Chemical synthesis of phosphorylated peptides of the carboxy-terminal domain of human p53 by a segment condensation method.
A segment condensation method was developed for the chemical synthesis of large (> 90 amino acid) phosphopeptides and was used to produce phosphorylated and non-phosphorylated derivatives of the C-terminal tetramerization and regulatory domains of human p53 (residues 303-393). Efficient condensation synthesis of the 91 residue p53 domain was achieved in two steps. The non-phosphorylated N-terminal segment p53(303-334) (1) and its derivative phosphorylated at serine 315 (1P315), and the non-phosphorylated middle segment p53(335-360) (2), were synthesized as partially protected peptide thioesters in the solid phase using Boc chemistry. The C-terminal segment p53(361-393) (3) and its derivative phosphorylated at serine 392 (3P392) were synthesized as partially protected peptides in the solid phase using Fmoc chemistry. Phosphoamino acid was incorporated into the N-terminal segment (1P315) at the residue corresponding to p53 serine 315 as Boc-Ser(PO3(Bzl)2)-OH during synthesis. Serine 392 in the C-terminal segment was selectively phosphorylated after synthesis by phosphitylation followed by oxidation. A derivative phosphorylated at serine 378 was synthesized in a one-step condensation of the unphosphorylated N-terminal segment (1) and the phosphorylated long C-terminal segment p53(335-393) (2-3P378). Yields of the ligated peptides after removal of the protecting groups and HPLC purification averaged 60% for the first condensation and 35% for the second condensation. All five p53 peptides exhibited monomer-tetramer association as determined by analytical ultracentrifugation. Circular dichroism spectroscopy revealed that phosphorylation at Ser315 increased the alpha-helical content, which was abolished when Ser392 also was phosphorylated, suggesting an interaction between N-terminal and C-terminal residues of the C-terminal domain of p53.
Sakamoto
H
H
Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.
Kodama
H
H
Higashimoto
Y
Y
Kondo
M
M
Lewis
M S
MS
Anderson
C W
CW
Appella
E
E
Sakaguchi
K
K
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis*; D010748:Phosphopeptides / Q000138:chemical synthesis*; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry*
D010446:Peptide Fragments; D010748:Phosphopeptides; D016159:Tumor Suppressor Protein p53