Metal Ion Binding Sites Research Articles

Examining the influence of bilayer structure on energy transfer and molecular photon upconversion in metal ion linked multilayers.

Metal ion linked multilayers is a unique motif to spatially control and geometrically restrict molecules on a metal oxide surface and is of interest in a number of promising applications. Here we use a bilayer composed of a metal oxide surface, an anthracene annihilator molecule, Zn(II) linking ion, and porphyrin sensitizers to probe the influence of the position of the metal ion binding site on energy transfer, photon upconversion, and photocurrent generation. Despite being energetically similar, varying the position of the carboxy metal ion binding group (i.e. ortho, meta, para) of the Pt(II) tetraphenyl porphyrin sensitizer had a large impact on energy transfer rates and upconverted photocurrent that can be attributed to differences in their geometries. From polarized attenuated total reflectance measurements of the bilayers on ITO, we found that the orientation of the first layer (anthracene) was largely unperturbed by subsequent layers. However, the tilt angle of the porphyrin plane varies dramatically from 41° to 64° to 57° for the para-, meta-, and ortho-COOH substituted porphyrin molecules, which is likely responsible for the variation in energy transfer rates. We go on to show using molecular dynamics simulations that there is considerable flexibility in porphyrin orientation, indicating that an average structure is insufficient to predict the ensemble behavior. Instead, even a small subset of the population with highly favorable energy transfer rates can be the primary driver in increasing the likelihood of energy transfer. Gaining control of the orientation and its distribution will be a critical step in maximizing the potential of the metal ion linked structures.

Characterisation and structural analysis of glyoxylate cycle enzymes of Teladorsagia circumcincta

A 1332 bp full length cDNA encoding Teladorsagia circumcincta isocitrate lyase (TciICL) and a 1575 bp full length cDNA encoding T. circumcincta malate synthase (TciMS) were cloned, expressed in Escherichia coli and the recombinant proteins purified. The predicted TciICL protein of 444 amino acids was present as a single band of about 52 kDa on SDS-PAGE and the recombinant TciMS of 525 amino acids formed a single band about 62 kDa. Multiple alignments of the combined bifunctional TciICL-MS protein sequence with homologues from other nematodes showed that the greatest similarity (89–92 %) to the homologues of Ancylostoma ceylanicum, Haemonchus contortus and Haemonchus placei and 71–87 % similarity to the other nematode sequences. The 3-dimensional structures, binding and catalytic sites were determined for TciICL and TciMS and shown to be highly conserved. Substrate and metal ion binding sites were identified and were completely conserved in other homologues. TciICL was confirmed as a functional enzyme. At 30 °C, the optimum pH was pH 7.5, the Vmax was 275 ± 23 nmoles.min−1. mg-1 protein and the apparent Km for the substrate isocitrate was 0.7 ± 0.01μM (mean ± SEM, n = 3). Addition of 10 mM metal ions (except Mg2+) or 1 mM inhibitors reduced the recombinant TciICL activity by 60–90 %. Antibodies in both serum and saliva from field-immune, but not nematode-naïve, sheep recognised recombinant TciICL in ELISA, supporting similar antigenicity to that of the native enzyme.

Copper binding by the cystatin C fragment. The role of histidine residues

Human cystatin C (hCC) is one of the amyloidogenic molecules involved in neurodegeneration processes. Fragment 85–94, with the FHDQPHLKRK sequence, is interesting from the point of view of coordination as histidine and aspartic acid residues are potential copper(II) ion donors. Our previous studies showed that two potential binding sites are possible within this peptide chain. Therefore, the coordination abilities of each of these two metal ion binding sites have been checked. We have analysed two analogues of the native peptide, where particular histidine residues were substituted with alanine – an amino acid without a coordinating side chain: Ac-H2A (with alanine in position 2) and Ac-H6A (with alanine in position 6). NMR, potentiometric titration, UV–Vis, CD, EPR and MS studies were carried out. Obtained results showed that both ligands finally form the 4 N copper(II) complexes in a basic range of pH. However, their coordination properties are slightly different. The copper(II) binding efficiency is also different in both cases. Up to pH 6–6.5, AcH2A is more effective, but the situation is quite reversed under more basic conditions. Considering the results obtained, it can be concluded that in the unmodified peptide, the N-terminal binding site seems to be more favourable than the C-terminal.

