An NMR study on nickel binding sites in Cap43 protein fragments

NMR spectroscopy was used to study the interaction of Ni(II) ions with C-terminal sequence of Cap43 protein where, from Thr341 to Gly 360 residue, a T1 R2 S3 R 4 S5 H6 T7 S8 E 9 G 10 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 -, 4OH2O} 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