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JBIC Journal of Biological Inorganic Chemistry

, Volume 12, Issue 2, pp 234–247 | Cite as

Phosphorylation-dependent metal binding by α-synuclein peptide fragments

Original Paper

Abstract

α-Synuclein (α-syn) is the major protein component of the insoluble fibrils that make up Lewy bodies, the hallmark lesions of Parkinson’s disease. Its C-terminal region contains motifs of charged amino acids that potentially bind metal ions, as well as several identified phosphorylation sites. We have investigated the metal-binding properties of synthetic model peptides and phosphopeptides that correspond to residues 119–132 of the C-terminal, polyacidic stretch of human α-syn, with the sequence Ac-Asp-Pro-Asp-Asn-Glu-Ala-Tyr-Glu-Met-Pro-Ser-Glu-Glu-Gly (α-syn119–132). The peptide pY125 replaces tyrosine with phosphotyrosine, whereas pS129 replaces serine with phosphoserine. By using Tb3+ as a luminescent probe of metal binding, we find a marked selectivity of pY125 for Tb3+ compared with pS129 and α-syn119–132, a result confirmed by isothermal titration calorimetry. Truncated or alanine-substituted peptides show that the phosphoester group on tyrosine provides a metal-binding anchor that is supplemented by carboxylic acid groups at positions 119, 121, and 126 to establish a multidentate ligand, while two glutamic acid residues at positions 130 and 131 contribute to binding additional Tb3+ ions. The interaction of other metal ions was investigated by electrospray ionization mass spectrometry, which confirmed that pY125 is selective for trivalent metal ions over divalent metal ions, and revealed that Fe3+ and Al3+ induce peptide dimerization through metal ion cross-links. Circular dichroism showed that Fe3+ can induce a partially folded structure for pY125, whereas no change was observed for pS129 or the unphosphorylated analog. The results of this study show that the type and location of a phosphorylated amino acid influence a peptide’s metal-binding specificity and affinity as well as its overall conformation.

Keywords

Peptide Binding affinity Mass spectrometry Luminescence Protein engineering 

Notes

Acknowledgements

We are grateful for support provided by a National Science Foundation CAREER award (CHE-0449699). We thank Eric J. Toone, Andrea Luteran, and Trine Christensen for help with ITC experiments, and David A. Franz of Lycoming College for many helpful discussions.

Supplementary material

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Copyright information

© SBIC 2006

Authors and Affiliations

  1. 1.Department of ChemistryDuke UniversityDurhamUSA

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