A doppel alpha-helix peptide fragment mimics the copper(II) interactions with the whole protein

The doppel protein (Dpi) is the first homologue of the prion protein (PrP C) to be discovered; it is overexpressed in transgenic mice that lack the prion gene, resulting in neurotoxicity. The whole prion protein is able to inhibit Dpi neurotoxicity, and its N-terminal domain is the determinant part of the protein function. This region represents the main copper(II) binding site of PrP C. Dpi is able to bind at least one copper ion, and the specific metalbinding site has been identified as the histidine residue at the beginning of the third helical region. However, a reliable characterization of copper(II) coordination features has not been reported. In a previous paper, we studied the copper(II) interaction with a peptide that encompasses only the loop region potentially involved in metal binding. Nevertheless, we did not find a complete match between the EPR spectroscopic parameters of the copper(II) complexes formed with the synthesized peptide and those reported for the copper(II) binding sites of the whole protein. Herein, the synthesis of the human Dpi peptide fragment hDpl(122-139) (Ac-KPDNKLHQQVLWRLVQEL-NH 2) and its copper(II) complex species are reported. This peptide encompasses the third a helix and part of the loop linking the second and the third helix of human doppel protein. The single-point-mutated peptide, hDpl(122-139)D124N, in which aspartate 124 replaces an asparagine residue, was also synthesized. This peptide was used to highlight the role of the carboxylate group on both the conformation preference of the Dpi fragment and its copper(II) coordination features. NMR spectroscopic measurements show that the hDpl( 122-139) peptide fragment is in the prevailing ahelix conformation. It is localized within the 127-137 amino acid residue region that represents a reliable conformational mimic of the related protein domain. A comparison with the singlepoint-mutated hDpl(122-139)D124N reveals the significant role played by the aspartic residue in addressing the peptide conformation towards a helical structure. It is further confirmed by CD measurements. Potentiometric titrations were carried out in aqueous solutions to obtain the stability constant values of the species formed by copper(II) with the hDpl peptides. Spectroscopic studies (EPR, NMR, CD, UV/Vis) were performed to characterize the coordination environments of the different metal complexes. The EPR parameters of the copper(II) complexes with hDpl( 122-139) match those of the previously reported copper(II) binding sites of the whole hDpl. Addition of the copper(II) ion to the peptide fragment does not alter the helical conformation of hDpl(122-139), as shown by CD spectra in the far-UV region. The aspartate-driven preorganized secondary structure is not significantly modified by the involvement of Aspl24 in the copper(II) complex species that form in the physiological pH range. To elaborate on the potential role of copper(II) in the recently reported interaction between the PrP C and Dpi, the affinity of the copper(II) complexes towards the prion N terminus domain and the binding site of Dpi was reported. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.

A doppel alpha-helix peptide fragment mimics the copper(II) interactions with the whole protein

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