Gianni Valensin

Selective and nonselective proton spin-lattice relaxation studies of enzyme-substrate interactions

Abstract The selective and nonselective longitudinal relaxation rates were measured for glycyl- l -tyrosine protons in the presence of Zn-carboxypeptidase A. Unlike the non-selective relaxation rates, the selective rate was found to be particularly sensitive to the presence of the enzyme. From the nonselective/selective ratio, the correlation times of the free and bound substrate were evaluated.

Bioinorganic Chemistry of Parkinson's Disease: Structural Determinants for the Copper-Mediated Amyloid Formation of Alpha-Synuclein

The aggregation of alpha-synuclein (AS) is a critical step in the etiology of Parkinsons disease (PD). A central, unresolved question in the pathophysiology of PD relates to the role of AS-metal interactions in amyloid fibril formation and neurodegeneration. Our previous works established a hierarchy in alpha-synuclein-metal ion interactions, where Cu(II) binds specifically to the protein and triggers its aggregation under conditions that might be relevant for the development of PD. Two independent, non-interacting copper-binding sites were identified at the N-terminal region of AS, with significant difference in their affinities for the metal ion. In this work we have solved unknown details related to the structural binding specificity and aggregation enhancement mediated by Cu(II). The high-resolution structural characterization of the highest affinity N-terminus AS-Cu(II) complex is reported here. Through the measurement of AS aggregation kinetics we proved conclusively that the copper-enhanced AS amyloid formation is a direct consequence of the formation of the AS-Cu(II) complex at the highest affinity binding site. The kinetic behavior was not influenced by the His residue at position 50, arguing against an active role for this residue in the structural and biological events involved in the mechanism of copper-mediated AS aggregation. These new findings are central to elucidate the mechanism through which the metal ion participates in the fibrillization of AS and represent relevant progress in the understanding of the bioinorganic chemistry of PD.

The dimeric and tetrameric octarepeat fragments of prion protein behave differently to its monomeric unit

Potentiometric and spectroscopic data have shown that octarepeat dimer and tetramer are much more effective ligands for Cu(II) ions than simple octapeptide. Thus, the whole N-terminal segment of prion protein due to cooperative effects, could be more effective in binding of Cu(II) than simple peptides containing a His residue. The gain of the Cu(II) binding by longer octarepeat peptides derives from the involvement of up to four imidazoles in the coordination of the first Cu(II) ion. This type of binding increases the order of the peptide structure, which allows successive metal ions for easier coordination.

fac-{Ru(CO)3}2+ Selectively Targets the Histidine Residues of the β-Amyloid Peptide 1-28. Implications for New Alzheimer's Disease Treatments Based on Ruthenium Complexes

The reaction of the ruthenium(II) complex fac-[Ru(CO)(3)Cl(2)(N(1)-thz)] (I hereafter; thz = 1,3-thiazole) with human beta-amyloid peptide 1-28 (Abeta(28)) and the resulting {Ru(CO)(3)}(2+) peptide adduct was investigated by a variety of biophysical methods. (1)H NMR titrations highlighted a selective interaction of {Ru(CO)(3)}(2+) with Abeta(28) histidine residues; circular dichroism revealed the occurrence of a substantial conformational rearrangement of Abeta(28); electrospray ionization mass spectrometry (ESI-MS) suggested a prevalent 1:1 metal/peptide stoichiometry and disclosed the nature of peptide-bound metallic fragments. Notably, very similar ESI-MS results were obtained when I was reacted with Abeta(42). The implications of the above findings for a possible use of ruthenium compounds in Alzheimers disease are discussed.

Copper Binding to the Neurotoxic Peptide PrP106–126: Thermodynamic and Structural Studies

The human prion protein fragment PrP106–126 is a highly fibrillogenic peptide, resistant to proteinases and toxic to neurons; it derives from the normal prion protein (PrPC), with which it can interact, thus inhibiting its superoxide dismutase‐like activity. The same properties are also shown by the abnormal isoform of the prion protein (PrPSc), and this similarity makes PrP106–126 an interesting model for the neurotoxic action of PrPSc. A role for copper in PrP106–126 aggregation and toxicity has recently been evidenced, and the interaction of terminal Lys, His and Met residues with the copper ion at neutral pH has been suggested. In order to shed more light on the complex‐formation equilibria of PrP106–126 with the copper ion, a thorough investigation has been carried out by means of several experimental techniques: potentiometry, solution calorimetry, VIS spectrophotometry, circular dichroism, EPR and NMR spectroscopy. A shorter and more soluble fragment—PrP106–113, which lacks the hydrophobic C‐terminal domain of PrP106–126 but contains all the potential donor groups—has also been considered for the sake of comparison. The involvement of terminal amino, imidazolic and amido nitrogens in complex formation has been confirmed, while no evidence was found for the interaction of side chains of Met and Lys residues with the copper ion. Solution structures for the main complexes are suggested.