Nanopore detection of metal ions: Current status and future directions.

In this review, we highlight recent research efforts that aimed at developing nanopore sensors for detection of metal ions, which play a crucial role in environmental safety and human health. Protein pores use three stochastic sensing-based strategies for metal ion detection. The first strategy is to construct engineered nanopores with metal ion binding sites, so that the interaction between the target analytes and the nanopore can slow the movement of metal ions in the nano-channel. Second, large molecules such as nucleic acids and especially peptides could be utilized as external selective molecular probes to detect metal ions based on the conformational change of the ligand molecules induced by the metal ion-ligand chelation / coordination interaction. Third, enzymatic reactions can also be used as an alternative to the molecule probe strategy in the situation that a sensitive and selective probe molecule for the target analyte is difficult to obtain. On the other hand, by taking advantage of steady-state analysis, synthetic nanopores mainly use two strategies (modification and modification-free) to detect metals. Given the advantages of high sensitivity & selectivity, and label-free detection, nanopore-based metal ion sensors should find useful application in many fields, including environmental monitoring, medical diagnosis, and so on.

Characterization of Putative Virulence Factors of Pseudomonas aeruginosa Strain RBS Isolated from a Saltern, Tunisia: Effect of Metal Ion Cofactors on the Structure and the Activity of LasB.

Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium able to survive in diverse environments such as soil, plants, freshwater, and seawater. P. aeruginosa can be an opportunistic pathogen to humans when their immune system is deficient. Its pathogenicity may be linked to the production of virulence factors. We isolated P. aeruginosa strain RBS from the saltern of Sfax in Tunisia. In this study, we characterized the halotolerance, antibiotic susceptibility, and some virulence factors of strain RBS. High NaCl concentrations inhibited growth and motility. However, biofilm formation was enhanced to protect bacteria against salt stress. Among the 18 antibiotics tested, quinolones and tetracycline showed a significant inhibitory effect on growth, motility, and biofilm formation of strain RBS. β-Lactams, however, did not have any inhibitory effect on neither bacterial growth nor motility. In some cases, resistance was due, in part, to biofilm formation. We also showed that RBS produces two proteases, LasB and AprA, which have been shown to be implicated in host infection. LasB was further characterized to study the role of metal ions in enzyme stability. It possesses two distinct metal ion-binding sites coordinating a calcium and a zinc ion. The effect of metal ion chelation was evaluated as well as substitutions of residues involved in metal ion binding. Impairing metal ion binding of LasB led to a loss of activity and a sharp decrease of stability. Our findings suggest that the binding of both metal ions is interdependent as the two metal ions' binding sites are linked via a hydrogen bond network.

Crystal Structure of Cytochrome cL from the Aquatic Methylotrophic Bacterium Methylophaga aminisulfidivorans MPT.

Cytochrome cL (CytcL) is an essential protein in the process of methanol oxidation in methylotrophs. It receives an electron from the pyrroloquinoline quinone (PQQ) cofactor of methanol dehydrogenase (MDH) to produce formaldehyde. The direct electron transfer mechanism between CytcL and MDH remains unknown due to the lack of structural information. To help gain a better understanding of the mechanism, we determined the first crystal structure of heme c containing CytcL from the aquatic methylotrophic bacterium Methylophaga aminisulfidivorans MPT at 2.13 Å resolution. The crystal structure of Ma-CytcL revealed its unique features compared to those of the terrestrial homologues. Apart from Fe in heme, three additional metal ion binding sites for Na+ , Ca+ , and Fe2+ were found, wherein the ions mostly formed coordination bonds with the amino acid residues on the loop (G93-Y111) that interacts with heme. Therefore, these ions seemed to enhance the stability of heme insertion by increasing the loop's steadiness. The basic N-terminal end, together with helix α4 and loop (G126 to Y136), contributed positive charge to the region. In contrast, the acidic C-terminal end provided a negatively charged surface, yielding several electrostatic contact points with partner proteins for electron transfer. These exceptional features of Ma-CytcL, along with the structural information of MDH, led us to hypothesize the need for an adapter protein bridging MDH to CytcL within appropriate proximity for electron transfer. With this knowledge in mind, the methanol oxidation complex reconstitution in vitro could be utilized to produce metabolic intermediates at the industry level.

