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Alzheimer's disease (AD) is becoming a rapidly growing health problem, as it is one of the main causes of dementia in the elderly. Interestingly, copper(II) (together with zinc and iron) ions are accumulated in amyloid deposits, suggesting that metal binding to Abeta could be involved in AD pathogenesis. In Abeta, the metal binding is believed to occur within the N-terminal region encompassing the amino acid residues 1-16. In this work, potentiometric, spectroscopic (UV-vis, circular dichroism, and electron paramagnetic resonance), and electrospray ionization mass spectrometry (ESI-MS) approaches were used to investigate the copper(II) coordination features of a new polyethylene glycol (PEG)-conjugated Abeta peptide fragment encompassing the 1-16 amino acid residues of the N-terminal region (Abeta(1-16)PEG). The high water solubility of the resulting metal complexes allowed us to obtain a complete complex speciation at different metal-to-ligand ratios ranging from 1:1 to 4:1. Potentiometric and ESI-MS data indicate that Abeta(1-16)PEG is able to bind up to four copper(II) ions. Furthermore, in order to establish the coordination environment at each metal binding site, a series of shorter peptide fragments of Abeta, namely, Abeta(1-4), Abeta(1-6), AcAbeta(1-6), and AcAbeta(8-16)Y10A, were synthesized, each encompassing a potential copper(II) binding site. The complexation properties of these shorter peptides were also comparatively investigated by using the same experimental approach.

Copper(II) complexes of the neurotoxic peptide fragments of human and chicken prion proteins were studied by potentiometric, UV-vis, CD, and EPR spectroscopic and ESI-MS methods. The peptides included the terminally blocked native and scrambled sequences of HuPrP106-126 (HuPrPAc106-126NH2 and ScrHuPrPAc106-126NH2) and also the nona- and tetrapeptide fragments of both the human and chicken prion proteins (HuPrPAc106-114NH2, ChPrPAc119-127NH2, HuPrPAc109-112NH2, and ChPrPAc122-125NH2). The histidyl imidazole-N donor atoms were found to be the major copper(II) binding sites of all peptides; 3N and 4N complexes containing additional 2 and 3 deprotonated amide-N donors, respectively, are the major species in the physiological pH range. The complex formation processes for nona- and tetrapeptides are very similar, supporting the fact that successive deprotonation and metal ion coordination of amide functions go toward the N-termini in the form of joined six- and five-membered chelates. As a consequence, the peptide sequences investigated here, related to the neurotoxic region of the human PrP106-126 sequence, show a higher metal-binding affinity than the octarepeat fragments. In the case of the HuPrP peptide sequences, a weak pH-dependent binding of the Met109 residue was also detected in the 3N-coordinated complexes.

The N-terminal octapeptide repeat region of human prion protein (PrPc) is known to bind Cu(II). To investigate the binding modes of copper in PrPc, an octapeptide Ac-PHGGGWGQ-NH2 (1), which corresponds to an octa-repeat sequence, and a tetrapeptide Ac-HGGG-NH2 (2) have been synthesised. The copper(II) complexes formed with 1 and 2 have been studied by circular dichroism (CD) and electron spin resonance (ESR) spectroscopy. Both peptides form 1:1 complexes with Cu(II) at neutral and basic pH. CD, ESR and visible absorption spectra suggest a similar co-ordination sphere of the metal ion in both peptides, which at neutral pH consists of a square pyramidal geometry with three peptidic nitrogens and the imidazole nitrogen as donor atoms. Cyclic voltammetric measurements were used to confirm the geometrical features of these copper(II) complexes: the observation of negative redox potentials are in good agreement with the inferred geometry. All these results taken together suggest that peptide 1 provides a single metal binding site to which copper(II) binds strongly at neutral and basic pH and that the binding of the metal induces the formation of a stiffened structure in the HGGG peptide fragment.

An aqueous solution spectroscopic (Vis and EPR) study of the copper(II) complexes with the Ac-HGGG-NH2 and Ac-PHGGGWGQ-NH2 polypeptides (generically designated as L) suggests square base pyramids ascribable to [Cu(L)H(-2)] complex species, which contain three nitrogen donor atoms, arising from imidazole and peptide groups, in the equatorial plane and for a pseudo-octahedral geometry in the case of [CuLH-3]- and [Cu(L)H-4]2- which have four nitrogen donor atoms in their equatorial plane. The coordination sphere of the copper complex in the [Cu(L)H(-2)] species, which is present at neutral pH values, is completed by two oxygen donor atoms. ESI-MS spectra ascertained that water molecules are not present in the coordination equatorial plane of this latter species, in comparison with other copper(II) complexes with ligands bearing nitrogen and oxygen donor atoms and surely having equatorial water molecules. This indicates the coordination of a carbonyl oxygen atom in the equatorial plane has to be invoked. However, no direct proof about the involvement of a carbonyl group oxygen donor atom apically linked to copper was obtained, due to the flexibility of these structures at room temperature. Additionally, the low A(ll) value leads one to consider another oxygen atom of a carbonyl group being involved in the apical bond to copper in a fast exchange fashion. This apical interaction, which may also involve a water molecule, is more pronounced in the Cu-Ac-HGGG-NH2 than in the analogous Cu-Ac-PHGGGWGQ-NH2 system, probably because of the presence of tryptophan and proline in the polypeptide sequence.

Aggregation of the amyloid beta-peptide (Abeta) into insoluble fibrils is a key pathological event in Alzheimer's Disease (AD). There is now compelling evidence that metal binding to Abeta is involved in AD pathogenesis. The amino acid region 1-16 is widely considered as the metal binding domain of Abeta. In this work, we used a combined potentiometric, NMR, and electrospray ionization mass spectrometry (ESI-MS) approach to study the zinc(II) binding to a new polyethylene glycol (PEG)-conjugated peptide fragment encompassing the 1-16 amino acid sequence of Abeta (Abeta(1-16)PEG). Our results demonstrate for the first time that the Abeta(1-16) is able to coordinate up to three zinc ions, all the histidyl residues acting as independent anchor sites. The study was complemented by systematically investigating the zinc(II) complexes of a series of shorter peptide fragments related to the Abeta(1-16) sequence, namely, Abeta(1-4), Abeta(1-6), AcAbeta(1-6), AcAbeta(8-16)Y10A. The comparison of the whole results allowed the identification of the zinc(II) preferred binding sites within the longer Abeta(1-16) amino acid sequence. Unlike copper(II) that prefers the N-terminal amino group as the main binding site, the zinc(II) is preferentially placed in the 8-16 amino acidic region of Abeta(1-16).

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