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We designed and synthesized a novel near-infrared (NIR) mitochondria-targeted fluorescent probe RQNA for the specific detection of mitochondrial Cu2+ because mitochondria are important reservoirs of intracellular copper. For the preparation of this probe, a novel π-extended fluorescent xanthene dye RQN was firstly synthesized via an intramolecular nucleophilic substitution of aromatic hydrogen (SNArH) strategy. Then, probe RQNA was prepared by the reaction of RQN and hydrazine hydrate, followed by treatment with acetone. RQNA exhibited selectivity, sensitivity (22 nM), and fast response time (20 s) for the detection of Cu2+via a specific Cu2+-triggered ring-opening and hydrolysis cascade reaction. RQNA is cell-membrane permeable and mitochondria-targetable, and can be used for monitoring mitochondrial Cu2+ in living cells.

Identifying and detecting similar target cations through combining "turn on" and "turn off" fluorescence mechanism is effective and challenging. Now a new colorimetric and ON-OFF-ON fluorescent probe N'-((7-(diethylamino)-2-oxo-2H-chromen-3-yl)methylene)-3-hydroxy-2-naphthohydrazide (L) was reported, which could detect Cu 2+ and Co 2+ in phosphate buffered CH 3 CH 2 OH-H 2 O solvent system. With the assistance of glutathione and pH adjustment, a unique ON-OFF-ON fluorescence detection strategy could be achieved for distinguishing Cu 2+ and Co 2+ . The emission of probe could recover from the L-Cu 2+ and L-Co 2+ system by addition of GSH or adjusting pH value to 4, respectively, which is due to the abolishment of paramagnetic Cu 2+ /Co 2+ . Based on fluorescence titration experiments, the limit of detection was determined as 3.84 × 10 -9  M and 4.55 × 10 -9  M for Cu 2+ and Co 2+ , respectively. Meanwhile, the detection limit reached 6.21 × 10 -8  M for Cu 2+ and 6.96 × 10 -8  M for Co 2+ according to absorbance signal output. Fast recognition of Cu 2+ /Co 2+ can be achieved by obvious color changes from green to colorless under UV light, as well as from yellow to orange-red in room light. The binding mode of L toward Cu 2+ and Co 2+ have been systematically studied by Job's plot analysis, ESI-MS, IR and density functional theory calculations. Most strikingly, further practical applications of the probe L in fluorescence imaging were investigated in MCF-7 cells and zebrafish due to its low cytotoxicity and good optical properties, suggesting that L could serve as a fluorescent sensor for tracking Cu 2+ and Co 2+ in vivo., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Oxidative aggregation of γ-crystallins induced by copper in aged lens increases the lens opacity and causes cataract formation. Therefore, chelation of free Cu 2+ by small molecules can inhibit metal-mediated aggregation of γ-crystallin. In this work, the inhibition potency of several naturally occurring flavonoid compounds was studied against aggregation of human γD-crystallin (HGD) mediated by copper ions. Among them, rutin demonstrated ~20% inhibition of HGD aggregation induced by Cu 2+ through its metal chelation ability. Not only that, the chaperone activity of lens chaperone, human αA-crystallin (HAA) was found to be enhanced in the presence of rutin. Subsequently, the molecular interactions between HAA and rutin were investigated using fluorescence and CD spectroscopy to understand the molecular basis of the chaperone activity enhancement by rutin. Quenching of HAA fluorescence by rutin with a quenching constant in the order of ~10 5  M -1 depicts a complexation between them. Entropy driven process of complexation between HAA and rutin suggests significant involvement of hydrophobic interactions. Fluorescence resonance energy transfer between protein and ligand can occur at a distance of 2.73 nm. Synchronous fluorescence and circular dichroism spectroscopy revealed that protein-ligand interaction does not cause any notable conformational changes in HAA. Experimental observations have been well substantiated by docking., (Copyright © 2019 Elsevier B.V. All rights reserved.)

We have constructed a new coumarin based fluorescence probe BENZPYR with ICT character through condensation of N, N-diethylamino-3-acetyl coumarin with 2-hydrazinobenzothiazole. The absorbance and fluorescence spectral characteristics of BENZPYR revealed that the chemosensor can specifically detect for Cu 2+ ions over other different metal ions and the lowest limit of detection was found in nano molar range. The turn off sensor of BENZPYR is related to chelation enhanced quenching (CHEQ) and intramolecular charge transfer (ICT) processes were serve as excellent fluorescent detection of Cu 2+ ions in DMF medium. Fluorescence microscopy experiments revealed that probe BENZPYR may have application as a fluorophore to detect the Cu 2+ in living cells. The simulated DFT analysis of electronic and structural properties and also UV-vis absorption spectra are in well accordance with the experimental UV-vis absorption spectra., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Copper is an important element indispensable for human life and health. Many copper-determining probes have been created for exploring its functional behavior in various cell types but few of them contains both fluorescent and colorimetric characters. In the present study, we developed a set of copper probes by synthesizing several novel thiophene-based Schiff bases in order to make a suitable sensor for quantifying and imaging copper in living cells. We find that the ligand FS-1 has a splendid selectivity and affinity toward Cu 2+ among the common divalent metal ions. Living cell imaging show that FS-1 has a robust and repetitive fluorescence response in the presence of Cu 2+ only in the cytosolic space of Hepg2 cell and not in the other cells examined. These data suggest that we have developed a new copper probe that can be used as a Cu 2+ fluorescent and colorimetric sensor for in vivo and in vitro copper studies., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Mycobacterium leprae uptakes various bivalent metal ions via different transporters in host species. Uptake of Cu 2+ and Zn 2+ are essential for generation of superoxide dismutases and catalases, which provide defense against reactive oxygen species mediated death of this pathogen in macrophages. Furthermore, it has also been noticed that levels of different bivalent metal ions (Ca 2+ , Mg 2+ , Cu 2+ and Zn 2+ ) in blood serum are altered in leprotic patients. Mycobacterium leprae HSP18 is an immunodominant antigen which helps in growth and survival of Mycobacterium leprae in host species. A possible link can exist between HSP18 and aberration of bivalent metal ion homeostasis. Therefore, we investigated the interaction of these four bivalent metal ions with HSP18 and found that the protein only interacts with Zn 2+ and Cu 2+ . Such association process is reversible and moderately high affinity in nature with unit binding stoichiometry. Theoretical studies revealed that the most probable site for Zn 2+ -binding lies in the N-terminal domain; While, the same for Cu 2+ -binding lies in the "α-crystallin domain" of HSP18. Binding of Zn 2+ /Cu 2+ to HSP18 brings about subtle changes in the secondary and tertiary structure of HSP18 but are distinctly opposite in nature. While Zn 2+ causes oligomeric association, Cu 2+ leads to oligomeric dissociation of HSP18. Structural stability, surface hydrophobicity and chaperone activity of HSP18 are enhanced on Zn 2+ binding, while all of them are reduced upon Cu 2+ binding. Altogether, metal ions binding to HSP18 regulate its function which may have far reaching effect on the survival and pathogenicity of Mycobacterium leprae in host species., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The mechanism of Aβ aggregation in the absence of metal ions is well established, yet the role that Zn 2+ and Cu 2+ , the two most studied metal ions, released during neurotransmission, paly in promoting Aβ aggregation in the vicinity of neuronal synapses remains elusive. Here we report the kinetics of Zn 2+ binding to Aβ and Zn 2+ /Cu 2+ binding to Aβ-Cu to form ternary complexes under near physiological conditions (nM Aβ, μM metal ions). We find that these reactions are several orders of magnitude slower than Cu 2+ binding to Aβ. Coupled reaction-diffusion simulations of the interactions of synaptically released metal ions with Aβ show that up to a third of Aβ is Cu 2+ -bound under repetitive metal ion release, while any other Aβ-metal complexes (including Aβ-Zn) are insignificant. We therefore conclude that Zn 2+ is unlikely to play an important role in the very early stages (i.e., dimer formation) of Aβ aggregation, contrary to a widely held view in the subject. We propose that targeting the specific interactions between Cu 2+ and Aβ may be a viable option in drug development efforts for early stages of AD.