Iminodiacetic acid-functionalized porous polymer for removal of toxic metal ions from water

The design of efficient adsorbent with abundant binding sites for heavy metal ions is crucial for developing innovative materials that will remove pollutant metal ions. The high uptake capacity, kinetics, and affinity towards the toxic metals are the key requirements that the materials under invesigation should accomplish. Here we report the synthesis of iminodiacetic acid-functionalized hypercrosslinked polymer (IDA-HCP) for purification of water polluted by toxic metal ions via coordination of carboxylate and amino active sites on the surface of porous polymer. The obtained porous polymer is stable under harsh conditions and the structural features on the polymer work together to help the removal of Pb(II) with 1138 mg g–1 uptake capacity. In the meanwhile, the IDA-HCP reveals reuseability and very promising capture efficiency not only for Pb2+, but also for Hg2+ and Cd2+ from a mixture of Pb2+, Hg2+, Cd2+, Co2+, Fe3+, Zn2+, Mg2+, and Na+ metal ions. This result gives us confidence that the polymer material can solve the pollution problem caused by various metal ions.

Dynamer and Metallodynamer Interconversion: An Alternative View to Metal Ion Complexation.

A bifunctional molecule containing both a bidentate binding site for metal ions and an aminopyrimidine H-bond donor-acceptor site has been synthesized, and its properties, in its free and coordinated forms, have been established in solution and in the solid state by analytical and spectroscopic methods as well as by X-ray structure determinations. Structural characterization has shown that it forms a one-dimensional H-bonded polymeric assembly in the solid state, while spectroscopic measurements indicate that it also aggregates in solution. The reaction of a simple Fe(II) salt with this assembly results in the emergence of two geometrical isomers of the complex: [FeL3](BF4)2·9H2O-C1 (meridional, mer) and [FeL3]2(SiF6)(BF4)2·12H2O-C2 (facial, fac). While, complex C1 in the solid state generates a one-dimensional H-bonded polymer involving just two ligands on each Fe center, with the chirality of the complex units alternating along the polymer chain, the structure of complex C2 shows NH···N interactions seen in both the ligand and mer complex (C1) structures to be completely absent. Physicochemical properties of the free and complexed ligand differ substantially.

A novel anthrapyridone diamine-based probe for selective and distinctive Cu2+ and Hg2+ sensing in aqueous solution; utility as molecular logic gates

A newly synthesized anthrapyridone-based diamine (probe 1) having two metal ion binding sites exhibited selective and sensitive sensing of Cu2+ or Hg2+ among various metal ions. Upon addition of the metal ion, the probe 1-containing solution vividly changed color, enabling visual detection of each metal ion (Cu2+ or Hg2+) without requiring any instruments. A ratiometric approach insensitive to environmental interference provided limit of detections (LODs) of 1.0 and 2.0 μM for Cu2+ and Hg2+ sensing, respectively. The green fluorescence emission of probe 1 was entirely (Cu2+) or partially (Hg2+) quenched in the presence of the metal ion, yielding an observed fluorescence-based LOD (5/200 nM for Cu2+/Hg2+). Thus, probe 1 showed a dual responsiveness for detection of the metal ion. A model probe (2) with only a single metal binding site was ineffective at sensing the metal ion (Cu2+ or Hg2+) under the same conditions, demonstrating that the presence of the two binding sites in probe 1 is essential for the sensitive detection of the metal ion. The DFT calculations suggest binding of the metal ion to the probe with a stoichiometry of 1:1 or 2:1 (M2+: probe). Probe 1 was also effective at detecting a small amount of Cu2+ in live HeLa cells or real samples such as tap and lake waters. The UV–Vis. absorbance results of probe 1 with Cu2+ and Hg2+ enabled us to build ‘NOR-YES-INHIBIT’ molecular logic gates. The metal ion sensing system presented here was extensively investigated by various techniques including UV–visible, fluorescence, NMR spectroscopy, DFT calculations, FE-SEM, and DLS.