Identification of a novel high affinity copper binding site in the APP(145–155) fragment of amyloid precursor protein

The copper(II) binding features of the APP(145-155) and APP(145-157) fragments of the amyloid precursor protein, Ac-Glu-Thr-His-Leu-His-Trp-His-Thr-Val-Ala-Lys-NH2 and Ac-Glu-Thr-His-Leu-His-Trp-His-Thr-Val-Ala-Lys-Glu-Thr-NH2 were studied by NMR spectroscopy and NMR findings were supported by UV-vis, CD and EPR spectra. Potentiometric measurements were performed only for the more soluble Ac-Glu-Thr-His-Leu-His-Trp-His-Thr-Val-Ala-Lys-Glu-Thr-NH2 peptide fragment. The following was shown: (i) the imidazole rings of all the three His residues are involved in metal coordination; (ii) metal binding induces ionisation of Leu-148 and His-149 amide nitrogens that complete the donor set to copper(II) in the species dominant at neutral pH; (iii) the unusual coordination scheme of the His-Xxx-His-Xxx-His consensus sequence justifies the high specificity for Cu(II) when compared to SOD-like or albumin-like peptides or even in amyloid Abeta fragments. The present findings may represent the key for interpreting the observed requirement of His residues conservation for the redox cycling between Cu(II) and Cu(I) by soluble APP.

Copper(I)-α-Synuclein Interaction: Structural Description of Two Independent and Competing Metal Binding Sites

The aggregation of α-synuclein (αS) is a critical step in the etiology of Parkinsons disease. Metal ions such as copper and iron have been shown to bind αS, enhancing its fibrillation rate in vitro. αS is also susceptible to copper-catalyzed oxidation that involves the reduction of Cu(II) to Cu(I) and the conversion of O(2) into reactive oxygen species. The mechanism of the reaction is highly selective and site-specific and involves interactions of the protein with both oxidation states of the copper ion. The reaction can induce oxidative modification of the protein, which generally leads to extensive protein oligomerization and precipitation. Cu(II) binding to αS has been extensively characterized, indicating the N terminus and His-50 as binding donor residues. In this study, we have investigated αS-Cu(I) interaction by means of NMR and circular dichroism analysis on the full-length protein (αS(1-140)) and on two, designed ad hoc, model peptides: αS(1-15) and αS(113-130). In order to identify and characterize the metal binding environment in full-length αS, in addition to Cu(I), we have also used Ag(I) as a probe for Cu(I) binding. Two distinct Cu(I)/Ag(I) binding domains with comparable affinities have been identified. The structural rearrangements induced by the metal ions and the metal coordination spheres of both sites have been extensively characterized.

NMR studies on Cu(II)–peptide complexes: exchange kinetics and determination of structures in solution

The interaction of copper(II) with histidine containing peptides has recently acquired renewed interest following the established link between abnormal protein behaviour in neurodegenerative processes and unpaired copper homeostasis. Five peptide sequences taken from the amyloid precursor protein and the prion protein were considered. Addition of paramagnetic Cu(II) ions to solutions of such peptides was not found to severely affect the appearance of NMR spectra, thus limiting the usual approach for structural determination. Exchange kinetics was shown to play a major role in determining the observed paramagnetic spin-lattice relaxation rates. Two independent methods were suggested for evaluating the exchange rates of His-containing peptides from the copper-coordination sphere and to calculate copper-proton distances. In such a way NMR was demonstrated to have the potential of providing detailed structures of the Cu(II)-peptide complexes in solution.

Exploring the reactions of β-amyloid (Aβ) peptide 1-28 with Al(III) and Fe(III) ions.

The reactions of human β-amyloid peptide 1-28 (Aβ28) with Al(III) and Fe(III) ions were investigated by (1)H NMR and electrospray ionization mass spectrometry (ESI-MS) under pH conditions close to physiological ones. (1)H NMR titrations, performed in the 5.3-8.0 pH range, revealed that no measurable amounts of Aβ28-Al(III) or Aβ28-Fe(III) adducts are formed; such metal adducts could not be obtained even by changing a number of experimental conditions, e.g., temperature, buffer, nature of the salt, etc. These observations were later confirmed by ESI-MS. It is thus demonstrated that Aβ28, at physiological pH, is not able to form binary complexes with Al(III) and Fe(III) ions of sufficient stability to compete with metal hydroxide precipitation. The biological implications of these findings are discussed in the frame of current literature.

The His-His sequence of the antimicrobial peptide demegen P-113 makes it very attractive ligand for Cu2+

Histatins are a family of histidine-rich, cationic peptides up to 38 amino acids long. As other antimicrobial peptides histatins exhibit in vitro activity against both bacteria and yeasts. A 12 amino acid amidated fragment of histatin 5, designated P-113 or demegen, has been identified as the smallest fragment retaining antimicrobial activity comparable to the parent compound. Demegen, AKRHHGYKRKFH, has three His and a N-terminal group known to participate in copper ion coordination. In this study potentiometric and spectroscopic (UV-vis, CD, EPR, NMR) measurements were used to evaluate the stability constants, stoichiometry and structures of Cu(2+) complexes with demegen P-113 and its analogues in aqueous solution. The main aim of this work was to understand the role of two adjacent histidine residues in metal ion binding. The comparison with results for modified ligands showed that two histydyl residues are basic for complex formation in the 4.5-7 pH range.