Synthesis of complex phosphopeptides as mimics of p53 functional domains.
A complete set of mono-, di- and triphosphorylated peptides comprising amino acids 10-27, the Mdm2 and p300 binding site(s) of p53, with and without a fluorescein label at the N-terminus, was synthesized by step-by-step solid phase synthesis. Fluorescence polarization analysis revealed that phosphorylation at Thr18 decreased binding to recombinant Mdm2 protein compared with the unphosphorylated and the two other single phosphorylated analogues. Unlabelled multiply phosphorylated peptides corresponding to this amino-terminal transactivation domain proved to be powerful tools in analysing the phosphate specificity of existing anti-p53 monoclonal and polyclonal antibodies using direct ELISA. The tetramerization domain of human p53 protein was modelled with a 53 residue-long unlabelled unphosphorylated and Ser315-phosphorylated peptide pair. CD analysis showed similar alpha-helical structures for both peptides and no major difference in the secondary structure could be observed upon phosphorylation. Size-exclusion HPLC indicated that these synthetic oligomerization domain mimics underwent a pH-dependent tetramerization process, but the presence of a phosphate group at Ser315 did not modify the oligomeric state of the 308-360 p53 fragments. Nevertheless, the fluorescein-labelled Ser315 phosphorylated peptide bound to the downstream signalling ligand DNA topoisomerase I protein with slightly higher affinity than did the unphosphorylated analogue.
Varadi
Gyorgyi
G
The Wistar Institute, 3601 Spruce Street, Philadelphia, Pennsylvania 19104, USA. varadigy@mdche.szote.u-szeged.hu
Otvos
Laszlo
L
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004797:Enzyme-Linked Immunosorbent Assay; D005453:Fluorescence; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000138:chemical synthesis* / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000276:immunology
D000906:Antibodies; D010748:Phosphopeptides; D016159:Tumor Suppressor Protein p53