RESUMO
The tumor suppressor protein p53 is a nuclear protein that serves as an important transcription factor. The region responsible for sequence-specific DNA interaction is located in its core domain (p53C). Although full-length p53 binds to DNA as a tetramer, p53C binds as a monomer since it lacks the oligomerization domain. It has been previously demonstrated that two core domains have a dimerization interface and undergo conformational change when bound to DNA. Here we demonstrate that the interaction with a consensus DNA sequence provides the core domain of p53 with enhanced conformational stability at physiological salt concentrations (0.15 M). This stability could be either increased or abolished at low (0.01 M) or high (0.3 M) salt concentrations, respectively. In addition, interaction with the cognate sequence prevents aggregation of p53C into an amyloid-like structure, whereas binding to a nonconsensus DNA sequence has no effect on p53C stability, even at low ionic strength. Strikingly, sequence-specific DNA binding also resulted in a large stabilization of full-length p53, whereas nonspecific sequence binding led to no stabilization. The effects of cognate DNA could be mimicked by high concentrations of osmolytes such as glycerol, which implies that the stabilization is caused by the exclusion of water. Taken together, our results show an enhancement in protein stability driven by specific DNA recognition. When cognate DNA was added to misfolded protein obtained after a pressurization cycle, the original conformation was mostly recovered. Our results may aid the development of therapeutic approaches to prevent misfolded species of p53.
Assuntos
DNA/farmacologia , Dobramento de Proteína/efeitos dos fármacos , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Naftalenossulfonato de Anilina/química , Sequência Consenso , DNA/metabolismo , Corantes Fluorescentes/química , Glicerol/química , Humanos , Pressão Hidrostática , Luz , Concentração Osmolar , Polidesoxirribonucleotídeos/química , Polidesoxirribonucleotídeos/metabolismo , Ligação Proteica/genética , Conformação Proteica/efeitos dos fármacos , Desnaturação Proteica , Estabilidade Proteica/efeitos dos fármacos , Proteínas Recombinantes/química , Espalhamento de Radiação , Espectrometria de Fluorescência , Proteína Supressora de Tumor p53/genética , Água/químicaRESUMO
We report ab initio calculations for positively charged fragments of dry poly(dC)-poly(dG) DNA, with up to 4 C-G pairs. We find a strong hole-lattice coupling and clear evidence for the formation of small polarons. The largest geometry distortions occur in only one or two base pairs. They involve the stretching of weak bonds within each base pair, increasing the distance of positive hydrogens, and decreasing that of negative oxygens, to the region in which the hole localizes. We obtain an energy of approximately 0.30 eV for the polaron formation, nearly independent of the chain size. From it, we can estimate an activation energy for polaron hopping of approximately 0.15 eV, consistent with the available experimental value.