ANALYSIS OF THE CONFORMATIONAL CHANGE OF RECOMBINANT HUMAN PAPILLOMA-VIRUS TYPE-18 E7 PROTEIN-INDUCED BY METAL-BINDING

Human papillomavirus (HPV) type 18 E7 gene was isolated by polymerase chain reaction (PCR) amplification from tissues of Korean cervical can cer patients and cloned into a plasmid vector, pET-3a, for the express ion of recombinant E7 protein (rE7) in Escherichia coli. The rE7 prote in was purified to the homogeneity and its purity was confirmed by HPL C. The purified protein was analyzed for the metal-binding properties by UV spectroscopy and it was shown that two Cd2+ or Zn2+ ions bind to one E7 protein by the metal-sulfur ligand formation via two Cys-X-X-C ys motifs in E7 protein. When the change of intrinsic fluorescence of tryptophan residue was analyzed for rE7-Zn complex, the blue shift of emission wavelength and the decrease in maximum intensity of emission were observed compared with rE7. These results suggest that Zn2+-bound rE7 has undergone conformational change, in which a tryptophan residu e located in the second Cys-X-X-Cys motif was moved into solvent-inacc essible or hydrophobic environment. The rE7-Zn complex was found to be resistant to chymotrypic digestion by comparing the digestion pattern s of rE7. Therefore, we showed the folding status of HPV 18 E7 could b e changed by metal binding resulting in a different conformation in wh ich a tryptophan residue was driven into more hydrophobic environment and the resistancy to chymotryptic digestion was conferred. (C) 1997 E lsevier Science B.V.