Site selection in tandem arrays of metal-binding domains

The metal binding properties of peptides corresponding to metal-binding sites spanning regions that normally function as linkers in tandem arrays of metal-binding domain-containing proteins were examined. For a peptide with two His residues from one TFIIIA-like zinc finger domain, a canonical TFIIIA-like linker, and two Cys residues from an adjacent zinc domain, the dissociation constant for the 1:1 peptide to cobalt(II) was found to be 15 +/- 10 microM, compared with 60 nM for the corresponding zinc finger domains themselves. Peptides overlapping two sets of metal-binding domains from human TRAF (tumor necrosis factor receptor-associated factor) proteins were examined. In one case, the affinity of the presumed metal-binding domain and that for the linker region were comparable, while in the second case, the affinity of the linker peptide was higher than that for the corresponding presumed metal-binding domain peptide. These studies revealed that cobalt(II) affinities in the micromolar range can occur even for peptides that do not correspond to natural zinc-binding domains and that the degree of distinction between authentic metal-binding domains and the corresponding linker-spanning peptides may be modest, at least for single domain peptide models.     

Characterisation by immobilised metal ion affinity chromatographic procedures of the binding behaviour of several synthetic peptides designed to have high affinity for Cu(II) ions.

In this investigation, several peptides containing an increasing number of histidine residues have been designed and synthesised. The peptides involved repeat units of either the pentameric EAEHA or the tetrameric HLLH sequence motifs. Adsorption isotherms for these synthetic peptides and hexahistidine (hexa-His) as a control substance were measured under batch equilibrium binding conditions with an immobilised Cu(II)-iminodiacetic acid (IDA) sorbent. The experimental data were analysed in terms of Langmuirean binding behaviour. In common with previous studies with synthetic peptides, these investigations have demonstrate that the sequential organisation of the histidine side chains in these peptides can affect the selectivity of the coordination interactions with borderline metal ions in immobilised metal ion affinity chromatographic systems. The results also confirm that peptides selected on the basis of their potential to form amphipathic secondary structures with their histidine residues presented on one face of the molecule can exhibit equivalent or higher affinity constants towards copper ions than hexa-His, although they contain fewer histidine residues. These findings are thus relevant to the selection of peptides produced inter alia by combinatorial synthetic procedures to have enhanced binding properties for Cu(II) or Ni(II) ions, or intended for use as peptide tags in the fusion handle approach for the affinity chromatographic purification of recombinant proteins.     

Design of a selective metal ion switch for self-assembly of peptide-based fibrils

The self-assembling peptide TZ1H, a structural variant of the trimeric isoleucine zipper GCN4-pII, contains histidine residues at core d-positions of alternate heptads that define three trigonal coordination sites within the coiled-coil trimer. Circular dichroism spectropolarimetry indicated that peptide TZ1H undergoes a random coil to alpha-helical conformational change upon binding of 1 equiv of silver(I) ion, but not zinc(II), copper(II), or nickel(II) ions. Isothermal titration calorimetry provided evidence for a single binding-site model in which each peptide contributes one net silver(I) coordination site, in agreement with the proposed structural model. Transmission electron microscopy revealed that TZ1H self-assembles into long aspect ratio helical fibers in the presence of silver(I) ion. These results demonstrate that the rational design of selective metal ion binding sites within de novo designed peptides represents a promising approach to the controlled fabrication of nanoscale, self-assembled materials.     

Metal-binding properties of the peptide APP170-188: a model of the ZnII-binding site of amyloid precursor protein (APP)