Fluorescence-based biosensors have shown themselves to be a powerful tool for the study of a variety of chemical species in biological systems, notably including metal ions. This chapter provides an overview of several important issues in using such sensors to study metallobiochemistry. These issues include selectivity for the analyte over potential interferents, including those that do not themselves induce a signal, the different forms in which metal ions are found (speciation), the utility of metal ion buffers, and the importance of kinetics in studying metal ion binding reactions. Finally, the chapter briefly discusses some of the issues in understanding whole-organism distribution of metal ions and its control., (© 2017 Elsevier Inc. All rights reserved.)

Divalent copper and zinc ions bind to the amyloid-β(40) and amyloid-β(42) alloforms and affect their structural stability as well as their chemical and physical properties. Current literature debates the impact of copper ions on amyloid-β alloforms. Recently, we reported the structural and thermodynamic properties of apo amyloid-β and divalent zinc ion bound amyloid-β alloforms (see, Wise-Scira et al. in J Biol Inorg Chem 17:927-938, 2012 and Coskuner et al. in ACS Chem Neurosci 4: 310-320, 2013). In our search for understanding the impacts of transition metal ions on disordered amyloid-β, we also developed and reported new potential functions using quantum mechanics, which are required for high-quality molecular dynamics simulations of divalent copper ion bound amyloid-β alloforms (see, Wise and Coskuner in J Comput Chem 35:1278-1289, 2014). The structures and thermodynamic properties of the divalent copper ion bound amyloid-β(40) and amyloid-β(42) alloforms in an aqueous medium are studied. The secondary and tertiary structures of divalent copper ion bound amyloid-β(40) and amyloid-β(42) along with their thermodynamic properties including enthalpy, entropy, Gibbs free energy and potential of mean force surface are investigated. Results are compared to those for apo amyloid-β and divalent zinc ion bound amyloid-β alloforms. Results demonstrate that copper binding to Aβ alloforms is thermodynamically less preferred rather than zinc binding. Less compact structures of copper ion bound amyloid-β alloforms possess reduced stability in comparison to zinc ion bound amyloid-β alloforms. Cu(II) binding impacts the thermodynamic properties, secondary and tertiary structural properties of Aβ40 and Aβ42.

An exceptional probe comprising indole-3-carboxaldehyde fluorescein hydrazone (FI) performs multiple tasks, namely, disaggregating amyloid β (Aβ) aggregates in different biomarker environments such as cerebrospinal fluid (CSF), Aβ1-40 fibrils, β-amyloid lysozyme aggregates (LA), and U87 MG human astrocyte cells. Additionally, the probe FI binds with Cu(2+) ions selectively, disrupts the Aβ aggregates that vary from few nanometers to micrometers, and prevents their reaggregation, thereby performing disaggregation and modulation of amyloid-β in the presence as well as absence of Cu(2+) ion. The excellent selectivity of probe FI for Cu(2+) was effectively utilized to modulate the assembly of metal-induced Aβ aggregates by metal chelation with the "turn-on" fluorescence via spirolactam ring opening of FI as well as the metal-free Aβ fibrils by noncovalent interactions. These results confirm that FI has exceptional ability to perform multifaceted tasks such as metal chelation in intracellular conditions using Aβ lysozyme aggregates in cellular environments by the disruption of β-sheet rich Aβ fibrils into disaggregated forms. Subsequently, it was confirmed that FI had the ability to cross the blood-brain barrier and it also modulated the metal induced Aβ fibrils in cellular environments by "turn-on" fluorescence, which are the most vital properties of a probe or a therapeutic agent. Furthermore, the morphology changes were examined by atomic force microscopy (AFM), polarizable optical microscopy (POM), fluorescence microscopy, and dynamic light scattering (DLS) studies. These results provide very valuable clues on the Aβ (CSF Aβ fibrils, Aβ1-40 fibrils, β-amyloid lysozyme aggregates) disaggregation behavior via in vitro studies, which constitute the first insights into intracellular disaggregation of Aβ by "turn-on" method thereby influencing amyloidogenesis.

Native electrospray ionization (ESI) mass spectrometry (MS) is often used to monitor noncovalent complex formation between peptides and ligands. The relatively low throughput of this technique, however, is not compatible with extensive screening. Laser ablation electrospray ionization (LAESI) MS combined with ion mobility separation (IMS) can analyze complex formation and provide conformation information within a matter of seconds. Islet amyloid polypeptide (IAPP) or amylin, a 37-amino acid residue peptide, is produced in pancreatic beta-cells through proteolytic cleavage of its prohormone. Both amylin and its precursor can aggregate and produce toxic oligomers and fibrils leading to cell death in the pancreas that can eventually contribute to the development of type 2 diabetes mellitus. The inhibitory effect of the copper(II) ion on amylin aggregation has been recently discovered, but details of the interaction remain unknown. Finding other more physiologically tolerated approaches requires large scale screening of potential inhibitors. Here, we demonstrate that LAESI-IMS-MS can reveal the binding stoichiometry, copper oxidation state, and the dissociation constant of human amylin-copper(II) complex. The conformations of hIAPP in the presence of copper(II) ions were also analyzed by IMS, and preferential association between the β-hairpin amylin monomer and the metal ion was found. The copper(II) ion exhibited strong association with the -HSSNN- residues of the amylin. In the absence of copper(II), amylin dimers were detected with collision cross sections consistent with monomers of β-hairpin conformation. When copper(II) was present in the solution, no dimers were detected. Thus, the copper(II) ions disrupt the association pathway to the formation of β-sheet rich amylin fibrils. Using LAESI-IMS-MS for the assessment of amylin-copper(II) interactions demonstrates the utility of this technique for the high-throughput screening of potential inhibitors of amylin oligomerization and fibril formation. More generally, this rapid technique opens the door for high-throughput screening of potential inhibitors of amyloid protein aggregation.

Prion diseases, known as Transmissible Spongiform Encephalopathies (TSEs), are a group of fatal neuronal, and to some extent infectious disorders, associated with a pathogenic protein agent called prion protein (PrP). The human prion protein (hPrP) fragment encompassing the 91-127 region, also known as the amyloidogenic domain, comprises two copper-binding sites corresponding to His-96 and His-111 residues that act as anchors for Cu(2+) binding. In this work, we investigated Cu(2+) interaction with hPrP91-127 in the presence of the anionic surfactant sodium dodecyl sulfate (SDS), which induces a partial α-helix folding of the peptide. Our data indicate that the Cu(2+) coordination ability of the amyloidogenic fragment in the presence of SDS micelles is significantly different to that observed in aqueous solution. This is mainly due to the fact that SDS micelles strongly stabilize the formation of the α-helical structure of the peptide backbone, which is well conserved also upon Cu(2+) binding, contrary to the random coil conformation mainly assumed by hPrP91-127 in aqueous solutions. Potentiometric and spectroscopic studies clearly indicate that in the case of SDS containing solutions, Cu(2+) ions coordinate simultaneously to both imidazoles, while in the case of water solutions, metal ion coordination involves only a single His side chain, which individually acts as an independent Cu(2+) anchoring site.

