Solvent-exposed residues located in the beta-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 (Tyr(327), Thr(329), and Gln(331)) located in thebeta-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 T(m) values between 27 and 83 degrees 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 beta-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.
Mora
Puig
P
Department of Medicinal Chemistry, Centro de Investigación Príncipe Felipe, E-46013 Valencia, Spain.
Carbajo
Rodrigo J
RJ
Pineda-Lucena
Antonio
A
Sánchez del Pino
Manuel M
MM
Pérez-Payá
Enrique
E
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics; D001703:Biophysics / Q000379:methods; D002942:Circular Dichroism; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D019281:Dimerization; D005998:Glycine / Q000737:chemistry; D019791:Guanidine / Q000494:pharmacology; D006358:Hot Temperature; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D019151:Peptide Library; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012997:Solvents / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013696:Temperature; D013816:Thermodynamics; D013912:Threonine / Q000737:chemistry; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000235:genetics; D014443:Tyrosine / Q000737:chemistry
D000596:Amino Acids; D019151:Peptide Library; D010455:Peptides; D012997:Solvents; D016159:Tumor Suppressor Protein p53; D013912:Threonine; D014443:Tyrosine; D019791:Guanidine; D005998:Glycine