Amyloid precursor protein (APP) plays a key role in Alzheimer's disease (AD), although the function of this membrane protein is still unclear. Metal ions are implicated in AD and they also interact with APP. APP possesses a strong ZnII binding site, which is evolutionary conserved. In this paper a synthetic peptide, APP170-188, with a sequence corresponding to the conserved ZnII-binding domain of APP, was synthesised and its metal-binding properties analysed. Titration experiments pointed to the binding of a stoichiometric amount of divalent ions. Further studies indicated that the binding of divalent metals like ZnII, CdII and CoII induces the dimerisation of the peptide. This dimer contains a dinuclear cluster in which the two divalent metals are bridged by two thiolate ligands from cysteine residues. The other two ligands of the tetrahedral coordination sites of each metal ion are terminal thiolate ligands. This structure was supported by the following arguments. The complex formed with CoII presents the characteristic features for tetrahedral tetrathiolate coordination in its UV-visible spectrum. The sequence of APP170-188 contains only three cysteine residues, which is incompatible with a monomeric CoII-APP170-188 complex. EPR measurements of the complex with one equivalent of CoII show almost no signal at 4 K, which is compatible with an antiferromagnetic spin-coupling of the metal ions in a cluster structure. Size-exclusion chromatography indicated that the elution time for the complexes with ZnII and CdII corresponds to the expected molecular weight of a dimer. The circular dichroism (CD) spectrum of the complex with one equivalent of CdII shows a band at 265 nm+, and an ellipticity similar to those observed for similar CdII-thiolate clusters. Possible biological implications of the ZnII binding site and the metal-induced dimerisation are discussed.     

A cyclodecapeptide ligand to vitamin B12

Libraries of cyclic decapeptides were screened with vitamin B(12) derivatives to give cyclic peptide ligands incorporating histidine and cysteine as coordinating residues and negatively charged amino acids. Two hits, cyclo-(HisAspGluProGlyIleAlaThrProdGln) and cyclo-(ValAspGluProGlyGluAspCysProdGln) were resynthesized in good yields for solution experiments. The peptides bind aquocobalamin with coordination of His or Cys to the cobalt with high affinities (K(a) approximately 10(5) M(-1)). Additional interactions between the peptide side chains and the vitamin B(12) corrin moiety were determined by studying the (1)H NMR solution structure. The cyclopeptide-cobalamin complex with the histidine residue showed enhanced stability towards cyanide exchange, demonstrating the shielding effect of the ligand on the metal center.     

Gas-phase investigation of Pd(II)-alanine complexes with small native and derivatized peptides containing histidine

We report here the generation of gas-phase complexes containing Pd(II), a ligand (deprotonated alanine, A-), and/or N-terminus derivatized peptides containing histidine as one of the amino acids. The species were produced by electrospray ionization, and their gas-phase reactions were investigated using ion-trap tandem mass spectrometry. Pd(II) forms a stable diaqua complex in the gas phase of the formula, [Pd(A-) (H(2)O)(2)]+, (where A- = deprotonated alanine) along with ternary complexes containing A- and peptide. The collision-induced dissociation (CID) patterns of the binary and ternary complexes were investigated, and the dissociation patterns for the ternary complexes suggest that: (a) the imidazole ring of the histidine side group may be the intrinsic binding site of the metal ion, and (b) the peptides fragment primarily by cleavage of the amide bond to the C-terminal side of the histidine residues. These observations are in accord with previous solution-state studies in which Pd(II) was shown to cause hydrolysis of an amide bond of a peptide at the same position.     

Spectroscopic identification of different types of copper centers generated in synthetic four-helix bundle proteins

Using a combined rational-combinatorial approach, stable copper binding sites were implemented in template-assembled synthetic four-helix bundle proteins constructed by three different helices with only 16 amino acid residues. These peptides include two histidines and one cysteine at positions appropriate for coordinating a copper ion. Sequence variations of the helices were made in the second coordination shell or even more remote from the copper binding site (i) to increase the overall stability of the metalloproteins and (ii) to fine-tune the structure and properties of the copper center. As a result, ca. 90% of the 180 proteins that were synthesized were capable to bind copper with a substantially higher specificity than those obtained in the first design cycle (Schnepf, R.; Horth, P.; Bill, E.; Wieghardt, K.; Hildebrandt, P.; Haehnel, W. J. Am. Chem. Soc. 2001, 123, 2186-2195). Furthermore, the stabilities of the copper protein complexes were increased by up to 2 orders of magnitude and thus allowed a UV-vis absorption, resonance Raman, electron paramagnetic resonance, and (magnetic) circular dichroism spectroscopic identification and characterization of three different types of copper binding sites. It could be shown that particularly steric perturbations in the vicinity of the His(2)Cys ligand set control the formation of either a tetragonal (type II) or a tetrahedral (type I) copper binding site. With the introduction of two methionine residues above the histidine ligands, a mixed-valent dinuclear copper binding site was generated with spectroscopic properties that are very similar to those of Cu(A) sites in natural proteins. The results of the present study demonstrate for the first time that structurally different metal binding sites can be formed and stabilized in four-helix bundle proteins.     

