Katalin Osz

The Metal Loading Ability of β-Amyloid N-Terminus: A Combined Potentiometric and Spectroscopic Study of Copper(II) Complexes with β-Amyloid(1−16), Its Short or Mutated Peptide Fragments, and Its Polyethylene Glycol (PEG)-ylated Analogue

Alzheimers 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.

Interaction of copper(II) with the prion peptide fragment HuPrP(76-114) encompassing four histidyl residues within and outside the octarepeat domain.

Complex formation processes between the 39-mer residue peptide fragment of human prion protein, HuPrP(76-114), and copper(II) ions have been studied by potentiometric, UV-vis, circular dichroism (CD), electron paramagnetic resonance, and electrospray ionization mass spectrometry methods. This peptide consists of 39 amino acid residues and contains two histidines (His77 and His85) belonging to the octarepeat domain and two histidines (His96 and His111) outside this domain. It was found that HuPrP(76-114) is able to bind 4 equiv of metal ions and all histidyl residues are independent, except nonequivalent metal binding sites in the oligonuclear species. Imidazole nitrogen donor atoms are the primary and exclusive metal binding sites below pH 5.5 in the form of various macrochelates. The macrochelation slightly suppresses, but cannot prevent, the deprotonation and metal ion coordination of amide functions, resulting in the formation of (N(im),N(-)), (N(im),N(-),N(-)), and (N(im),N(-),N(-),N(-))-coordinated copper(II) complexes in the pH range from 5.5 to 9. CD spectroscopy results gave clear evidence for the differences in the metal binding affinity of the histidyl sites according to the following order: His111 > His96 >> His77 approximately His85. Among the oligonuclear complexes, the formation of di- and tetranuclear species seems to be favored over the trinuclear ones, at pH values beyond the physiological ones. This phenomenon was not observed in the complex formation reactions of HuPrP(84-114), a peptide fragment containing only one histidyl residue from the octarepeat. As a consequence, the data support the existence of cooperativity in the metal binding ability of this peptide probably due to the presence of two octarepeat sequences of the dimeric octarepeat domain of HuPrP(76-114) at basic pH values.

Metal Loading Capacity of Aβ N-Terminus: a Combined Potentiometric and Spectroscopic Study of Zinc(II) Complexes with Aβ(1−16), Its Short or Mutated Peptide Fragments and Its Polyethylene Glycol−ylated Analogue

Aggregation of the amyloid beta-peptide (Abeta) into insoluble fibrils is a key pathological event in Alzheimers 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).

Zn2+’s Ability to Alter the Distribution of Cu2+ among the Available Binding Sites of Aβ(1–16)-Polyethylenglycol-ylated Peptide: Implications in Alzheimer’s Disease

The formation of mixed copper(II) and zinc(II) complexes with Aβ(1-16)-PEG has been investigated. The peptide fragment forms stable mixed metal complexes at physiological pH in which the His13/His14 dyad is the zinc(II)s preferred binding site, while copper(II) coordination occurs at the N-terminus also involving the His6 imidazole. Copper(II) is prevented by zinc(II) excess from the binding to the two His residues, His13 and His14. As the latter binding mode has been recently invoked to explain the redox activity of the copper-Aβ complex, the formation of ternary metal complexes may justify the recently proposed protective role of zinc(II) in Alzheimers disease. Therefore, the reported results suggest that zinc(II) competes with copper for Aβ binding and inhibits copper-mediated Aβ redox chemistry.

Copper(II), nickel(II) and zinc(II) complexes of amino acids containing bis(imidazol-2-yl)methyl residues

Abstract Copper(II), nickel(II) and zinc(II) complexes of Phe-BIMA and His-BIMA were studied by potentiometric, UV–Vis and EPR spectroscopic methods. The nitrogen donor atoms of the bis(imidazol-2-yl)methyl residues were described as the primary metal binding sites in all systems studied. Deprotonation and coordination of the terminal amino and amide nitrogen atoms took place in the copper(II) complexes of both ligands and resulted in the formation of dinuclear complexes ([Cu2H−2L2]2+) containing [NH2, N−, N(Im)] tridentate ligands and imidazole bridging. The presence of the histidyl side chain provides a great versatility in the complex formation reactions of His-BIMA. The existence of the species [Cu2L2]4+ was detected in slightly acidic solution and its structure was described as a mixture of three isomeric forms. Deprotonation of the imidazole-N(1)H donor functions was detected under slightly alkaline conditions with pK values of 8.13 and 8.93. An excess of copper(II) ions shifted this reactions even into the slightly acidic pH range and resulted in the formation of a trinuclear complex ([Cu3H−4L2]2+).

A new, model-free calculation method to determine the coordination modes and distribution of copper(II) among the metal binding sites of multihistidine peptides using circular dichroism spectroscopy

A new calculation method to determine microscopic protonation processes from CD spectra measured at different pH and Cu(II):ligand ratios was developed and used to give the relative binding strengths for the three histidines of hsPrP(84-114), a 31-mer polypeptide modeling the N-terminal copper(II) binding region of human (homo sapiens) prion protein. Mutants of hsPrP(84-114) with two or one histidyl residues have also been synthesized and their copper(II) complexes studied by CD spectroscopy. The 1-His models were analyzed first, and the molar CD spectra for the different coordination modes on the different histidines were calculated using the general computational program PSEQUAD. These spectra were deconvoluted into the sum of Gaussian curves and used as a first parameter set to calculate the molar spectra for the different coordination modes (3N and 4N coordination) and coordination positions (His85, His96 and His111) of the 2-His peptides. The calculation method therefore does not require the direct use of CD spectra measured in the smaller peptide models. This is a significant improvement over earlier calculation methods. In the same runs, the stepwise deprotonation pK(mic) values were refined and the pH-dependent distribution of copper(II) between the two histidines was determined. The results revealed the high, but different copper(II) binding affinities of the three separate histidines in the following order: His85 << His96His111. The calculation also showed that molar CD spectra which belong to the same coordination mode and coordination position in different ligands have very similar transition energies but different intensities. For this reason, direct transfer of molar CD spectra between different ligands may be a source of error, but the pK(mic) values and the copper(II) binding preferences are transferable from the 2-His peptides to the 3-His hsPrP(84-114).

Copper(II) complexes of amino acids and peptides containing chelating bis(imidazolyl) residues.

Copper(II) complexes of amino acids and peptides containing the chelating bis(imidazolyl) residues have been reviewed. The results reveal that bis(imidazolyl) analogues of these biomolecules are very effective ligands for metal binding. The nitrogen donor atoms of the chelating agent are the major metal binding sites under acidic conditions. In the presence of terminal amino group the multidentate character of the ligands results in the formation of various polynuclear complexes including the ligand and the imidazole bridged dimeric species. The most intriguing feature of the coordination chemistry of these ligands is that the deprotonation of the coordinated imidazole-N(1)H groups results in the appearance of a new chelating site in the molecules. It leads to the formation of stable trinuclear complexes via negatively charged imidazolato bridges.