A Chromone Based Fluorescent Probe for the Effective Detection of Aluminium Ion.

A new chromone moiety fused with phenyl hydrazine carboxamide as a binding site for metal ion was designed and synthesized as a colorimetric/fluorimetric probe for selective sensing of Al3+ ion. The absorption and fluorescence spectral results showed selective and sensitive recognition of Al3+ with no significant interference from other competitive metal ions. The addition of Al3+ to R brought a prompt color change from colorless to yellow and the turn-on fluorescence response with Al3+ among different cations was studied. The visible color change and turn-on fluorescence Al3+ ion with R was attributed to the intra molecular charge transfer transition. The plausible binding nature of the receptor with Al3+ has also been anticipated by the results of 1H NMR and mass spectral analysis. For the practical effectiveness, test strip prepared using probe R was successfully applied to monitor the presence of Al3+ ion in aqueous medium.

Crystallographic characterization of a tri-Asp metal-binding site at the three-fold symmetry axis of LarE

Detailed crystallographic characterization of a tri-aspartate metal-binding site previously identified on the three-fold symmetry axis of a hexameric enzyme, LarE from Lactobacillus plantarum, was conducted. By screening an array of monovalent, divalent, and trivalent metal ions, we demonstrated that this metal binding site stoichiometrically binds Ca2+, Mn2+, Fe2+/Fe3+, Co2+, Ni2+, Cu2+, Zn2+, and Cd2+, but not monovalent metal ions, Cr3+, Mg2+, Y3+, Sr2+ or Ba2+. Extensive database searches resulted in only 13 similar metal binding sites in other proteins, indicative of the rareness of tri-aspartate architectures, which allows for engineering such a selective multivalent metal ion binding site into target macromolecules for structural and biophysical characterization.

Paramagpy: software for fitting magnetic susceptibility tensors using paramagnetic effects measured in NMR spectra.

Paramagnetic metal ions with fast-relaxing electrons generate pseudocontact shifts (PCSs), residual dipolar couplings (RDCs), paramagnetic relaxation enhancements (PREs) and cross-correlated relaxation (CCR) in the nuclear magnetic resonance (NMR) spectra of the molecules they bind to. These effects offer long-range structural information in molecules equipped with binding sites for such metal ions. Here we present the new open-source software Paramagpy, which has been written in Python 3 with a graphic user interface. Paramagpy combines the functionalities of different currently available programs to support the fitting of magnetic susceptibility tensors using PCS, RDC, PRE and CCR data and molecular coordinates in Protein Data Bank (PDB) format, including a convenient graphical user interface. Paramagpy uses efficient fitting algorithms to avoid local minima and supports corrections to back-calculated PCS and PRE data arising from cross-correlation effects with chemical shift tensors. The source code is available from 10.5281/zenodo.3594568 .

Adsorption of heavy metal from industrial wastewater onto low-cost Malaysian kaolin clay-based adsorbent.

