title: Binding of Cu(II) ions to peptides studied by fluorescence spectroscopy and isothermal titration calorimetry.
abstract: Steady-state and time-resolved fluorescence quenching measurements supported by Isothermal Titration Calorimetry (ITC) were used to study the interactions of Cu(2+) 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 298.15K in 20mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution with a pH of 6. The fluorescence of all peptides was quenched by Cu(2+) ions. The stoichiometry, conditional stability constants and thermodynamic parameters for the interactions of the Cu(2+) 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 Cu(2+) ions are discussed.
authors.affiliation: Faculty of Chemistry, University of Gdańsk, ul. Wita Stwosza 63, 80-308 Gdańsk, Poland.
authors.forename: Joanna
authors.initials: J
authors.lastname: Makowska
authors.affiliation: Faculty of Chemistry, University of Gdańsk, ul. Wita Stwosza 63, 80-308 Gdańsk, Poland. Electronic address: krzysztof.zamojc@ug.edu.pl.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Żamojć
authors.affiliation: Faculty of Chemistry, University of Gdańsk, ul. Wita Stwosza 63, 80-308 Gdańsk, Poland.
authors.forename: Dariusz
authors.initials: D
authors.lastname: Wyrzykowski
authors.affiliation: Faculty of Chemistry, University of Gdańsk, ul. Wita Stwosza 63, 80-308 Gdańsk, Poland.
authors.forename: Dorota
authors.initials: D
authors.lastname: Uber
authors.affiliation: Faculty of Chemistry, University of Gdańsk, ul. Wita Stwosza 63, 80-308 Gdańsk, Poland.
authors.forename: Małgorzata
authors.initials: M
authors.lastname: Wierzbicka
authors.affiliation: Faculty of Chemistry, University of Gdańsk, ul. Wita Stwosza 63, 80-308 Gdańsk, Poland.
authors.forename: Wiesław
authors.initials: W
authors.lastname: Wiczk
authors.affiliation: Faculty of Chemistry, University of Gdańsk, ul. Wita Stwosza 63, 80-308 Gdańsk, Poland.
authors.forename: Lech
authors.initials: L
authors.lastname: Chmurzyński
mesh_terms: D002151:Calorimetry / Q000379:methods*; D003300:Copper / Q000378:metabolism*; D007477:Ions; D010455:Peptides / Q000378:metabolism*; D012996:Solutions; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
chemical_list: D007477:Ions; D010455:Peptides; D012996:Solutions; D003300:Copper
keywords: FBP28 protein fragments; Fluorescence quenching; Isothermal titration calorimetry; Metal–peptide binding; Protein G fragments