Synthesis of complex phosphopeptides as mimics of p53 functional domains

✍ Scribed by Dr Gyorgyi Varadi; Laszlo Otvos Jr

• Book ID: 105360232
• Publisher: John Wiley and Sons
• Year: 2002
• Tongue: English
• Weight: 148 KB
• Volume: 8
• Category: Article
• ISSN: 1075-2617
• DOI: 10.1002/psc.422

✦ Synopsis

Abstract

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 Thr^18^ 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 Ser^315^‐phosphorylated peptide pair. CD analysis showed similar α‐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 Ser^315^ did not modify the oligomeric state of the 308–360 p53 fragments. Nevertheless, the fluorescein‐labelled Ser^315^ phosphorylated peptide bound to the downstream signalling ligand DNA topoisomerase I protein with slightly higher affinity than did the unphosphorylated analogue. Copyright © 2002 European Peptide Society and John Wiley & Sons, Ltd.