Electron Paramagnetic Resonance Study of Retention of Chromium(III), Chromate or Dichromate(VI) and Copper(II) ions by Thermotolerant Hansenula polymorpha

We report on CrIII and CrVI interaction with Hansenula polymorpha, a thermotolerant yeast, studied by means of EPR spectroscopy at room temperature. CrIII retention results in a reduced mobility of the ion, probably due to the formation of complexes immobilized on a polymeric matrix of cell. CrVI interaction produces stable, mobile CrV species, which can eventually partially decay to CrIII species. Different reducing agents in the cell may be responsible for this behaviour, especially on cell wall components or on cytoplasmic space. A comparison with a previous study on CuII ion retention is also made

Coordination features of prion protein domains

Many systemic and neurodegenerative disorders, collectively termed as “protein conformational diseases” are characterized by the accumulation of intracellular or extracellular protein aggregates. Abnormal metal-protein interactions have now been implicated in some of these degenerative disorders including Alzheimer’s disease, cataracts, Parkinson’s, Creutzfeldt-Jakob disease, etc.). Prion diseases provide a typical example in which the conversion of the normal folded α-helical PrPC isoform into a β-sheet rich conformation PrPSc, results in neurodegeneration. The physiological function of PrPC has not yet been identified, even though it is emerging that this highly conserved protein is important for a healthy brain. Increasing evidence indicates that PrPc is a copper-binding protein, and its localisation at pre and postsynaptic levels suggests an involvement in copper uptake and transmembrane signalling. Copper(II)-PrPC studies can be problematic due to the formation of PrPc insoluble species, therefore different protein fragments have been used to determine the metalprotein stoichiometry, the copper(II) affinity of different regions, the binding sites and the coordination features of the resulting metal complexes. Conflicting results have been reported, probably due to an oversimplification of metal complex speciation. A combined thermodynamic and spectroscopic approach has been recently used to characterize the coordination features of different prion domains. The results questioned the role of octarepeats present in the unstructured N-terminus region as the main binding site for copper(II), rather suggesting preferential metal coordination outside of the octarepeat region. Furthermore, comparative analysis of the human and chicken PrP copper(II) binding sites may provide new insights into the prion protein structure-function relationship and the conversion process of PrP

Copper(II) complexes with peptide fragments encompassing the sequence 122-130 of human doppel protein

Copper(II) complexes of the peptide fragment (Dpl122–130) encompassing the sequence 122–130 of human doppel protein were characterized by potentiometric, UV–Visible, CD and EPR spectroscopic methods. An analogous peptide, in which the aspartate residue was substituted by an asparagine amino acid, was synthesized in order to provide evidence on the possible role of carboxylate group in copper(II) coordination. It was found that the carboxylic group is directly involved in copper(II) coordination at acidic pH, forming the CuLH2 species with Dpl122–130. This copper(II) complex displayed EPR parameters very similar to those of the analogous complex with the whole doppel protein. At pH higher than 7, the complexes showed magnetic parameters similar to those of the major species of protein formed in the pH range 7–8, with the metal coordination environment consisting of one imidazole and three amide nitrogen atoms. The comparison of Cu-Dpl122–130 binding constant values with those of the prion peptide fragments (PrP106–114), showed that doppel peptide had a higher metal binding affinity at acidic pH whereas the prion peptide fragment binds the metal tightly at physiological pH

EPR Study on Vanadyl and Vanadate Ion Retention by a Thermotolerant Yeast

The retention of vanadyl(IV) and vanadate(V) ions by Hansenula polymorpha, a thermotolerant yeast, has been studied by EPR spectroscopy. Vanadyl ions were retained as mobile complexes associated with relatively low molecular weight ligands as well as immobilized complexes formed with high molecular weight residues, the donor atoms of which are probably oxygens or deprotonated hydroxyls. The distribution of vanadyl ions between mobile and immobilized complex species is a function of the metal concentration as well as of the contact time. Vanadate ions were reduced by cellular components to vanadyl ions

Water-Soluble Calixarenes as Synthetic Receptors. Remarkable Influence of Stereochemistry on the Coordination Properties of Two New Conformational Isomers of a Calix[4]arene Tetracarboxylate

Two new water-soluble calix[4]arene tetracarboxylic acids in the cone and partial cone blocked conformations were synthesized. Conformational assignment was made on the basis of their Ar-CH2-Ar H-1 and C-13 NMR patterns. Their proton and Cu(II) coordination properties were studied by several techniques to evaluate the role played by the different stereochemistry of the two isomers. The combination of potentiometric, calorimetric and spectroscopic (NMR, ESR and UV-visible) results gives a detailed and coherent description of the complex formation processes. The NMR analysis of the partial-cone isomer highlights the role played by solvophobic forces on the degree of inclusion of the carboxylic group inside the apolar calixarene cavity. The results show that the protonation of the cone isomer significantly differs from that of the partial cone isomer, indicating that hydrogen bonding, involving oxygen atoms of two adjacent carboxylate groups, occurs only in the cone isomer. Also the complex formation with copper(II) is strongly affected by the different conformations of the two calixarenes which produce not only species with different stabilities but also different species

Copper(II) complexes with chicken prion repeats: influence of proline and tyrosine residues on the coordination features

The prion protein (PrPc) is a copper-binding glycoprotein that can misfold into a beta-sheet-rich and pathogenic isoform (PrPSc) leading to prion diseases. The first non-mammalian PrPc was identified in chicken and it was found to keep many structural motifs present in mammalian PrPc, despite the low sequence identity (approximately 40%) between the two primary structures. The present paper describes the synthesis and the coordination properties of some hexapeptide fragments (namely, PHNPGY, HNPGYP and NPGYPH) as well as a bishexapeptide (PHNPGYPHNPGY), which encompasses two hexarepeats. The copper(II) complexes were characterized by means of potentiometric, UV-vis, circular dichroism and electron paramagnetic resonance techniques. We also report the synthesis of three hexapeptides (PHNPGF, HNPGFP and NPGFPH), in which one tyrosine was replaced by phenylalanine as well as two bishexapeptides in which either one (PHNPGFPHNPGY and PHNPGYPHNPGF), or two tyrosines were replaced by phenylalanine, in order to check whether tyrosine was involved in copper(II) binding. Overall, the results indicate that the major copper(II) species formed by the chicken PrP dodecapeptides are stabler than the analogous species reported for the peptide fragments containing two octarepeat peptides from the mammalian prion protein. It is concluded that the presence of four prolyl residues, that are break points in copper coordination, induces the metal-assisted formation of macrochelates as well as the formation of binuclear species. Furthermore, it has been shown that the phenolic group is directly involved in the formation of copper binuclear species

A doppel α-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

Synthetic Models for Biological Trinuclear Copper Clusters. Trinuclear and Binuclear Complexes Derived from an Octadentate Tetraamine-Tetrabenzimidazole Ligand

Synthetic Models for Biological Trinuclear Copper Clusters. Trinuclear and Binuclear Complexes Derived from an Octadentate Tetraamine-Tetrabenzimidazole Ligan

METAL BINDING TO PRION PROTEIN

METAL BINDING TO PRION PROTEI