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

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Abstract
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, ΔGH2O, was found to be 9.8 kcal/mol at pH 5.6, with the corresponding equilibrium unfolding/dissociation constant, Ku, being 6.5 → 10−8 M. Equilibrium sedimentation experiments give a Kd 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 Ku 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.