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 dim eric DNA-binding domain of human papillomavirus (HPV) strain 16 E2 pro tein has been investigated using fluorescence, far-UV CD, and equilibr ium sedimentation. The stability of the HPV-16 E2 DNA-binding domain i s concentration-dependent, and the unfolding reaction is well describe d as a two-state transition from folded dimer to unfolded monomer. The conformational stability of the protein, Delta G(H2O), was found to b e 9.8 kcal/mol at pH 5.6, with the corresponding equilibrium unfolding /dissociation constant, K-u, being 6.5 x 10(-8) M. Equilibrium sedimen tation experiments give a K-d of 3.0 x 10(-8) M, showing an excellent agreement between the two different techniques. Denaturation by temper ature followed by the change in ellipticity also shows a concomitant d isappearance of secondary and tertiary structures. The K-u changes dra matically at physiologically relevant pH's: with a change in pH from 6 .1 to 7.0, it goes from 5.5 x 10(-8) M to 4.4 x 10(10) M. Our results suggest that, at the very low concentration of protein where DNA bindi ng is normally measured(e.g., 10(-11) M), the protein is predominantly monomeric and unfolded. They also stress the importance of the coupli ng between folding and DNA binding.