Adsorption capacity and percentage removal efficiency of Cu(II) and Ni(II) ions were studied and compared between raw kaolinite and acid-activated kaolinite. Acid-activated kaolin was prepared by refluxing raw kaolinite with concentrated sulphuric acid followed by calcination to enhance its surface properties and adsorption ability. Both raw and acid-activated kaolinite samples were characterized by Fourier transform infrared spectroscopy, energy dispersive X-ray, scanning electron micrograph and zeta potential analysis. Upon acid treatment, acid-activated kaolinite was discovered to have altered chemical composition and larger BET surface area as compared with raw kaolinite. The batch adsorption studies on aqueous solution were performed under different factors such as contact time, pH condition, adsorbent dosage, initial metal ion concentration and temperature. The optimum condition was selected for each factor including a contact time of 60min, pH of 7.0, adsorbent dosage of 0.1g, initial metal ion concentration of 100mg/L and temperature of 25°C. Then, the adsorption studies on wastewater samples were carried out at the selected optimum conditions. Acid-activated kaolinite always had better adsorption capacity and percentage removal efficiency than raw kaolinite due to the increasing amount of negative charges on the adsorbent surface and the number of metal ion binding sites upon acid treatment. The adsorption kinetic obtained was well described by the pseudo-second-order model, whereas the adsorption isotherms obtained were well described by either the Freundlich or the Langmuir adsorption model. The results showed that acid-activated kaolinite adsorbent is a better option as a favourable and feasible commercial low-cost adsorbent for wastewater treatment.

Partial Metal Ion Saturation of C2 Domains Primes Synaptotagmin 1-Membrane Interactions

Synaptotagmin 1 (Syt1) is an integral membrane protein whose phospholipid-binding tandem C2 domains, C2A and C2B, act as Ca2+ sensors of neurotransmitter release. Our objective was to understand the role of individual metal-ion binding sites of these domains in the membrane association process. We used Pb2+, a structural and functional surrogate of Ca2+, to generate the protein states with well-defined protein-metal ion stoichiometry. NMR experiments revealed that binding of one divalent metal ion per C2 domain results in loss of conformational plasticity of the loop regions, potentially pre-organizing them for additional metal-ion and membrane-binding events. In C2A, a divalent metal ion in site 1 is sufficient to drive its weak association with phosphatidylserine-containing membranes, whereas in C2B, it enhances the interactions with the signaling lipid phosphatidylinositol-4,5-bisphosphate. In full-length Syt1, both Pb2+-complexed C2 domains associate with phosphatidylserine-containing membranes. Electron paramagnetic resonance experiments show that the extent of membrane insertion correlates with the occupancy of the C2 metal ion sites. Together, our results indicate that upon partial metal ion saturation of the intra-loop region, Syt1 adopts a dynamic, partially membrane-bound state. The properties of this state, such as conformationally restricted loop regions and positioning of C2 domains in close proximity to anionic lipid headgroups, “prime” Syt1 for cooperative binding of a full complement of metal ions and deeper membrane insertion.

Poorly differentiated thyroid carcinoma of childhood and adolescence: a distinct entity characterized by DICER1 mutations.

Poorly differentiated thyroid carcinomas (PDTC) in young individuals are rare and their clinical and histopathologic features, genetic mechanisms, and outcomes remain largely unknown. Here, we report a detailed characterization of a series of 6 PDTC in patients ≤ 21 years old defined by Turin diagnostic criteria studied for mutations and gene fusions characteristic of thyroid cancer using targeted next-generation sequencing (NGS) and whole exome sequencing (WES). All tumors had solid, insular or trabecular growth pattern and high mitotic rate, and 5 out of 6 tumors showed tumor necrosis. Targeted NGS assay identified somatic mutations in the DICER1 gene in 5 of 6 (83%) tumors, all of which were “hotspot” mutations encoding the metal-ion binding sites of the RNase IIIb domain of DICER1. WES was performed in 5 cases which confirmed all hotspot mutations and detected 2 tumors with additional inactivating DICER1 alterations. Of these two, one was a germline pathogenic DICER1 variant and the other had loss of heterozygosity for DICER1. No other mutations or gene fusions characteristic of adult well-differentiated thyroid cancer and PDTC (BRAF, RAS, TERT, RET/PTC and other) were detected. On follow-up, available for 5 patients, 3 patients died of disease 8-24 months after diagnosis, whereas 2 were alive with no disease. The results of our study demonstrate that childhood- and adolescent-onset PDTC are genetically distinct from adult-onset PDTC in that they are strongly associated with DICER1 mutations and may herald DICER1 syndrome in a minority. As such, all young persons with PDTC may benefit from genetic counselling. Furthermore, their clinically aggressive behavior contrasts sharply with the indolent nature of the great majority of thyroid tumors with DICER1 mutations reported to date.

