Biochemical characterization of different conformational states of the Sf9 cell-purified p53His175 mutant protein.
In this study, we expressed and purified the p53 mutant encoded by the His175 allele (p53His175) in a baculovirus expression system in order to study the folding and the DNA binding activity of the protein. A two-site ELISA revealed that purified p53His175 protein preferentially displayed a PAb1620 conformation, which appeared to be not sufficient to interact specifically with DNA. The cryptic DNA binding activity of this mutant was then investigated by electrophoretic mobility shift assay in the presence of anti-p53 antibodies, and shown to be refractory to significant activation by PAb421 (a potent allosteric activator of wild-type p53's DNA binding activity). Nevertheless, p53His175 DNA binding was regulated by antibodies targeting the N-terminal region of the protein. Furthermore, while the protein preferentially displayed a PAb1620 conformation, our data suggested the existence of an equilibrium between at least two folding states of the protein (PAb1620 and PAb240 conformations). A model rationalizing the conformation, antibody-interacting ability and DNA binding regulation potential of p53His175 is presented.
Cohen
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PA
CRC Laboratories, Department of Biochemistry, Medical Sciences Institute, Dundee University, Dundee, UK. p.cohen@valdorel.fnclcc.fr
Hupp
T R
TR
Lane
D P
DP
Daniels
D A
DA
D000911:Antibodies, Monoclonal; D016367:Baculoviridae / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004586:Electrophoresis; D004797:Enzyme-Linked Immunosorbent Assay; D006639:Histidine / Q000737:chemistry; D006801:Humans; D016296:Mutagenesis; D011485:Protein Binding; D011487:Protein Conformation; D014162:Transfection; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; D016159:Tumor Suppressor Protein p53; D006639:Histidine