RESUMO
A flexible polymer chain in the presence of inert macromolecular crowders will experience a loss of configurational entropy due to the crowder excluded volume. This entropy reduction will be most pronounced in good solvent conditions where the chain assumes an expanded coil conformation. For polymers that undergo a folding transition from a coil to a compact ordered state, as is the case for many globular proteins, macromolecular crowding is expected to stabilize the folded state and thereby shift the transition location. Here, we study such entropic stabilization effects for a tangent square-well sphere chain (monomer diameter σ) in the presence of hard-sphere (HS) crowders (diameter D ≥ σ). We use the Wang-Landau simulation algorithm to construct the density of states for this chain in a crowded environment and are thus able to directly compute the reduction in configurational entropy due to crowding. We study both a chain that undergoes all-or-none folding directly from the coil state and a chain that folds via a collapsed-globule intermediate state. In each case, we find an increase in entropic stabilization for the compact states with an increase in crowder density and, for fixed crowder density, with a decrease in crowder size (concentrated, small crowders have the largest effect). The crowder significantly reduces the average size for the unfolded states while having a minimal effect on the size of the folded states. In the athermal limit, our results directly provide the confinement free energy due to crowding for a HS chain in a HS solvent.
RESUMO
The p53 tumor suppressor protein is susceptible to oxidation, which prevents it from binding to its DNA response element. The goal of the current research was to determine the nature of the cysteine residue thiol oxidation that prevents p53 from binding its DNA target and its effect on p53 structure. Recombinant p53, purified in the presence of the reducing agent dithiothreitol (DTT), contains five free thiol groups on the surface of the protein. In the absence of DTT, p53 contains only four thiol groups, indicating that an average of one surface thiol group is readily susceptible to oxidation. Sulfite-mediated disulfide bond cleavage followed by reaction with 2-nitro-5-thiosulfobenzoate showed that oxidized p53 contains a single disulfide bond per monomer. By atomic force microscopy, we determined that reduced p53 binds to a double-stranded DNA containing the p53 promoter element of the MDM2 gene. The DNA-bound reduced p53 has an average cross-sectional diameter of 8.61 nm and a height of 4.12 nm. The amount of oxidized p53 that bound to the promoter element was ninefold lower, and it has an 18% larger average cross-sectional diameter. Electromobility shift assays showed that binding of oxidized p53 to DNA was enhanced upon addition of DTT, indicating that oxidation is reversible. The possibility that oxidized p53 contained significant amounts of sulfenic (-SOH), sulfinic (-SO2H), or sulfonic acid (-SO3H) was ruled out. Gel filtration chromatography indicated that oxidation increases the percentage of p53 monomers and high-molecular-weight oligomers (>1,000 kDa) relative to tetrameric p53. Protein modeling studies suggest that a mixed disulfide glutathione adduct on Cys182 could account for the observed stoichiometry of oxidized thiols and structural changes. The glutathione adduct may prevent proper helix-helix interaction within the DNA binding domain and contribute to tetramer dissociation.