Insulin-copper quantum clusters preparation and receptor targeted bioimaging

Protein embedded fluorescence quantum clusters (QCs) have received a great amount of interest among the researchers because of their high aqueous solubility, stability, cost efficiency, and target specificity. Considerable advancement has happened in making functional quantum clusters with target specificity. This work reports the simple synthesis of insulin protected copper quantum clusters (ICuQCs) and its receptor-targeted bioimaging applications. The preparation of copper quantum clusters (CuQCs) was done simply by one-pot synthesis method by changing the pH of the insulin protein firstly to 10.5 basic pH than physiological pH. At physiological pH, the mixture incubated in oven 37 ⁰C at 240 rpm has been developed to process initially polydisperse, non-fluorescent, and unstable CuDs into monodispersed (∼2−3 nm), highly fluorescent, and extremely stable ICuQCs in the same phase (aqueous) using insulin as protein. HRTEM image show uniform distribution of CuDs within the protein matrix. Metal ion binding site prediction and docking server (MIB) results show that chain B of insulin contains 3 templates contains 5 amino acid residues which bind with Cu2+ metal ion. Groove 1 contains GLY8 and HIS10 bind has the highest binding potential towards Cu metal ions. The methodology adopted in this study should largely contribute to the practical applications of this new class of QCs. In view of the protein protection, coupled with direct synthesis and easy functionalization, this hybrid QC-protein system is expected to have numerous optical and bioimaging applications in the future.

The yeast cell wall protein Pry3 inhibits mating through highly conserved residues within the CAP domain.

ABSTRACTMembers of the CAP/SCP/TAPS superfamily have been implicated in many different physiological processes, including pathogen defense, sperm maturation and fertilization. The mode of action of this class of proteins, however, remains poorly understood. The genome of Saccharomyces cerevisiae encodes three CAP superfamily members, Pry1-3. We have previously shown that Pry1 function is required for the secretion of sterols and fatty acids. Here, we analyze the function of Pry3, a GPI-anchored cell wall protein. Overexpression of Pry3 results in strong reduction of mating efficiency, providing for a cell-based readout for CAP protein function. Mating inhibition is a conserved function of the CAP domain and depends on highly conserved surface exposed residues that form part of a putative catalytic metal-ion binding site. Pry3 displays polarized cell surface localization adjacent to bud scars, but is absent from mating projections. When overexpressed, however, the protein leaks onto mating projections, suggesting that mating inhibition is due to mislocalization of the protein. Trapping of the CAP domain within the cell wall through a GPI-anchored nanobody results in a dose-dependent inhibition of mating, suggesting that a membrane proximal CAP domain inhibits a key step in the mating reaction, which is possibly related to the function of CAP domain proteins in mammalian fertilization.This article has an associated First Person interview with the first author of the paper.

The L-platform/L-scaffold framework: a blueprint for RNA-cleaving nucleic acid enzyme design.

