Interaction of Copper(II) with the PEG-ylated derivative peptide fragment encompassing residues 60-114 of prion protein

Prion diseases are a group of neurodegenerative disease based upon the conformational conversion of the normal form of protein (PrPC) to the disease-related scrapie isoform (PrPSc). The physiological function of PrP has not been identified unambiguously yet, but increasing evidence indicates that it is a copper-binding protein. A series of publications support now that mammalian prion protein contains at least six copper(II) binding sites located at the repeating motif of eight aminoacids between residues 58 and 91, with each repeat containing a histidine residue (tetraoctarepeats domain), and the region between the octarepeat and the C-terminal structured domain containing His96 and His111. Several studies have separately been focused on the octarepeat domain or on the aminoacid region between residues 90 and 126 disregarding the possibility to study the copper(II) complexes with peptide fragments containing both domains. As a matter of fact, we recently reported the speciation and structures of the copper(II) complexes with peptides fragments that allowed us to determine the metal binding affinities of histidyl residues inside and outside the octarepeat domain.3 Our systematic studies of the copper(II) interaction with peptide fragments of prion protein represented a necessary step on the way to a detailed thermodynamic and structural characterization of the metal complexes formed within the entire N-terminal domain. In the present work we synthesized the peptide fragment encompassing the residues 60-114 of prion protein with the aim to unambiguously define the complexation properties the N-terminal domain.To overcome the low solubility constrain, a PEG-ylated derivative of the 60-114 peptide sequence, Ac-(PEG11)3PrP60-114, was synthesized. We investigated the complexation properties of the peptide by UV-vis, circular dichroism (CD), and electrospray ionization mass spectrometry methods and compared these spectra with those of the copper(II) complexes of our previous peptide fragments. This comparative study supported the predominance of macrochelates in the physiological pH range and at low copper occupancy. At increased copper(II) ratios, however, amide bonded species form at the expenses of macrochelate species. At basic pH, the amide bonded species predominate at any ratios and are preferably formed with the binding sites of His96 and His111.