Metal ions, i.e., Zn(2+) and Cu(2+), are released from neuron terminals in the hippocampus, which plays important roles in spatial and declarative memory, and may serve as a signal factor. Synaptic homeostasis of metal ions is critical for cognitive activity in the hippocampus. Amyloid-β (Aβ) is a causative candidate for the pathogenesis of Alzheimer's disease (AD) and Aβ-induced synapse dysfunction is easy to emerge along with normal aging and leads to the cognitive decline and memory loss in the pre-dementia stage of AD. Because Aβ interacts with Zn(2+) and Cu(2+), it is likely that these metal ions are involved in the Aβ-induced modification of the synaptic function. There is evidence to indicate that the inhibition of the interaction of Aβ with Zn(2+) and Cu(2+) may ameliorate the pathophysiology of AD. Interaction of extracellular Zn(2+) with Aβ in the hippocampus is involved in transiently Aβ-induced cognition deficits, while the interaction of extracellular Cu(2+) reduces bioavailability of intracellular Cu(2+), followed by an increase in oxidative stress, which may lead to cognitive deficits. It is likely that Zn(2+) and Cu(2+) play as a key-mediating factor in pathophysiology of the synaptic dysfunction in which Aβ is involved. Based on the idea that understating Aβ-induced changes in synaptic plasticity is important to prevent AD, the present paper summarizes the interaction of Aβ with metal ions in cognition.

Herein, the apparent equilibrium dissociation constant, K(Dapp), between Cu(2+) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS), a negatively charged phospholipid, was measured as a function of PS concentrations in supported lipid bilayers (SLBs). The results indicated that K(Dapp) for Cu(2+) binding to PS-containing SLBs was enhanced by a factor of 17,000 from 110 nM to 6.4 pM as the PS density in the membrane was increased from 1.0 to 20 mol %. Although Cu(2+) bound bivalently to POPS at higher PS concentrations, this was not the dominant factor in increasing the binding affinity. Rather, the higher concentration of Cu(2+) within the double layer above the membrane was largely responsible for the tightening. Unlike the binding of other divalent metal ions such as Ca(2+) and Mg(2+) to PS, Cu(2+) binding does not alter the net negative charge on the membrane as the Cu(PS)2 complex forms. As such, the Cu(2+) concentration within the double layer region was greatly amplified relative to its concentration in bulk solution as the PS density was increased. This created a far larger enhancement to the apparent binding affinity than is observed by standard multivalent effects. These findings should help provide an understanding on the extent of Cu(2+)-PS binding in cell membranes, which may be relevant to biological processes such as amyloid-β peptide toxicity and lipid oxidation.

Vitamin D (vit D) is a known anticancer molecule, and cancer cells are reported to have elevated levels of Cu(II) ions. In this study, we show that interaction of vit D and Cu(II) leads to the formation of hydroxyl free radicals, superoxide anion and hydrogen peroxide, which causes severe oxidative stress, selectively in malignant cells. We show that the production of these reactive oxygen species causes cellular DNA fragmentation which may cause cell death. A novel putative chemical mechanism explaining how vit D causes cell death by DNA damage, selectively in malignant cells, is proposed.

A novel and highly sensitive colorimetric and fluorescence assay for the accurate determination of alkaline phosphatase (ALP) activity has been developed. The assay takes advantage of the inhibition of the peroxidase activity of Cu(2+) ions caused by complexation with pyrophosphate (PPi), a natural substrate for ALP. This inhibition disappears when PPi undergoes ALP catalyzed hydrolysis to generate phosphate, which does not bind to Cu(2+) ions. Thus, ALP causes generation of uncomplexed Cu(2+) ions, which promote multiple oxidation reactions of Amplex UltraRed in the presence of hydrogen peroxide in conjunction with the production of intense fluorescence and colorimetric signals. By employing the fluorescence and colorimetric assay strategies, ALP can be detected at respective concentrations as low as 4.3 pM and 5.4 pM, detection limits that are much lower than those associated with previously described methods. The practical diagnostic capability of the assay system has been demonstrated by its use to detect ALP in human blood serum.

Amylin or islet amyloid polypeptide (IAPP) is a 37-residue peptide hormone secreted from the pancreatic islets into the blood circulation and is cleared by peptidases in the kidney. IAPP aggregates are strongly associated with β-cell degeneration in type 2 diabetes, as demonstrated by the fact that more than 95% of patients exhibit IAPP amyloid upon autopsy. Recently, it has been reported that metal ions such as copper(II) and zinc(II) are implicated in the aggregation of IAPP as well as able to modulate the proteolytic activity of IAPP degrading enzymes. For this reason, in this work, the role of the latter metal ions in the degradation of IAPP by insulin-degrading enzyme (IDE) has been investigated by a chromatographic and mass spectrometric combined method. The latter experimental approach allowed not only to assess the overall metal ion inhibition of the human and murine IAPP degradation by IDE but also to have information on copper- and zinc-induced changes in IAPP aggregation. In addition, IDE cleavage site preferences in the presence of metal ions are rationalized as metal ion-induced changes in substrate accessibility., (Copyright © 2014 John Wiley & Sons, Ltd.)

Extracellular polymeric substances (EPS) were extracted from Aspergillus fumigatus using cationic exchange resin technique. The EPS were mainly composed of polysaccharide and low quantities of protein and nucleic acid. Biosorption of Cd(II), Pb(II), and Cu(II) of EPS was investigated as a function of pH using differential pulse polarography and the Ruzic model. Results showed that the EPS biosorption capacity determined using either the direct titration curves i = f(C M) or the method proposed by Ruzic (Analytica Chimica Acta 140:99-113, 1982) were coincident. Cu(II) had the highest affinity with EPS followed by Pb(II) and Cd(II). The total number of binding sites for Cu(II) and Cd(II) increased with pH in the range of 4.0-7.0. Similar trend was observed for Pb(II) at pH 4.0-5.0, while precipitates were observed at pH 6.0 and 7.0. The conditional binding constants of these three metals displayed low levels of fluctuation with pH and ranged from 4.02 ± 0.02 to 5.54 ± 0.05.

This is the first report on using Catathelasma ventricosum for production of fruiting body and lignocellulosic enzymes. To improve the laccase activity and productivity of mushroom, the substrate was added with different supplements (eight aromatic compounds, Mn2+, and Cu2+). Based on the results, all these supplements can improve the laccase activity and productivity of C. ventricosum, and it seems that there is a critical value of laccase activity that affects the productivity of C. ventricosum. In addition, when Penicillium decumbens was inoculated into the substrate that had been cultivated with C. ventricosum for 20 days, the highest values of laccase activity, FPA activity, and productivity of C. ventricosum were obtained. Moreover, the laccase activity showed a positive correlation with the productivity of C. ventricosum. Finally, the effect of Mn2+, Cu2+, and P. decumbens on laccase activity was investigated by response surface methodology (RSM).