We develop an L-platform/L-scaffold framework we hypothesize may serve as a blueprint to facilitate site-specific RNA-cleaving nucleic acid enzyme design. Building on the L-platform motif originally described by Suslov and coworkers, we identify new critical scaffolding elements required to anchor a conserved general base guanine (“L-anchor”) and bind functionally important metal ions at the active site (“L-pocket”). Molecular simulations, together with a broad range of experimental structural and functional data, connect the L-platform/L-scaffold elements to necessary and sufficient conditions for catalytic activity. We demonstrate that the L-platform/L-scaffold framework is common to five of the nine currently known naturally occurring ribozyme classes (Twr, HPr, VSr, HHr, Psr), and intriguingly from a design perspective, the framework also appears in an artificially engineered DNAzyme (8–17dz). The flexibility of the L-platform/L-scaffold framework is illustrated on these systems, highlighting modularity and trends in the variety of known general acid moieties that are supported. These trends give rise to two distinct catalytic paradigms, building on the classifications proposed by Wilson and coworkers and named for the implicated general base and acid. The “G + A” paradigm (Twr, HPr, VSr) exclusively utilizes nucleobase residues for chemistry, and the “G + M + ” paradigm (HHr, 8–17dz, Psr) involves structuring of the “L-pocket” metal ion binding site for recruitment of a divalent metal ion that plays an active role in the chemical steps of the reaction. Finally, the modularity of the L-platform/L-scaffold framework is illustrated in the VS ribozyme where the “L-pocket” assumes the functional role of the “L-anchor” element, highlighting a distinct mechanism, but one that is functionally linked with the hammerhead ribozyme.

Structural analysis of the manganese transport regulator MntR from Bacillus halodurans in apo and manganese bound forms.

The manganese transport regulator MntR is a metal-ion activated transcriptional repressor of manganese transporter genes to maintain manganese ion homeostasis. MntR, a member of the diphtheria toxin repressor (DtxR) family of metalloregulators, selectively responds to Mn2+ and Cd2+ over Fe2+, Co2+ and Zn2+. The DtxR/MntR family members are well conserved transcriptional repressors that regulate the expression of metal ion uptake genes by sensing the metal ion concentration. MntR functions as a homo-dimer with one metal ion binding site per subunit. Each MntR subunit contains two domains: an N-terminal DNA binding domain, and a C-terminal dimerization domain. However, it lacks the C-terminal SH3-like domain of DtxR/IdeR. The metal ion binding site of MntR is located at the interface of the two domains, whereas the DtxR/IdeR subunit contains two metal ion binding sites, the primary and ancillary sites, separated by 9 Å. In this paper, we reported the crystal structures of the apo and Mn2+-bound forms of MntR from Bacillus halodurans, and analyze the structural basis of the metal ion binding site. The crystal structure of the Mn2+-bound form is almost identical to the apo form of MntR. In the Mn2+-bound structure, one subunit contains a binuclear cluster of manganese ions, the A and C sites, but the other subunit forms a mononuclear complex. Structural data about MntR from B. halodurans supports the previous hypothesizes about manganese-specific activation mechanism of MntR homologues.

Copper(II) Coordination Abilities of the Tau Protein's N-Terminus Peptide Fragments: A Combined Potentiometric, Spectroscopic and Mass Spectrometric Study.

Copper(II) complexes of the N-terminal peptide fragments of tau protein have been studied by potentiometric and various spectroscopic techniques (UV-vis, CD, ESR and ESI-MS). The octapeptide Tau(9-16) (Ac-EVMEDHAG-NH2 ) contains the H14 residue of the native protein, while Tau(26-33) (Ac-QGGYTMHQ-NH2 ) and its mutants Tau(Q26K-Q33K) (Ac-KGGYTMHK-NH2 ) and Tau(Q26K-Y29A-Q33K) (Ac-KGGATMHK-NH2 ) include the H32 residue. To compare the binding ability of H14 and H32 in a single molecule the decapeptide Ac-EDHAGTMHQD-NH2 (Tau(12-16)(30-34)) has also been synthesized and studied. The histidyl residue is the primary metal binding site for metal ions in all the peptide models studied. In the case of Tau(9-16) the side chain carboxylate functions enhance the stability of the M-Nim coordinated complexes compared to Tau(26-33) (logK(Cu-Nim )=5.04 and 3.78, respectively). Deprotonation and metal ion coordination of amide groups occur around the physiological pH range for copper(II). The formation of the imidazole- and amide-coordinated species changes the metal ion preference and the complexes formed with the peptides containing the H32 residue predominate over those of H14 at physiological pH values (90 %-10 %) and in alkaline samples (96 %-4 %).