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 olig omerization domain (residues 303-366) of the tumor suppressor p53 has been studied using high-precision differential scanning calorimetry (D SC) and circular dichroism spectroscopy (CD). Previous NMR solution st ructural determinations have revealed that the fragment forms a symmet ric 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 tetr amer is reversible and can be described as a two-state transition in w hich the folded tetramer is converted directly to unfolded monomers (N -4 <----> 4U). According to the DSC and CD data, the population of int ermediate species consisting of folded monomers or dimers is insignifi cant, indicating that isolated dimeric or monomeric structures have a much lower stability than the dimer and do not become Populated during thermal denaturation under the conditions studied. The transition tem perature of unfolding is found to be highly dependent on protein conce ntration and to follow the expected behavior for a tetramer that disso ciates upon unfolding. Experiments conducted at pH 4.0 in 25 mM sodium acetate at a tetramer concentration of 145.8 mu M have a transition t emperature (T-m) of 75.3 degrees C while at 0.5 mu M the value drops t o 39.2 degrees C. The enthalpy change of unfolding at 60 degrees C is 26 kcal (mol of monomer)(-1) with a heat capacity change of 387 cal (K .mol of monomer)(-1). The stability of p53tet is dependent on pH and s alt concentration. Decreasing the pH from 7.0 to 3.0 lowered the stabi lity of the tetramer significantly (T-m's of 84.5 and 34.3 degrees C, respectively) while higher salt concentrations increased the stability , especially at low pH values. The results of these studies indicate t hat the tetramer is stabilized primarily by intersubunit interactions rather than intrasubunit interactions. In fact, more than 58% of the t otal area buried from the solvent in the folded tetramer corresponds t o the intersubunit interfaces, and 70% of this area is hydrophobic. Th ese results emphasize the role of quaternary structure in the stabiliz ation of small oligomeric proteins.