Promoting self-assembly of collagen-related peptides into various higher-order structures by metal-histidine coordination

Collagen is an important and widely used biomaterial and therapeutic. The construction of large-scale collagen structures via the self-assembly of small collagen-related peptides has been extensively studied in the past decade. Here, we report a highly effective and simple means to assemble small synthetic collagen-related peptides into various higher-order structures by utilizing metal-histidine coordination. In this work, two short collagen-related peptides in which histidine residues were incorporated as metal binding sites were designed and chemically synthesized: HG(PPG)(9)GH (X9) and HG(PPG)(4)(PHG)(PPG)(4)GH (PHG). Circular dichroism measurements indicated that these two peptides form only marginally stable collagen triple helices but that their stability can be increased upon the addition of metal ions. Dynamic light scattering analyses, turbidity measurements, TEM, and SEM results demonstrated the metal ion-dependent self-assembly of X9 and PHG into supramolecular structures ranging from various nanofibrils to microscale spherical, laminated, and granulated assemblies. The topology and size of these higher-order structures depends both on the metal ion identity and the location of the binding sites. Most intriguingly, the assembled fibrils show similar D-periodicity to that of natural collagen. Our results demonstrate that metal-histidine coordination can serve as an effective force to induce the self-assembly of unstable collagen-related peptides into higher-order structures.     

Contribution of individual zinc ligands to metal binding and peptide folding of zinc finger peptides

Little is known about the contribution of individual zinc-ligating amino acid residues for coupling between zinc binding and protein folding in zinc finger domains. To understand such roles of each zinc ligand, four zinc finger mutant peptides corresponding to the second zinc finger domain of Sp1 were synthesized. In the mutant peptides, glycine was substituted for one of four zinc ligands. Their metal binding and folding properties were spectroscopically characterized and compared to those of the native zinc finger peptide. In particular, the electronic charge-transfer and d-d bands of the Co(II)-substituted peptide complexes were used to examine the metal coordination number and geometry. Fluorescence emission studies revealed that the mutant peptides are capable of binding zinc despite removing one ligand. Circular dichroism results clearly showed the induction of an alpha-helix by zinc binding. In addition, the structures of certain mutant zinc finger peptides were simulated by molecular dynamics calculation. The information indicates that His23 and the hydrophobic core formed between the alpha-helix and the beta-sheet play an essential role in alpha-helix induction. This report demonstrates that each ligand does not contribute equally to alpha-helix formation and coordination geometry in the zinc finger peptide.     

Products of Cu(II)-catalyzed oxidation of alpha-synuclein fragments containing M1-D2 and H50 residues in the presence of hydrogen peroxide

Metal-catalyzed oxidation (MCO) of proteins is mainly a site-specific process in which one or a few amino acids at metal-binding sites on the protein are preferentially oxidized. The oxidation of proteins by MCO can lead to oxidation of amino acid residue side chains, cleavage of the peptide bonds and formation of covalent protein-protein cross-linked derivatives. In an attempt to elucidate the products of the copper(II)-catalyzed oxidation of the 29-56, M29-D30-56 and Ac-M29-D30-56 fragments of alpha-synuclein, high performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) methods and Cu(II)/hydrogen peroxide as a model oxidizing system were employed. The peptide solution (0.50 mM) was incubated at 37 degrees C for 24 h with metal : peptide : hydrogen peroxide 1 : 1 : 4 molar ratio in phosphate buffer, pH 7.4. Oxidation targets for all studied peptides are the histidine residues coordinated to the metal ions. For the M29-D30-56 and Ac-M29-D30-56 peptides the oxidation of the methionine residue to methionine sulfoxide and sulfone is observed. The cleavage of the peptide bond M29-D30 for the M29-D30-56 peptide was detected as metal binding residues. The fragmentations of the M29-D30-56 peptide near the Lys residues were observed supporting the participation of this (Lys) residue in the coordination of the copper(II) ions.     

