An NMR study on nickel binding sites in Cap43 protein fragments

Abstract

NMR spectroscopy was used to study the interaction of Ni(II) ions with C-terminal sequence of Cap43 protein where, from Thr₃₄₁ to Gly₃₆₀ residue, a T₁R₂S₃R₄S₅H₆T₇S₈E₉G₁₀ ten-amino acid fragment is consecutively repeated three times. The behaviour of ends-blocked Ac-RSRSHTSEG-Am (pept1), Ac-TRSRSHTSEG-Am (pept2), and the three repeats Ac-TRSRSHTSEG-TRSRSHTSEG-TRSRSHTSEG-Am (pept3) peptides towards Ni(II) ions was examined at different pH values and, for pept3, at different ligand-to-metal molar ratios. ¹H-¹H TOCSY, ¹H-¹³C HSQC, ¹H-¹H NOESY and ¹H-¹H ROESY multidimensional NMR techniques were performed to understand the details of metal binding sites and the conformational behaviour of the peptides. The results confirmed that each mono-histidinic sequence of pept3 is able to independently coordinate one, two or three Ni(II) ions for 1:1, 1:2 and 1:3 ligand-to-metal molar ratios, respectively. At higher pH values, the coordination of Ni(II) involves imidazole Nδ of His₆ and three preceding deprotonated peptide nitrogens from the backbone, giving a {Nδ, 3N⁻} chromophore in a square planar geometry. In addition, at lower pH values, the involvement of γ-O of carboxyl group from Glu₉ residue with the formation of a macrochelate giving a {Nδ, γ-O⁻, 4O_H2O} chromophore in an octahedral geometry, was evidenced. NMR results allowed us to build a model for the structure of the major complex. Structural changes in the conformation of the peptide with organized Arg₄ and Thr₇ side chain orientation promoted by nickel coordination, were detected.