We have identified QDR2 in a screening for genes able to confer tolerance to sodium and/or lithium stress upon overexpression. Qdr2 is a multidrug transporter of the major facilitator superfamily, originally described for its ability to transport the antimalarial drug quinidine and the herbicide barban. To identify its physiological substrate, we have screened for phenotypes dependent on QDR2 and found that Qdr2 is able to transport monovalent and divalent cations with poor selectivity, as shown by growth tests and the determination of internal cation content. Moreover, strains overexpressing or lacking QDR2 also exhibit phenotypes when reactive oxygen species- producing agents, such as hydrogen peroxide or menadione were added to the growth medium. We have also found that the presence of copper and hydrogen peroxide repress the expression of QDR2. In addition, the copper uptake of a qdr2 mutant strain is similar to a wild type, but the extrusion is clearly impaired. Based on our results, we propose that free divalent copper is the main physiological substrate of Qdr2. As copper is a substrate for several redox reactions that occur within the cytoplasm, its function in copper homeostasis explains its role in the oxidative stress response., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

The aim of this study was to investigate the biosorption characteristics of Cd(2+), Cu(2+), and Pb(2+) by the fruiting body of jelly fungus Auricularia polytricha. Batch experiments were conducted to characterize the kinetics, equilibrium, and mechanisms of the biosorption process. Optimum values of pH 5, biomass dosage 4 g L(-1), and contact time 60 min provided maximum biosorption capacities of A. polytricha for Cd(2+), Cu(2+), and Pb(2+) of 63.3, 73.7, and 221 mg g(-1), respectively. The maximum desorption was achieved using 0.05 mol L(-1) HNO(3) as an elute. The fruiting body was reusable at least for six cycles of operations. The pseudo-second-order model was the best to describe the biosorption processes among the three kinetic models tested. Freundlich and Dubinin-Radushkevich models fitted the equilibrium data well, indicating a heterogeneous biosorbent surface and the favorable chemisorption nature of the biosorption process. A Fourier transform infrared spectroscopy analysis indicated that carboxyl, amine/hydroxyl, amino, phosphoryl, and C-N-C were the main functional groups to affect the biosorption process. Synergistic ion exchange and surface complexation were the dominant mechanisms in the biosorption process. The present work revealed the potential of jelly fungus (fruiting body of A. polytricha) to remove toxic heavy metals from contaminated water.

Expression of the periplasmic protein PcoE of Escherichia coli is induced strongly by cupric salts under the control of the chromosomal copper tolerance system cusRS. Its isolation and study were complicated by de-amidation of Asn 54 and 103 at alkaline pH. Its apo form is essentially unstructured in solution and can be likened to a large unstructured multidentate ligand carrying multiple metal binding sites (15 Met; 10 His; 13 Asp, Glu; 10 Asn; 6 Lys). As expected, it binds multiple soft metal ions Cu(+) and Ag(+) non-cooperatively with the highest affinity for Cu(I) in the picomolar range (K(D)~10(-12) M). Binding of multiple soft ions induced dimerization and formation of some α-helical structure. PcoE also binds the harder metal ions Cu(2+) or Zn(2+) but with lower affinities and in smaller numbers. Cu(II) bound in PcoE is reduced readily to more tightly bound Cu(I). Overall, these properties mean that it is difficult to characterize individual species of defined metal content. Similar properties and difficulties have been reported for the homologous silver-binding protein SilE from Salmonella. However, the properties are consistent with a role for PcoE as a 'metal sponge' acting as a first line of defence against metal toxicity (under the control of the copper tolerance system cusRS) until the copper resistance operon pcoABCD is expressed., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Transient receptor potential melastatin 2 (TRPM2) is a Ca(2+)-permeable cationic channel in the TRP channel family. The channel activity can be regulated by reactive oxygen species (ROS) and cellular acidification, which has been implicated to the pathogenesis of diabetes and some neuronal disorders. However, little is known about the effect of redox-active metal ions, such as copper, on TRPM2 channels. Here we investigated the effect of divalent copper on TRPM2. TRPM2 channel was over-expressed in HEK-293 cells and the whole-cell current was recorded by patch clamp. We found the whole-cell current evoked by intracellular ADP-ribose was potently inhibited by Cu(2+) with a half maximal inhibitory concentration (IC(50)) of 2.59 μM. The inhibitory effect was irreversible. The single channel activity was abolished in the outside-out patches, and intracellular application of Cu(2+) did not prevent the channel activation, suggesting that the action site of Cu(2+) is located in the extracellular domains of the channel. TRPM2 current was also blocked by Hg(2+), Pb(2+), Fe(2+) and Se(2+). We concluded that Cu(2+) is a potent TRPM2 channel blocker. The sensitivity of TRPM2 channel to heavy metal ions could be a new mechanism for the pathogenesis of some metal ion-related diseases., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Metal ions like Cu(II) and Zn(II) are accumulated in Alzheimer's disease amyloid plaques. The amyloid-β (Aβ) peptide involved in the disease interacts with these metal ions at neutral pH via ligands provided by the N-terminal histidines and the N-terminus. The present study uses high-resolution NMR spectroscopy to monitor the residue-specific interactions of Cu(II) and Zn(II) with (15)N- and (13)C,(15)N-labeled Aβ(1-40) peptides at varying pH levels. At pH 7.4 both ions bind to the specific ligands, competing with one another. At pH 5.5 Cu(II) retains its specific histidine ligands, while Zn(II) seems to lack residue-specific interactions. The low pH mimics acidosis which is linked to inflammatory processes in vivo. The results suggest that the cell toxic effects of redox active Cu(II) binding to Aβ may be reversed by the protective activity of non-redox active Zn(II) binding to the same major binding site under non-acidic conditions. Under acidic conditions, the protective effect of Zn(II) may be decreased or changed, since Zn(II) is less able to compete with Cu(II) for the specific binding site on the Aβ peptide under these conditions., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Phosphatidylserine (PS) embedded within supported lipid bilayers was found to bind Cu(2+) from solution with extraordinarily high affinity. In fact, the equilibrium dissociation constant was in the femtomolar range. The resulting complex formed in a 1:2 Cu(2+)-to-PS ratio and quenches a broad spectrum of lipid-bound fluorophores in a reversible and pH-dependent fashion. At acidic pH values, the fluorophores were almost completely unquenched, while at basic pH values significant quenching (85-90%) was observed. The pH at which the transition occurred was dependent on the PS concentration and ranged from approximately pH 5 to 8. The quenching kinetics was slow at low Cu(2+) concentrations and basic pH values (up to several hours), while the unquenching reaction was orders of magnitude more rapid upon lowering the pH. This was consistent with diffusion-limited complex formation at basic pH but rapid dissociation under acidic conditions. The tight binding of Cu(2+) to PS may have physiological consequences under certain circumstances.