Copper(II)‐Mediated Self‐Assembly of Hairpin Peptides and Templated Synthesis of CuS Nanowires

The self‐assembly of peptides and proteins under well‐controlled conditions underlies important nanostructuring processes that could be harnessed in practical applications. Herein, the synthesis of a new hairpin peptide containing four histidine residues is reported and the self‐assembly process mediated by metal ions is explored. The work involves the combined use of circular dichroism, NMR spectroscopy, UV/Vis spectroscopy, AFM, and TEM to follow the structural and morphological details of the metal‐coordination‐mediated folding and self‐assembly of the peptide. The results indicate that by forming a tetragonal coordination geometry with four histidine residues, copper(II) ions selectively trigger the peptide to fold and then self‐assemble into nanofibrils. Furthermore, the copper(II)‐bound nanofibrils template the synthesis of CuS nanowires, which display a near‐infrared laser‐induced thermal effect.     

Thiocyanate hydrolase is a cobalt-containing metalloenzyme with a cysteine-sulfinic acid ligand

Thiocyanate hydrolase (SCNase) purified from Thiobacillus thioparus THI115 hydrolyzes thiocyanate to carbonyl sulfide and ammonia. DNA sequences of the cloned genes revealed the close relation of SCNase to nitrile hydratase (NHase). The consensus sequences for coordination of the metal ion found in NHases were also conserved in the gamma subunit of SCNase. Here, we showed that the SCNase contained one cobalt atom per alphabetagamma heterotrimer. UV-vis absorption spectrum suggested that the cobalt exists as a non-corrin ion. Reduced SCNase showed an ESR signal characteristic of low-spin Co2+, which closely resembled that of the Co-type NHases. Mass spectrometry for the peptide fragment containing the metal-binding motif of the SCNase gamma subunit indicated that the cysteine residue at position 131 was post-translationally oxidized to a cysteine-sulfinic acid. From these results, we concluded that SCNases and NHases form a novel non-corrin and/or non-heme protein family having post-translationally modified cysteine ligands.     

Substantial contribution of the two imidazole rings of the His13-His14 dyad to Cu(II) binding in amyloid-β(1-16) at physiological pH and its significance

The interaction of amyloid-β (Aβ) peptide with Cu(II) appears to play an important role in the etiology of Alzheimer's disease. At physiological pH, the Cu(II) coordination in Aβ is heterogeneous, and there exist at least two binding modes in which Cu(II) is coordinated by histidine residues. Electron spin resonance studies have revealed a picture of the Cu(II) binding at a higher or lower pH, where only one of the two binding modes is almost exclusively present. We describe a procedure to directly examine the coordination of Cu(II) to each histidine residue in the dominant binding mode at physiological pH. We use nonlabeled and residue-specifically (15)N-labeled Aβ(1-16). For quantitative analysis, the intensities of three-pulse electron spin-echo envelope modulation (ESEEM) spectra are analyzed. Spectral simulations show that ESEEM intensities provide information about the contribution of each histidine residue. Indeed, the ESEEM experiments at pH 6.0 confirm the dominant contribution of His6 to the Cu(II) coordination as expected from the work of other researchers. Interestingly, however, the ESEEM data obtained at pH 7.4 reveal that the contributions of the three residues to the Cu(II) coordination are in the order of His14 ≈ His6 > His13 in the dominant binding mode. The order indicates a significant contribution from the simultaneous coordination by His13 and His14 at physiological pH, which has been underappreciated. These findings are supported by hyperfine sublevel correlation spectroscopy experiments. The simultaneous coordination by the two adjacent residues is likely to be present in a non-β-sheet structure. The coexistence of different secondary structures is possibly the molecular origin for the formation of amorphous aggregates rather than fibrils at relatively high concentrations of Cu(II). Through our approach, precise and useful information about Cu(II) binding in Aβ(1-16) at physiological pH is obtained without any side-chain modification, amino acid residue replacement, or pH change, each of which might lead to an alteration in the peptide structure or the coordination environment.     

