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Divalent Metal Ions (divalent + metal_ion)
Selected AbstractsCloning, crystallization and preliminary X-ray studies of XC2981 from Xanthomonas campestris, a putative CutA1 protein involved in copper-ion homeostasisACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 11 2006Chien-Hung Lin Divalent metal ions play key roles in all living organisms, serving as cofactors for many proteins involved in a variety of electron-transfer activities. However, copper ions are highly toxic when an excessive amount is accumulated in a cell. CutA1 is a protein found in all kingdoms of life that is believed to participate in copper-ion tolerance in Escherichia coli, although its specific function remains unknown. Several crystal structures of multimeric CutA1 with different rotation angles and degrees of interaction between trimer interfaces have been reported. Here, the cloning, expression, crystallization and preliminary X-ray analysis of XC2981, a possible CutA1 protein present in the plant pathogen Xanthomonas campestris, are reported. The XC2981 crystals diffracted to a resolution of 2.6,Å. They are cubic and belong to space group I23, with unit-cell parameters a = b = c = 130.73,Å. [source] Determination of the metal ion dependence and substrate specificity of a hydratase involved in the degradation pathway of biphenyl/chlorobiphenylFEBS JOURNAL, Issue 4 2005Pan Wang BphH is a divalent metal ion-dependent hydratase that catalyzes the formation of 2-keto-4-hydroxypentanoate from 2-hydroxypent-2,4-dienoate (HPDA). This reaction lies on the catabolic pathway of numerous aromatics, including the significant environmental pollutant, polychlorinated biphenyls (PCBs). BphH from the PCB degrading bacterium, Burkholderia xenoverans LB400, was overexpressed and purified to homogeneity. Atomic absorption spectroscopy and Scatchard analysis reveal that only one divalent metal ion is bound to each enzyme subunit. The enzyme exhibits the highest activity when Mg2+ was used as cofactor. Other divalent cations activate the enzyme in the following order of effectiveness: Mg2+ > Mn2+ > Co2+ > Zn2+ > Ca2+. This differs from the metal activation profile of the homologous hydratase, MhpD. UV-visible spectroscopy of the Co2+,BphH complex indicates that the divalent metal ion is hexa-coordinated in the enzyme. The nature of the metal ion affected only the kcat and not the Km values in the BphH hydration of HPDA, suggesting that cation has a catalytic rather than just a substrate binding role. BphH is able to transform alternative substrates substituted with methyl- and chlorine groups at the 5-position of HPDA. The specificity constants (kcat/Km) for 5-methyl and 5-chloro substrates are, however, lowered by eight- and 67-fold compared with the unsubstituted substrate. Significantly, kcat for the chloro-substituted substrate is eightfold lower compared with the methyl-substituted substrate, showing that electron withdrawing substituent at the 5-position of the substrate has a negative influence on enzyme catalysis. [source] Conformational states of human H-Ras detected by high-field EPR, ENDOR, and 31P NMR spectroscopy ,MAGNETIC RESONANCE IN CHEMISTRY, Issue S1 2005Michael Spoerner Abstract Ras is a central constituent of the intracellular signal transduction that switches between its inactive state with GDP bound and its active state with GTP bound. A number of different X-ray structures are available. Different magnetic resonance techniques were used to characterise the conformational states of the protein and are summarised here. 31P NMR spectroscopy was used as probe for the environment of the phosphate groups of the bound nucleotide. It shows that in liquid solution additional conformational states in the GDP as well as in the GTP forms coexist which are not detected by X-ray crystallography. Some of them can also be detected by solid-state NMR in the micro crystalline state. EPR and ENDOR spectroscopy were used to probe the environment of the divalent metal ion (Mg2+ was replaced by Mn2+) bound to the nucleotide in the protein. Here again different states could be observed. Substitution of normal water by 17O-enriched water allowed the determination of the number of water molecules in the first coordination sphere of the metal ion. In liquid solution, they indicate again the existence of different conformational states. At low temperatures in the frozen state ENDOR spectroscopy suggests that only one state exists for the GDP- and GTP-bound form of Ras, respectively. Copyright © 2005 John Wiley & Sons, Ltd. [source] Affinity cleavage at the divalent metal site of porcine NAD-specific isocitrate dehydrogenasePROTEIN SCIENCE, Issue 1 2000Yu-Chu Huang Abstract A divalent metal ion, such as Mn2+, is required for the catalytic reaction and allosteric regulation of pig heart NAD-dependent isocitrate dehydrogenase. The enzyme is irreversibly inactivated and cleaved by Fe2+ in the presence of O2 and ascorbate at pH 7.0. Mn2+ prevents both