Binding of Cu(II) ions to peptides studied by fluorescence spectroscopy and isothermal titration calorimetry

Joanna Makowska , Krzysztof Żamojć , Dariusz Wyrzykowski , Dorota Uber , Małgorzata Wierzbicka , Wiesław Wiczk , Lech Chmurzyński


Steady-state and time-resolved fluorescence quenching measurements supported by Isothermal Titration Calorimetry (ITC) were used to study the interactions of Cu2 + with four peptides. Two of them were taken from the N-terminal part of the FBP28 protein (formin binding protein) WW domain: Tyr-Lys-Thr-Ala-Asp-Gly-Lys-Thr-Tyr-NH2 (D9) and its mutant Tyr-Lys-Thr-Ala-Asn-Gly-Lys-Thr-Tyr-NH2 (D9_M) as well as two mutated peptides from the B3 domain of the immunoglobulin binding protein G derived from Streptococcus: Asp-Val-Ala-Thr-Tyr-Thr-NH2 (J1) and Glu-Val-Ala-Thr-Tyr-Thr-NH2 (J2). The measurements were carried out at 298.15 K in 20 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution with a pH of 6. The fluorescence of all peptides was quenched by Cu2 + ions. The stoichiometry, conditional stability constants and thermodynamic parameters for the interactions of the Cu2 + ions with D9 and D9_M were determined from the calorimetric data. The values of the conditional stability constants were additionally determined from fluorescence quenching measurements and compared with those obtained from calorimetric studies. There was a good correlation between data obtained from the two techniques. On the other hand, the studies revealed that J1 and J2 do not exhibit an affinity towards metal ions. The obtained results prove that fluorescence quenching experiments may be successfully used in order to determine stability constants of complexes with fluorescent ligands. Finally, based on the obtained results, the coordinating properties of the peptides towards the Cu2 + ions are discussed.
Author Joanna Makowska (FCh / DGICh / LPCC)
Joanna Makowska,,
- Laboratory of Physicochemistry of Coordination Complexes
, Krzysztof Żamojć (FCh / DGICh / LPCC)
Krzysztof Żamojć,,
- Laboratory of Physicochemistry of Coordination Complexes
, Dariusz Wyrzykowski (FCh / DGICh / LPCC)
Dariusz Wyrzykowski,,
- Laboratory of Physicochemistry of Coordination Complexes
, Dorota Uber (FCh / DGICh / LPCC)
Dorota Uber ,,
- Laboratory of Physicochemistry of Coordination Complexes
, Małgorzata Wierzbicka (FCh / DBCh / LP)
Małgorzata Wierzbicka,,
- Laboratory of Photobiophysics
, Wiesław Wiczk (FCh / DBCh / LP)
Wiesław Wiczk,,
- Laboratory of Photobiophysics
, Lech Chmurzyński (FCh / DGICh / LPCC)
Lech Chmurzyński,,
- Laboratory of Physicochemistry of Coordination Complexes
Journal seriesSpectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, ISSN 1386-1425, (A 30 pkt)
Issue year2016
Publication size in sheets0.5
Keywords in Englishprotein G fragments, FBP28 protein fragments, fluorescence quenching, metal-peptide binding, isothermal titration calorimetry
Languageen angielski
Score (nominal)30
ScoreMinisterial score = 30.0, ArticleFromJournal
Ministerial score (2013-2016) = 30.0, ArticleFromJournal
Publication indicators WoS Impact Factor: 2016 = 2.536 (2) - 2016=2.346 (5)
Citation count*
Share Share

Get link to the record

* presented citation count is obtained through Internet information analysis and it is close to the number calculated by the Publish or Perish system.