Molecular Simulations of Human and Mouse Aβ1–16 at Different pH Values: Structural Characteristics toward Understanding Cu2+‐Coordinated Amyloid Beta Spheres

As the main sequence responsible for metal ion coordination in the amyloid beta (Aβ) peptide, Aβ_1–16 plays a key role in the understanding of the aggregation of Aβ induced by Cu²⁺ ions. There is no consensus on the nature of the coordination sphere of the Cu²⁺–Aβ complex so far due to the amorphous conformation of the Aβ_1–16 peptide itself and the pH dependence of Cu²⁺–Aβ coordination. The simulation reported here reveals that human Aβ_1–16 monomer has a U‐shape morphology, which is preserved at any pH. This morphology accommodates Cu²⁺ ions with several binding sites and is also the basis for establishing a center‐distance statistical method (CDSM). Based on this CDSM, specific histidine residues for a Cu²⁺‐coordinated sphere are identified and the corresponding accurate pH range is established, indicating that the CDSM can be used as a reference to predict the potential coordination sites of metal ions in other amorphous peptides. By contrast, mouse Aβ_1–16 monomer has a more open and random morphology than human Aβ_1–16 due to the differences of three sequence positions. These mutations not only reduce the number of binding sites required by a stable Cu²⁺‐binding sphere but also diminish the capacity to generate salt bridges compared to the human peptide. These observations offer insights into the roles of three residues that differ in the mouse Aβ_1–16 and perhaps into the reasons mice seldom develop Alzheimer's disease.     

The effect of point mutations on copper(II) complexes with peptide fragments encompassing the 106–114 region of human prion protein

Abstract  

The tetrapeptides Ac-SKHM-NH₂, Ac-TKHM-NH₂, Ac-MKHS-NH₂, Ac-S(OMe)KHM-NH₂, and Ac-MKHS(OMe)-NH₂ and the nonapeptides Ac-KTNSKHMAG-NH₂ and Ac-KTNMKHSAG-NH₂ were synthesized and their copper(II) complexes were studied by potentiometric, UV–Vis, circular dichroism (CD), and electron paramagnetic resonance (EPR) spectroscopic methods. These peptides mimic the 109–112 and 106–114 residues of the sequence of human prion protein. The imidazole-N donor atoms of histidyl residues were found to be the primary metal binding sites of all peptide fragments. This binding mode provides a good possibility for the cooperative deprotonation and metal ion coordination of two amide functions preceding histidine. The (N_im,N⁻,N⁻)-bonded species predominate in the pH range 5.5–7.0 and the free coordination sites of these species make possible the metal binding of weakly coordinating side chains. The comparison of the potentiometric and spectroscopic results revealed the stabilizing role of the oxygen donors of seryl, threonyl, or methoxyseryl residues of Ac-SKHM-NH₂, Ac-TKHM-NH₂, Ac-S(OMe)KHM-NH₂, and Ac-KTNSKHMAG-NH₂ containing the mutations in position 109. These interactions were, however, not observed in the peptides containing the specific amino acids in other locations of the peptide sequence.     

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.     

Design of thiolate rich metal binding sites within a peptidic framework

A de novo protein design strategy provides a powerful tool to elucidate how heavy metals interact with proteins.Cysteine derivatives of the TRI peptide family (Ac-G(LKALEEK)4G-NH2) have been shown to bind heavy metals in an unusual trigonal geometry. Our present objective was to design binding sites in R-helical scaffolds that are able to form higher coordination number complexes with Cd(II) and Hg(II). Herein, we evaluate the binding of Cd(II) and Hg(II) to double cysteine substituted TRI peptides lacking intervening leucines between sulfurs in the heptads. We compare a -Cysd-X-X-X-Cysa- binding motif found in TRIL12CL16C to the more common -Cysa-X-X-Cysd- sequence of native proteins found in TRIL9CL12C. Compared to TRI, these substitutions destabilize the helical aggregates,leading to mixtures of two- and three-stranded bundles. The three-stranded coiled coils are stabilized by the addition of metals. TRIL9CL12C forms distorted tetrahedral complexes with both Cd(II) and Hg(II), as supported by UV-vis,CD, 113Cd NMR, 199Hg NMR and 111mCd PAC spectroscopy. Additionally, these signatures are very similar to those found for heavy metal substituted rubredoxin. These results suggest that in terms of Hg(II) binding, TRIL9CL12Ccan be considered as a good mimic of the metallochaperone HAH1, that has previously been shown to form protein dimers. TRIL12CL16C has limited ability to generate homoleptic tetrahedral complexes (Cd(SR)42-). These type of complexes were identified only for Hg(II). However, the spectroscopic signatures suggest a different geometry around the metal ion, demonstrating that effective metal sequestration into the hydrophobic interior of the bundle requires more than simply adding two sulfur residues in adjacent layers of the peptide core. Thus, proper design of metal binding sites must also consider the orientation of cysteine sidechains in a vs d positions of the heptads.     

