Characterization of a partially folded monomer of the DNA-binding domain of human papillomavirus E2 protein obtained at high pressure.

The pressure-induced dissociation of the dimeric DNA binding domain of the E2 protein of human papillomavirus (E2-DBD) is a reversible process with a Kd of 5.6 x 10(-8) M at pH 5.5. The complete exposure of the intersubunit tryptophans to water, together with the concentration dependence of the pressure effect, is indicative of dissociation. Dissociation is accompanied by a decrease in volume of 76 ml/mol, which corresponds to an estimated increase in solvent-exposed area of 2775 A2. There is a decrease in fluorescence polarization of tryptophan overlapping the red shift of fluorescence emission, supporting the idea that dissociation of E2-DBD occurs in parallel with major changes in the tertiary structure. The dimer binds bis(8-anilinonaphthalene-1-sulfonate), and pressure reduces the binding by about 30%, in contrast with the almost complete loss of dye binding in the urea-unfolded state. These results strongly suggest the persistence of substantial residual structure in the high pressure state. Further unfolding of the high pressure state was produced by low concentrations of urea, as evidenced by the complete loss of bis(8-anilinonaphthalene-1-sulfonate) binding with less than 1 M urea. Following pressure dissociation, a partially folded state is also apparent from the distribution of excited state lifetimes of tryptophan. The combined data show that the tryptophans of the protein in the pressure-dissociated state are exposed long enough to undergo solvent relaxation, but the persistence of structure is evident from the observed internal quenching, which is absent in the completely unfolded state. The average rotational relaxation time (derived from polarization and lifetime data) of the pressure-induced monomer is shorter than the urea-denatured state, suggesting that the species obtained under pressure are more compact than that unfolded by urea.