Solvent-exposed residues located in the β-sheet modulate the stability of the tetramerization domain of p53-A structural and combinatorial approach

The role of hydrophobic amino acids in the formation of hydrophobic cores as one of the major driving forces in protein folding has been extensively studied. However, the implication of neutral solvent‐exposed amino acids is less clear and available information is scarce. We have used a combinatorial approach to study the structural relevance of three solvent‐exposed residues (Tyr327, Thr329, and Gln331) located in theβ‐sheet of the tetramerization domain of the tumor suppressor p53 (p53TD). A conformationally defined peptide library was designed where these three positions were randomized. The library was screened for tetramer stability. A set of p53TD mutants containing putative stabilizing or destabilizing residue combinations was synthesized for a thermodynamic characterization. Unfolding experiments showed a wide range of stabilities, with Tm values between 27 and 83°C. Wild type p53TD and some highly destabilized and stabilized mutants were further characterized. Thermodynamic and biophysical data indicated that these proteins were folded tetramers, with the same overall structure, in equilibrium with unfolded monomers. An NMR study confirmed that the main structural features of p53TD are conserved in all the mutants analyzed. The thermodynamic stability of the different p53TD mutants showed a strong correlation with parameters that favor formation and stabilization of the β‐sheet. We propose that stabilization through hydrophobic interactions of key secondary structure elements might be the underlying mechanism for the strong influence of solvent‐exposed residues in the stability of p53TD. Proteins 2008. © 2007 Wiley‐Liss, Inc.