structure in the stabilization of small oligomeric proteins.