Investigations of copper(II) complexation by fragments of the FBP28 protein using isothermal titration (ITC) and differential scanning calorimetry (DSC)

Abstract

Isothermal titration calorimetry (ITC) was used to study the interactions between copper(II) ions and peptides with sequences taken from the N-terminal loop of the FBP28 protein (formin-binding protein) WW domain: Ac-Lys-Thr-Ala-Asp-Gly-Lys-Thr-NH2 (D7) and Ac-Tyr-Lys-Thr-Ala-Asn-Gly-Lys-Thr-Tyr-NH2 (D9 M), respectively. Measurements were taken at 298.15 K in 20 mM 2-(N-morpholino)ethanesulfonic acid buffer solution at a pH of 6. The stoichiometry, conditional stability constants and thermodynamic parameters (ΔITC G, ΔITC H and ΔITC S) for the pertinent complexation reactions were determined. Furthermore, the thermal stability of peptide conformations in the presence and absence of copper(II) in the system was investigated using differential scanning calorimetry. Finally, a general procedure on how to include the effect of buffer competition with the peptide for the metal as well as proton competition with the metal for the peptide and the buffer's component during ITC data analysis is described.