Biophysical characterization of the HPV-16 E7 oncoprotein

LEONARDO G.ALONSO; MARIA M. GARCIA ALAI; ALICIA N.LAPEÑA; ALEJANDRO NADRA; GONZALO DE PRAT GAY

Philadelphia, PA, USA

Congreso; The Protein Society 15th Annual Symposium; 2001

The Protein Society

The E7 protein from human papillomavirus exerts its oncogenic properties through targeting of the cellular tumor suppressor retinoblastoma, mainly, another proteins. E7 from the high risk strain HPV16 is a 11 kDa Zn containing acidic protein which, despite yelding a single band in silver stained SDS PAGE, displays two conformers as revealed by gel filtration. We find that these conformers (40 and 60 kDa apparent size0. Are not in equilibrium. We have separated the two forms and concerted on the analyses of the 40 kDa running dimeric species. As known E7 runs as a 19 kDa in a SDS-PAGE experiment and we found that this is related to the existence to a very stable residual structure, were the protein runs as the actual molecular weight in the presence of high urea concentration. The existence of an extended conformation is reflected in the gel filtration behavior. In dilution experiments monitored by circular dichroism we obtained a dissociation constant of 4 uM, in agreement with equilibrium sedimentation experiments. These species remind invariant after treatment at 90 degrees and 0.1 % SDS, as judged by Far-UV CD. Despite this large stability the protein displays a substantial dispersion of the amide backbone protons by NMR, indicative of structural disorder. Conversely, guanidine chloride does denature the protein judged by a large change in molecular ellipticity. A dissociation transition at less than 2M denaturant is followed by unfolding of the monomer ending at 6M denaturant, indicated by concentration dependence of Gdm.Cl denaturation. E7 is dimeric, rigid, extended and termostable.