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 cancer patients and cloned into a plasmid vector, pET-3a, for the expression of recombinant E7 protein (rE7) in Escherichia coli. The rE7 protein was purified to the homogeneity and its purity was confirmed by HPLC. 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-Cys 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 Zn(2+)-bound rE7 has undergone conformational change, in which a tryptophan residue located in the second Cys-X-X-Cys motif was moved into solvent-inaccessible or hydrophobic environment. The rE7-Zn complex was found to be resistant to chymotrypic digestion by comparing the digestion patterns of rE7. Therefore, we showed the folding status of HPV 18 E7 could be changed by metal binding resulting in a different conformation in which a tryptophan residue was driven into more hydrophobic environment and the resistancy to chymotryptic digestion was conferred.
Kang
J H
JH
Peptide Engineering Research Unit, Korea Research Institute of Bioscience and Biotechnology, KIST, Taejon, South Korea.
Jin
S W
SW
Yoon
H S
HS
Yoo
W D
WD
Kim
H S
HS
Hahm
K S
KS
Park
S N
SN
D002104:Cadmium / Q000378:metabolism*; D004268:DNA-Binding Proteins; D006801:Humans; D009856:Oncogene Proteins, Viral / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D027383:Papillomaviridae / Q000737:chemistry / Q000235:genetics*; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism; D015032:Zinc / Q000378:metabolism*
D004268:DNA-Binding Proteins; C052633:E7 protein, Human papillomavirus type 18; D009856:Oncogene Proteins, Viral; D011994:Recombinant Proteins; D002104:Cadmium; D011505:Protein-Tyrosine Kinases; D015032:Zinc