Yk. Mok et al., EQUILIBRIUM DISSOCIATION AND UNFOLDING OF THE DIMERIC HUMAN PAPILLOMAVIRUS STRAIN-16 E2 DNA-BINDING DOMAIN, Protein science, 5(2), 1996, pp. 310-319
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.