Copper(ii) binding by fragments of α-synuclein containing M1-D2- and -H50-residues; a combined potentiometric and spectroscopic study

Abstract

Stability constants and ligand donor sets of the copper(II) complexes of the NH₂-29–56(L¹)(AA³⁰GKTKEGVLYV⁴⁰GSKTKEGVVH⁵⁰GVATVA⁵⁶-NH₂), NH₂-M²⁹-D³⁰-56(L²) and Ac-M²⁹-D³⁰-56(L³) fragments of α-synuclein were determined in aqueous solution for 1 : 1 metal-to-ligand molar ratio in the pH range 2.5–10.5. The tyrosine residue in the 39th position of the α-synuclein fragments does not take part in the coordination of the metal ion. The potentiometric and spectroscopic data (UV-Vis, CD, EPR) show that acetylation of the amino terminal group induces significant changes in the coordination properties of the L³ fragment compared to that of the L² peptide. When the amino group is blocked (L³) the imidazole nitrogen of the histidine residue acts as an anchoring site and at higher pH the 3N {N_Im,2N⁻} and 4N {N_Im,3N⁻} complexes are formed. The L¹ peptide at physiological pH forms in equilibrium 3N {NH₂,N⁻,CO,N_Im} and 4N {NH₂,2N⁻,N_Im} complexes. For the L² peptide the coordination of the copper(II) ions starts from the N-terminal Met residue and with increasing of pH the Asp residue in second position of amino acid sequence coordinates and stabilizes significantly the 2N complex as a result of chelation through the β-carboxylate group. At physiological pH the 3N {NH₂,N⁻,β-COO⁻,N_Im} coordination mode dominates. At pH above 6 the results for the L² fragment suggest the formation of 3N and 4N complexes (in equatorial plane) and the involvement of the lateral NH₂ group of Lys residue in the axial coordination of Cu(II) ion. In CD spectra σ (ε-NH₂-Lys) → Cu(II) charge transfer transition is observed. The stability constants for the L² fragment of α-synuclein of the 4N {NH₂,2N⁻,N_Im} and {NH₂,3N⁻} complexes are higher about 1.5 and 0.7 orders of magnitude, respectively, by comparison to those of the L¹ peptide. This increase may be explained by the involvement of the ε-NH₂ group of Lys residue in the coordination sphere of metal ion.