Equilibrium dissociation and unfolding of the dimeric human papillomavirus strain‐16 E2 DNA‐binding domain

The equilibrium unfolding reaction of the C‐terminal 80‐amino‐acid dimeric DNA‐binding domain of human papillomavirus (HPV) strain 16 E2 protein has been investigated using fluorescence, far‐UV CD, and equilibrium sedimentation. The stability of the HPV‐16 E2 DNA‐binding domain is concentration‐dependent, and the unfolding reaction is well described as a two‐state transition from folded dimer to unfolded monomer. The conformational stability of the protein, ΔG_H2O, was found to be 9.8 kcal/mol at pH 5.6, with the corresponding equilibrium unfolding/dissociation constant, K_u, being 6.5 → 10⁻⁸ M. Equilibrium sedimentation experiments give a K_d of 3.0 → 10⁻⁸ M, showing an excellent agreement between the two different techniques. Denaturation by temperature followed by the change in ellipticity also shows a concomitant disappearance of secondary and tertiary structures. The K_u changes dramatically at physiologically relevant pH's: with a change in pH from 6.1 to 7.0, it goes from 5.5 → 10⁻⁸ M to 4.4 → 10¹⁰ M. Our results suggest that, at the very low concentration of protein where DNA binding is normally measured (e.g., 10⁻¹¹ M), the protein is predominantly monomeric and unfolded. They also stress the importance of the coupling between folding and DNA binding.