Unlabelled: The acquisition, delivery, and incorporation of metals into their respective metalloproteins are important cellular processes. These processes are tightly controlled in order to prevent exposure of cells to free-metal concentrations that could yield oxidative damage. Copper (Cu) is one such metal that is required as a cofactor in a variety of proteins. However, when present in excessive amounts, Cu is toxic due to its oxidative capability. Cytochrome c oxidases (Coxs) are among the metalloproteins whose assembly and activity require the presence of Cu in their catalytic subunits. In this study, we focused on the acquisition of Cu for incorporation into the heme-Cu binuclear center of the cbb(3)-type Cox (cbb(3)-Cox) in the facultative phototroph Rhodobacter capsulatus. Genetic screens identified a cbb(3)-Cox defective mutant that requires Cu(2+) supplementation to produce an active cbb(3)-Cox. Complementation of this mutant using wild-type genomic libraries unveiled a novel gene (ccoA) required for cbb(3)-Cox biogenesis. In the absence of CcoA, the cellular Cu content decreases and cbb(3)-Cox assembly and activity become defective. CcoA shows homology to major facilitator superfamily (MFS)-type transporter proteins. Members of this family are known to transport small solutes or drugs, but so far, no MFS protein has been implicated in cbb(3)-Cox biogenesis. These findings provide novel insights into the maturation and assembly of membrane-integral metalloproteins and on a hitherto-unknown function(s) of MFS-type transporters in bacterial Cu acquisition., Importance: Biogenesis of energy-transducing membrane-integral enzymes, like the heme copper-containing cytochrome c oxidases, and the acquisition of transition metals, like copper, as their catalytic cofactors are vital processes for all cells. These widespread and well-controlled processes are poorly understood in all organisms, including bacteria. Defects in these processes lead to severe mitochondrial diseases in humans and poor crop yields in plants. In this study, using the facultative phototroph Rhodobacter capsulatus as a model organism, we report on the discovery of a novel major facilitator superfamily (MFS)-type transporter (CcoA) that affects cellular copper content and cbb(3)-type cytochrome c oxidase production in bacteria.

The human small heat-shock protein αB-crystallin (αB) rescues misfolded proteins from irreversible aggregation during cellular stress. Binding of Cu(II) was shown to modulate the oligomeric architecture and the chaperone activity of αB. However, the mechanistic basis of this stimulation is so far not understood. We provide here first structural insights into this Cu(II)-mediated modulation of chaperone function using NMR spectroscopy and other biophysical approaches. We show that the α-crystallin domain is the elementary Cu(II)-binding unit specifically coordinating one Cu(II) ion with picomolar binding affinity. Putative Cu(II) ligands are His(83), His(104), His(111), and Asp(109) at the dimer interface. These loop residues are conserved among different metazoans, but also for human αA-crystallin, HSP20, and HSP27. The involvement of Asp(109) has direct implications for dimer stability, because this residue forms a salt bridge with the disease-related Arg(120) of the neighboring monomer. Furthermore, we observe structural reorganization of strands β2-β3 triggered by Cu(II) binding. This N-terminal region is known to mediate both the intermolecular arrangement in αB oligomers and the binding of client proteins. In the presence of Cu(II), the size and the heterogeneity of αB multimers are increased. At the same time, Cu(II) increases the chaperone activity of αB toward the lens-specific protein β(L)-crystallin. We therefore suggest that Cu(II) binding unblocks potential client binding sites and alters quaternary dynamics of both the dimeric building block as well as the higher order assemblies of αB.

The diets of two-thirds of the world's population are deficient in one or more essential elements and one of the approaches to enhance the levels of mineral elements in food crops is by developing plants with ability to accumulate them in edible parts. Besides conventional methods, transgenic technology can be used for enhancing metal acquisition in plants. Copper is an essential element, which is often deficient in human diet. With the objective of developing plants with improved copper acquisition, a high-affinity copper transporter gene (tcu-1) was cloned from fungus Neurospora crassa and introduced into a model plant (Nicotiana tabacum). Integration of the transgene was confirmed by Southern blot hybridization. Transgenic tobacco plants (T(0) and T(1)) expressing tcu-1, when grown in hydroponic medium spiked with different concentrations of copper, showed higher acquisition of copper (up to 3.1 times) compared with control plants. Transgenic plants grown in soil spiked with copper could also take up more copper compared with wild-type plants. Supplementation of other divalent cations such as Cd(2+) and Zn(2+) did not alter uptake of Cu by transgenic plants. The present study has shown that expression of a heterologous copper transporter in tobacco could enhance acquisition of copper.

Cu(II) ions are implicated in the pathogenesis of Alzheimer disease by influencing the aggregation of the amyloid-β (Aβ) peptide. Elucidating the underlying Cu(II)-induced Aβ aggregation is paramount for understanding the role of Cu(II) in the pathology of Alzheimer disease. The aim of this study was to characterize the qualitative and quantitative influence of Cu(II) on the extracellular aggregation mechanism and aggregate morphology of Aβ(1-40) using spectroscopic, microelectrophoretic, mass spectrometric, and ultrastructural techniques. We found that the Cu(II):Aβ ratio in solution has a major influence on (i) the aggregation kinetics/mechanism of Aβ, because three different kinetic scenarios were observed depending on the Cu(II):Aβ ratio, (ii) the metal:peptide stoichiometry in the aggregates, which increased to 1.4 at supra-equimolar Cu(II):Aβ ratio; and (iii) the morphology of the aggregates, which shifted from fibrillar to non-fibrillar at increasing Cu(II):Aβ ratios. We observed dynamic morphological changes of the aggregates, and that the formation of spherical aggregates appeared to be a common morphological end point independent on the Cu(II) concentration. Experiments with Aβ(1-42) were compatible with the conclusions for Aβ(1-40) even though the low solubility of Aβ(1-42) precluded examination under the same conditions as for the Aβ(1-40). Experiments with Aβ(1-16) and Aβ(1-28) showed that other parts than the Cu(II)-binding His residues were important for Cu(II)-induced Aβ aggregation. Based on this study we propose three mechanistic models for the Cu(II)-induced aggregation of Aβ(1-40) depending on the Cu(II):Aβ ratio, and identify key reaction steps that may be feasible targets for preventing Cu(II)-associated aggregation or toxicity in Alzheimer disease.

The terrestrial biotic ligand model (BLM) for metal toxicity in soil postulates that metal toxicity depends on the free metal ion activity in solution and on ions competing for metal sorption to the biotic ligand. Unequivocal evidence for the BLM assumptions is most difficult to obtain for native soil microorganisms because the abiotic and biotic compartments cannot be experimentally separated. Here, we report copper (Cu) toxicity to a bioluminescent Nitrosomonas europaea reporter strain that was used in a solid phase-contact assay and in corresponding soil extracts and artificial soil solutions. The Cu(2+) ion activities that halve bioluminescence (EC50) in artificial solutions ranged 10(-5) to 10(-7) M and increased with increasing activities of H(+), Ca(2+) and Mg(2+) according to the BLM concept. The solution based Cu(2+) EC50 values of N. europaea in six contaminated soils ranged 2 × 10(-6) to 2 × 10(-9) M and these thresholds for both solid phase or soil extract based assays were well predicted by the ion competition model fitted to artificial solution data. In addition, solution based Cu(2+) EC50 of the solid phase-contact assay were never smaller than corresponding values in soil extracts suggesting no additional solid phase toxic route. By restricting the analysis to the same added species, we show that the Cu(2+) in solution represents the toxic species to this bacterium.