Structural characterization of metallopeptides designed as scaffolds for the stabilization of nickel(II)-Fe(4)S(4) bridged assemblies by X-ray absorption spectroscopy

In earlier work, de novo designed peptides with a helix-loop-helix motif and 63 residues have been synthesized as potential scaffolds for stabilization of the [Ni(II)-X-Fe(4)S(4)] bridged assembly that is the spectroscopically deduced structure of the A-Cluster in clostridial carbon monoxide dehydrogenase. The 63mers contain a consensus tricysteinyl ferredoxin domain in the loop for binding an Fe(4)S(4) cluster and Cys and His residues proximate to the loop for binding Ni(II), with one Cys residue designed as the bridge X. The metallopeptides HC(4)H(2)-[Fe(4)S(4)]-Ni and HC(5)H-[Fe(4)S(4)]-M, containing three His and one Cys residue for Ni(II) coordination and two His and two Cys residues for binding M = Ni(II) and Co(II), have been examined by Fe-, Ni-, and Co-K edge spectroscopy and EXAFS. All peptides bind an [Fe(4)S(4)](2+) cubane-type cluster. Interpretation of the Ni and Co data is complicated by the presence of a minority population of six-coordinate species with low Z ligands, designated for simplicity as [M(OH(2))(6)](2+). Best fits of the data were obtained with ca. 20% [M(OH(2))(6)](2+) and ca. 80% M(II) with mixed N/S coordination. The collective XAS results for HC(4)H(2)-[Fe(4)S(4)]-Ni and HC(5)H-[Fe(4)S(4)]-M demonstrate the presence of an Fe(4)S(4) cluster and support the existence of the distorted square-planar coordination units [Ni(II)(S.Cys)(N.His)(3)] and [Ni(II)(S.Cys)(2)(N.His)(2)] in the HC(4)H(2) and HC(5)H metallopeptides, respectively. In the HC(5)H metallopeptide, tetrahedral [Co(II)(S.Cys)(2)(N.His)(2)] is present. We conclude that the designed scaffolded binding sites, including Ni-(mu(2)-S.Cys)-Fe bridges, have been achieved. This is the first XAS study of a de novo designed metallopeptide intended to stabilize a bridged biological assembly, and one of a few XAS analyses of metal derivatives of designed peptides. The scaffolding concept should be extendable to other bridged metal assemblies.     

The metal loading ability of beta-amyloid N-terminus: a combined potentiometric and spectroscopic study of copper(II) complexes with beta-amyloid(1-16), its short or mutated peptide fragments, and its polyethylene glycol (PEG)-ylated analogue

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.     

Interactions between Ni ions and tetrapeptides containing (Asp/Lys)HisGly(l/d)His sequence

A series of linear tetrapeptides containing two histidyl residues in positions 2 and 4 with different chirality: DHGH, DHG(d-His), KHGH, KHG(d-His), Ac-DHGH-NH₂, Ac-DHG(d-His)-NH₂, Ac-KHGH-NH₂, and Ac-KHG(d-His)-NH₂ were synthesized, characterized and their binding properties towards Ni²⁺ were investigated. To establish the stoichiometry and the stability of the resulting Ni²⁺ complexes, potentiometric titrations were carried out. The coordination mode of the complexes formed was investigated by performing extensive spectroscopic analyses (UV–Vis, CD) in strict correlation with the potentiometric results. The effects of the nature of the first amino acid (Lys versus Asp) and of the N-terminal amino group acetylation were determined. A careful comparison of the Ni²⁺ coordination abilities of the linear peptides provides a specific insight into the impact of the chirality of the C-terminal histidine residue (His⁴) on the metal binding properties.