Dyshomeostasis of transition metals iron and copper as well as accumulation of oxidative DNA damage have been implicated in multitude of human neurodegenerative diseases, including Alzheimer disease and Parkinson disease. These metals oxidize DNA bases by generating reactive oxygen species. Most oxidized bases in mammalian genomes are repaired via the base excision repair pathway, initiated with one of four major DNA glycosylases: NTH1 or OGG1 (of the Nth family) or NEIL1 or NEIL2 (of the Nei family). Here we show that Fe(II/III) and Cu(II) at physiological levels bind to NEIL1 and NEIL2 to alter their secondary structure and strongly inhibit repair of mutagenic 5-hydroxyuracil, a common cytosine oxidation product, both in vitro and in neuroblastoma (SH-SY5Y) cell extract by affecting the base excision and AP lyase activities of NEILs. The specificity of iron/copper inhibition of NEILs is indicated by a lack of similar inhibition of OGG1, which also indicated that the inhibition is due to metal binding to the enzymes and not DNA. Fluorescence and surface plasmon resonance studies show submicromolar binding of copper/iron to NEILs but not OGG1. Furthermore, Fe(II) inhibits the interaction of NEIL1 with downstream base excision repair proteins DNA polymerase beta and flap endonuclease-1 by 4-6-fold. These results indicate that iron/copper overload in the neurodegenerative diseases could act as a double-edged sword by both increasing oxidative genome damage and preventing their repair. Interestingly, specific chelators, including the natural chemopreventive compound curcumin, reverse the inhibition of NEILs both in vitro and in cells, suggesting their therapeutic potential.

Accumulating evidence suggests that hepcidin, a 25-residue peptide hormone, is the master regulator of iron metabolism. Further evidence suggests that the five N-terminal amino acids are crucial for mediating its biological function. With a histidine residue at position 3, this region also has the potential to bind bivalent metal ions. To characterize this hepcidin-metal interaction in detail, the present study utilizes electrospray MS to measure the binding of a range of metal ions to wild-type and mutant human and murine hepcidins. In addition, the biological effects of these point mutations were tested on Caco-2 and HEK-293T human cell lines and in mice. Our results show that hepcidin-25 can form complexes with copper, nickel and zinc; however, we failed to detect any hepcidin-25 binding to either ferric or ferrous ions. The greatest affinity observed was between hepcidin-25 and copper with a dissociation constant <<1 microM. Substituting the histidine residue at position 3 in human hepcidin-25 and comparably the asparagine residue at position 3 in murine hepcidin-25 with an alanine residue markedly diminished the affinity for copper. The amino acid substitutions also decreased the biological activity of hepcidin-25; namely repression of ferroportin protein levels and hypoferraemia. In summary, the high affinity of hepcidin for copper suggests that hepcidin could bind copper in vivo and this may be of biological relevance.

Copper is an essential element in a variety of biological processes, but it can be toxic when present in excessive amounts. The central regulators of cellular copper metabolism include copper-binding proteins, copper transporters, metal membrane active transporters and copper-dependent enzymes. However, the way in which cupric ions (Cu(2+)) cause cellular changes in proteins and lead to toxic effects is less well-known. The aim of this study is to identify the proteins related to Cu(2+) toxicity or detoxification mechanisms in tilapia (Oreochromis niloticus) using a proteomic approach. A cell line derived from the liver of tilapia, Hepa-T1, was used as a model and exposed to two sub-lethal concentrations of waterborne copper for 96 h. The proteins expressed in Hepa-T1 were investigated by differential protein profiling using two-dimensional gel electrophoresis (2DE). It was found that Cu(2+) (120 and 300 microM) caused the differential expression of 93 different proteins, 18 of which were further verified by real-time quantitative polymerase chain reaction (PCR) analysis. Following analysis with ingenuity pathway software, several proteins were found to be involved in lipid metabolism, tissue connective development and cell cycle control, thus indicating that copper toxicity affects these cellular functions.

As a consequence of human activity, various toxicants - especially metal ions - enter aquatic ecosystems and many fish are exposed to considerable levels. As the free ion and in some complexes, there is no doubt that copper promotes damage to cellular molecules and structures through radical formation. Therefore, we have investigated the influence of copper uptake by the red blood of the sea bass (Dicentrarchus labrax), and its oxidative action and effects on cells in the presence of complexed and uncomplexed Fe3+ ions. Erythrocytes were exposed to various concentrations of CuSO4, Fe(NO3)3, and K3Fe(CN)6 for up to 5h, and the effects of copper ions alone and in the combination with iron determined. The results show that inside the cells cupric ion interacts with hemoglobin, causing methemoglobin formation by direct electron transfer from heme Fe2+ to Cu2+. Potassium ferricyanide as a source of complexed iron decreases Met-Hb formation induced by copper ions unlike Fe(NO3)3. We also found that incubation of fish erythrocytes with copper increased hemolysis of cells. But complexed and uncomplexed iron protected the effect of copper. CuSO4 increased the level of lipid peroxidation and a protective effect on complexed iron was observed. Incubation of erythrocytes with copper ions resulted in the loss of a considerable part of thiol content at 10 and 20 microM. This effect was decreased by potassium ferricyanide and Fe(NO3)3 only after 1 and 3h of incubation. The level of nuclear DNA damage assayed by comet assay showed that 20 microM CuSO4 as well as 20 microM Fe(NO3)3 and 10 mM K3Fe(CN)6 induce single- and double-strand breaks. The lower changes were observed after the exposure of cells to K3Fe(CN)6. The data suggest that complexed iron can act protectively against copper ions in contrast to Fe(NO3)3.

Contryphans are bioactive peptides, isolated from the venom of marine snails of the genus Conus, which are characterized by the short length of the polypeptide chain and the high degree of unusual post-translational modifications. The cyclization of the polypeptide chain through a single disulphide bond, the presence of two conserved Pro residues, and the epimerization of a Trp/Leu residue confer to Contryphans a stable and well-defined structure in solution, conserved in all members of the family, and tolerant to multiple substitutions. The potential of Contryphans as scaffolds for the design of redox-active (macro)molecules was tested by engineering a copper-binding site on two different variants of the natural peptide Contryphan-Vn. The binding site was designed by computational modeling, and the redesigned peptides were synthesized and characterized by optical, fluorescence, electron spin resonance, and nuclear magnetic resonance spectroscopy. The novel peptides, named Cupryphan and Arg-Cupryphan, bind Cu(2+) ions with a 1:1 stoichiometry and a K(d) in the 100 nM range. Other divalent metals (e.g., Zn(2+) and Mg(2+)) are bound with much lower affinity. In addition, Cupryphans catalyze the dismutation of superoxide anions with an activity comparable to other nonpeptidic superoxide dismutase mimics. We conclude that the Contryphan motif represents a natural robust scaffold which can be engineered to perform different functions, providing additional means for the design of catalytically active mini metalloproteins.

It has been shown for the first time by the method of isothermal calorimetry of titration that the N-terminal amino group of the beta-amyloid protein, which plays a key role in the binding of copper ion, ceases to form the proper coordinating bond as a result of the isomerization of aspartate-7.

The C-terminal 20 and 30 amino acid sequences of Cap43 protein were chosen as models to study their interactions with Cu(II) ions. The behaviour of the 20 amino acid Ac-TRSRSH6TSEG-TRSRSH16TSEG and 30 amino acid Ac-TRSRSH6TSEG-TRSRSH16TSEG-TRSRSH26TSEG peptides towards Cu(II) ions at different pH values and different ligand-to-metal molar ratios, was examined. Spectroscopic (EPR, UV-Vis) and potentiometric techniques were performed to understand the details of metal binding to the peptides. The study showed that, starting from pH 4.0, each 10 amino acid fragment T1R2S3R4S5H6T7S8E9G10 was able to independently coordinate a single Cu(II) ion. The coordination mode involved the imidazole nitrogen of histidine H6 residue, and three amidic nitrogens from histidine H6, serine S5, and arginine R4 residues, respectively.

Microbial biomass represents a potentially cost-effective sorbent for water treatment applications. High sorption capacities for both cations and anions are demonstrated here for viable and autoclaved cell suspensions of the Gram-negative bacterium Shewanella putrefaciens. FTIR absorption spectra and pH-dependent zeta-potentials are similar for the viable and killed bacterial cells. Potentiometric titrations, however, reveal a two to three times higher OH(-) buffering capacity for the living cells. The Cu(2+) sorption capacity of the viable cells is also about twice that of the autoclaved cells. Sorption of fluoride and phosphate is not pH-dependent, although an initial addition of acid or base was needed to activate the anion binding sites. Uptake of fluoride is comparable for viable and killed cells. For the viable cells, the isotherms of Zn(2+) and Cu(2+) indicate the presence of at least two distinct populations of cell wall binding sites. In competitive sorption experiments, Cu(2+) completely inhibits the binding of Zn(2+) to the cells at aqueous concentrations above 150 mg L(-1). The release of dissolved organic compounds by the viable cells depends on the concentrations of metal cations or fluoride to which the cells are exposed. In particular, the presence of Cu(2+) nearly completely suppresses the release of protein-like substances, possibly reflecting Cu(2+) toxicity.

As in other P-type ATPases, metal binding to transmembrane metal-binding sites (TM-MBS) in Cu(+)-ATPases is required for enzyme phosphorylation and subsequent transport. However, Cu(+) does not access Cu(+)-ATPases in a free (hydrated) form but is bound to a chaperone protein. Cu(+) transfer from Cu(+) chaperones to regulatory cytoplasmic metal-binding domains (MBDs) present in these ATPases has been described, but there is no evidence of a proposed subsequent Cu(+) movement from the MBDs to the TM-MBS. Alternatively, we postulate the parsimonious Cu(+) transfer by the chaperone directly to TM-MBS. Testing both models, the delivery of Cu(+) by Archaeoglobus fulgidus Cu(+) chaperone CopZ to the corresponding Cu(+)-ATPase, CopA, was studied. As expected, CopZ interacted with and delivered the metal to CopA MBDs. Cu(+)-loaded MBDs, acting as metal donors, were unable to activate CopA or a truncated CopA lacking MBDs. Conversely, Cu(+)-loaded CopZ activated the CopA ATPase and CopA constructs in which MBDs were rendered unable to bind Cu(+). Furthermore, under nonturnover conditions, CopZ transferred Cu(+) to the TM-MBS of a CopA lacking MBDs. These data are consistent with a model where MBDs serve a regulatory function without participating in metal transport and the chaperone delivers Cu(+) directly to transmembrane transport sites of Cu(+)-ATPases.

There is evidence that binding of metal ions like Zn2+ and Cu2+ to amyloid beta-peptides (Abeta) may contribute to the pathogenesis of Alzheimer's disease. Cu2+ and Zn2+ form complexes with Abeta peptides in vitro; however, the published metal-binding affinities of Abeta vary in an enormously large range. We studied the interactions of Cu2+ and Zn2+ with monomeric Abeta(40) under different conditions using intrinsic Abeta fluorescence and metal-selective fluorescent dyes. We showed that Cu(2+) forms a stable and soluble 1 : 1 complex with Abeta(40), however, buffer compounds act as competitive copper-binding ligands and affect the apparent K(D). Buffer-independent conditional K(D) for Cu(II)-Abeta(40) complex at pH 7.4 is equal to 0.035 micromol/L. Interaction of Abeta(40) with Zn2+ is more complicated as partial aggregation of the peptide occurs during zinc titration experiment and in the same time period (within 30 min) the initial Zn-Abeta(40) complex (K(D) = 60 micromol/L) undergoes a transition to a more tight complex with K(D) approximately 2 micromol/L. Competition of Abeta(40) with ion-selective fluorescent dyes Phen Green and Zincon showed that the K(D) values determined from intrinsic fluorescence of Abeta correspond to the binding of the first Cu2+ and Zn2+ ions to the peptide with the highest affinity. Interaction of both Zn2+ and Cu2+ ions with Abeta peptides may occur in brain areas affected by Alzheimer's disease and Zn2+-induced transition in the peptide structure might contribute to amyloid plaque formation.

The binding of divalent copper ions to the full-length recombinant murine prion protein PrP23-231 at neutral pH was studied using vibrational Raman optical activity (ROA) and ultraviolet circular dichroism (UV CD). The effect of the Cu2+ ions on PrP structure depends on whether they are added after refolding of the protein in water or are present during the refolding process. In the first case ROA reveals that the hydrated alpha-helix is lost, with UV CD revealing a drop from approximately 25% to approximately 18% in the total alpha-helix content. The lost alpha-helix could be that comprising residues 145-156, located within the region associated with scrapie PrP formation. In the second case, ROA reveals the protein's structure to be almost completely disordered/irregular, with UV CD revealing a drop in total alpha-helix content to approximately 5%. Hence, although Cu2+ binding takes place exclusively within the unfolded/disordered N-terminal region, it can profoundly affect the structure of the folded/alpha-helical C-terminal region. This is supported by the finding that refolding in the presence of Cu2+ of a mutant in which the first six histidines associated with copper binding to the N-terminal region are replaced by alanine has a similar alpha-helix content to the metal-free protein. In contrast, when the protein is refolded in the presence of divalent manganese ions, ROA indicates the alpha-helix is reinforced, with UV CD revealing an increase in total alpha-helix content to approximately 30%. The very different influence of Cu2+ and Mn2+ ions on prion protein structure may originate in the different stability constants and geometries of their complexes.

The prion protein (PrP) is a cell-surface Cu(2+)-binding glycoprotein that when misfolded is responsible for a number of transmissible spongiform encephalopathies. Full-length PrP-(23-231) and constructs in which the octarepeat region has been removed, or His(95) and His(110) is replaced by alanine residues, have been used to elucidate the order and mode of Cu(2+) coordination to PrP-(23-231). We have built on our understanding of the appearance of visible CD spectra and EPR for various PrP fragments to characterize Cu(2+) coordination to full-length PrP. At physiological pH, Cu(2+) initially binds to full-length PrP in the amyloidogenic region between the octarepeats and the structured domain at His(95) and His(110). Only subsequent Cu(2+) ions bind to single histidine residues within the octarepeat region. Ni(2+) ions are used to further probe metal binding and, like Cu(2+), Ni(2+) will bind individually to His(95) and His(110), involving preceding main chain amides. Competitive chelators are used to determine the affinity of the first mole equivalent of Cu(2+) bound to full-length PrP; this approach places the affinity in the nanomolar range. The affinity and number of Cu(2+) binding sites support the suggestion that PrP could act as a sacrificial quencher of free radicals generated by copper redox cycling.

Amyloid precursor protein (APP) plays a central role in the pathogenesis of Alzheimer's disease, as its cleavage generates the Abeta peptide that is toxic to cells. APP is able to bind Cu2+ and reduce it to Cu+ through its copper-binding domain (CuBD). The interaction between Cu2+ and APP leads to a decrease in Abeta production and to alleviation of the symptoms of the disease in mouse models. Structural studies of CuBD have been undertaken in order to better understand the mechanism behind the process. Here, the crystal structure of CuBD in the metal-free form determined to ultrahigh resolution (0.85 A) is reported. The structure shows that the copper-binding residues of CuBD are rather rigid but that Met170, which is thought to be the electron source for Cu2+ reduction, adopts two different side-chain conformations. These observations shed light on the copper-binding and redox mechanisms of CuBD. The structure of CuBD at atomic resolution provides an accurate framework for structure-based design of molecules that will deplete Abeta production.

To further the understanding of the biological importance of metal-binding by avian prion proteins, we have investigated the affinity and selectivity of peptides Hx1 [Ac-HNPGYP-nh] and Hx2 [Ac-NPGYPHNPGYPH-nh] with a range of physiological metals via electrospray ionization mass spectrometry and tyrosine fluorescence emission spectroscopy. Both the hexamer Hx1 and the "dimer" peptide Hx2 bind only one equivalent of Cu(II), although only the latter peptide binds copper with significant affinity (Hx1 K(d)=150+/-35 microM; Hx2 K(d)=1.07+/-0.78 microM, pH 7.0 in 3-(N-morpholino)propanesulfonic acid (MOPS) buffer). Both peptides are selective for Cu(II) over divalent Ca, Co, Mg, Mn, Ni, and Zn. Cyclic voltammetry was used to estimate Cu(II/I) solution potentials at pH 6.8, which were very similar for the two peptides (CuHx1 E degrees'=+350 mV, CuHx2 E degrees'=+320 mV vs. normal hydrogen electrode). These results suggest similar binding modes for the two peptides, and relative stabilization of Cu(I) relative to similar His-Gly-rich peptides in the literature.

This study evaluated the potential use of brown seaweed Sargassum sp to sequester lead and copper (Pb(II) and Cu(ll)) from urban runoff based on batch as well as column experiments. The equilibrium data exhibited Langmuir isotherms. The adsorption capacity of this seaweed was found to be 196.1 mgg(-1) and 84.0 mg g(-1) for Pb(ll) and Cu(ll), respectively, which are in good agreement with those values obtained for the aqueous solution (188.6 mg g(-1) for Pb(ll) and 86.9 mg g(-1) for Cu(II)). The functional group analysis of the seaweed using FTIR demonstrated that the carboxyl functional groups are mainly responsible for biosorption. The cation exchange capacity of the biosorbent was 2.25 meq/g. This observation suggested that ion exchange mechanism is predominantly responsible for the metal ion uptake. The column study showed that the highest bed height and the lowest flow rate result in a substantial enhancement of the metals uptake with the biosorption uptake capacities being 264.3 mg Pb(ll) g(-1) and 86.0 mg Cu(ll) g(-1). In the binary system, the biosorption capacity was observed to be 208.7 mg Pb(ll) g(-1) and 61.0 mg Cu(II) g(-1). The predicted breakthrough curves by the Thomas adsorption model gave a good fit of the experimental data with r2 ranging from 0.92 to 0.99.

The divalent metal ion transporter DMT1 is localized in the brush border membrane (BBM) of the upper small intestine and has been shown to be able to transport Mn2+, Fe2+, Co2+, Ni2+, and Cu2+. Belgrade rats have a glycine-to-arginine (G185R) mutation in DMT1, which affects its function. We investigated copper transport with BBM vesicles of Belgrade rats loaded with calcein, which exhibits fluorescence quenching by various metal ions. Transport of copper was disrupted in unenergized BBM vesicle of b/b Belgrade rats, as had been described for iron transport, while +/b vesicles exhibited normal transport by DMT1. When either b/b or +/b vesicles were loaded with ATP and magnesium, similar high-affinity accumulation of copper was observed in both types of vesicles. Thus, brush border membranes possess an ATP-driven, high-affinity copper transport system which could serve as the primary route for copper uptake by the intestine.

Prion protein consists of an N-terminal domain containing a series of octapeptide repeats with the consensus sequence PHGGGWGQ and a C-terminal domain composed of three alpha-helices and two short beta-strands. Several studies have shown that the N-terminal domain binds five Cu2+ ions. In the present study, we have investigated copper-catalysed oxidation of a recombinant mouse prion protein, PrP23-231. The copper-loaded PrP23-231 was found to be carbonylated by incubation with dopamine. Besides the formation of carbonyls, a cross-linked species with the dimeric size and C-terminally truncated species were generated. These reactions were retarded in the presence of Cu+- and Cu2+-specific copper chelators, catalase, and SOD (superoxide dismutase), but not in the presence of various bivalent metal ions. Together, these results indicate that the copper bound to prion protein undergoes catalytic cycling in the presence of catecholamines and causes the oxidation of the protein.

A small metal-binding protein (SmbP) with no known similarity to other proteins in current databases was isolated and characterized from the periplasm of Nitrosomonas europaea. The primary structure of this small (9.9 kDa) monomeric protein is characterized by a series of 10 repeats of a seven amino acid motif and an unusually high number of histidine residues. The protein was isolated from N. europaea with Cu(II) bound but was found to be capable of binding multiple equivalents of a variety of divalent and trivalent metals. The protein was overexpressed in Escherichia coli and used for the study of its metal-binding properties by UV/vis, circular dichroism (CD), and electron paramagnetic resonance (EPR) spectroscopy and equilibrium dialysis and isothermal titration calorimetry. The protein was found to bind up to six Cu(II) atoms with dissociation constants of approximately 0.1 microM for the first two metal ions and approximately 10 microM for the next four. Binding of Cu(II) resulted in spectroscopic features illustrating two distinctive geometries, as determined by EPR spectroscopy. The levels of SmbP in the periplasm were found to increase by increasing the levels of copper in the growth media. This protein is proposed to have a role in cellular copper management in the ammonia-oxidizing bacterium N. europaea.

The potentiometric and spectroscopic (EPR, UV-Vis, CD) data have shown that the chicken prion hexa-repeat (Ac-His-Asn-Pro-Gly-Tyr-Pro-NH(2)) is a very specific ligand for Cu(2+) ions. The His imidazole is an anchoring binding site, then the adjacent amide nitrogen coordinates as a second donor. The presence of Pro at position 3 induces binding of phenolate oxygen as a third donor atom. The tridentate coordination dominates around physiological pH. Similar to human octapeptide fragments, chicken tandem repeats exhibit a cooperative effect in binding Cu(2+) ions, although chicken peptides are much less effective in metal ion coordination.

Thermodynamics of the binding of Ni(2+), Cu(2+) and Zn(2+) to bacitracin A(1) was studied by capillary electrophoresis measuring the peptide effective mobility at different pH in the presence of increasing concentration of the three ligands. The affinity follows the order Ni(2+) > Cu(2+) > Zn(2+), with association constant values of (2.3 +/- 0.1)x10(4), (4.9 +/- 0.2)x10(3), and (1.5 +/- 0.1)x10(3) M(-1), respectively. The only model able to rationalize mobility data implies that metal ion binds to the P(0) peptide form. Moreover, mobility values indicated a change of bacitracin A(1) acidic properties on Ni(2+) and Cu(2+) binding, with a shift of the pK(a) of N-terminal Ile-1 from 7.6 to about 5 and of the pK(a) of the delta-amino group of D-Orn-7 from 9.7 to about 7. Even though on Zn(2+) binding a shift of the N-terminal Ile-1 pK(a) was observed, restrictions in the pH range suitable for investigation, due to precipitation phenomena, did not allow establish if the shift of D-Orn-7 lateral chain pK(a) also occurred. Nonetheless, if present, the shift should be limited to the 7.8-9.7 range. Mobility data indicated that the Stokes radius of the complexes is ca. 3 A lower than that of the free peptide. The present results indicate that metal-ion binding to bacitracin A(1) is more complex than previously assumed.