inactivation and cleavage. Nucleotide ligands, such as NAD, NADPH, and ADP, neither prevent nor promote inactivation or cleavage of the enzyme by Fe2+. The NAD-specific isocitrate dehydrogenase is composed of three distinct subunits in the ratio 2,:1 ,:1 ,. The results indicate that the oxidative inactivation and cleavage are specific and involve the 40 kDa , subunit of the enzyme. A pair of major peptides is generated during Fe2+ inactivation: 29.5 + 10.5 kDa, as determined by SDS-PAGE. Amino-terminal sequencing reveals that these peptides arise by cleavage of the Val262-His263 bond of the , subunit. No fragments are produced when enzyme is incubated with Fe2+ and ascorbate under denaturing conditions in the presence of 6 M urea, indicating that the native structure is required for the specific cleavage. These results suggest that His263 of the , subunit may be a ligand of the divalent metal ion needed for the reaction catalyzed by isocitrate dehydrogenase. Isocitrate enhances the inactivation of enzyme caused by Fe2+ in the presence of oxygen, but prevents the cleavage, suggesting that inactivation occurs by a different mechanism when metal ion is bound to the enzyme in the presence of isocitrate: oxidation of cysteine may be responsible for the rapid inactivation in this case. Affinity cleavage caused by Fe2+ implicates , as the catalytic subunit of the multisubunit porcine NAD-dependent isocitrate dehydrogenase. [source] Structure and conformational stability of a tetrameric thermostable N -succinylamino acid racemaseBIOPOLYMERS, Issue 9 2009Joaquín Pozo-Dengra Abstract The N-succinylamino acid racemases (NSAAR) belong to the enolase superfamily and they are large homooctameric/hexameric species that require a divalent metal ion for activity. We describe the structure and stability of NSAAR from Geobacillus kaustophilus (GkNSAAR) in the absence and in the presence of Co2+ by using hydrodynamic and spectroscopic techniques. The Co2+, among other assayed divalent ions, provides the maximal enzymatic activity at physiological pH. The protein seems to be a tetramer with a rather elongated shape, as shown by AU experiments; this is further supported by the modeled structure, which keeps intact the largest tetrameric oligomerization interfaces observed in other homooctameric members of the family, but it does not maintain the octameric oligomerization interfaces. The native functional structure is mainly formed by ,-helix, as suggested by FTIR and CD deconvoluted spectra, with similar percentages of structure to those observed in other protomers of the enolase superfamily. At low pH, the protein populates a molten-globule-like conformation. The GdmCl denaturation occurs through a monomeric intermediate, and thermal denaturation experiments indicate a high thermostability. The presence of the cofactor Co2+ did alter slightly the secondary structure, but it did not modify substantially the stability of the protein. Thus, GkNSAAR is one of the few members of the enolase family whose conformational propensities and stability have been extensively characterized. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 757,772, 2009. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] Metal,Organic Perovskites: Synthesis, Structures, and Magnetic Properties of [C(NH2)3][MII(HCOO)3] (M=Mn, Fe, Co, Ni, Cu, and Zn; C(NH2)3= Guanidinium)CHEMISTRY - A EUROPEAN JOURNAL, Issue 44 2009Ke-Li Hu Abstract We report the synthesis, crystal structures, and spectral, thermal, and magnetic properties of a family of metal,organic perovskite ABX3, [C(NH2)3][MII(HCOO)3], in which A=C(NH2)3 is guanidinium, B=M is a divalent metal ion (Mn, Fe, Co, Ni, Cu, or Zn), and X is the formate HCOO,. The compounds could be synthesized by either diffusion or hydrothermal methods from water or water-rich solutions depending on the metal. The five members (Mn, Fe, Co, Ni, and Zn) are isostructural and crystallize in the orthorhombic space group Pnna, while the Cu member in Pna21. In the perovskite structures, the octahedrally coordinated metal ions are connected by the anti,anti formate bridges, thus forming the anionic NaCl-type [M(HCOO)3], frameworks, with the guanidinium in the nearly cubic cavities of the frameworks. The Jahn,Teller effect of Cu2+ results in a distorted anionic Cu,formate framework that can be regarded as Cu,formate chains through short basal CuO bonds linked by the long axial CuO bonds. These materials show higher thermal stability than other metal,organic perovskite series of [AmineH][M(HCOO)3] templated by the organic monoammonium cations (AmineH+) as a result of the stronger hydrogen bonding between guanidinium and the formate of the framework. A magnetic study revealed that the five magnetic members (except Zn) display spin-canted antiferromagnetism, with a Néel temperature of 8.8 (Mn), 10.0 (Fe), 14.2 (Co), 34.2 (Ni), and 4.6,K (Cu). In addition to the general spin-canted antiferromagnetism, the Fe compound shows two isothermal transformations (a spin-flop and a spin-flip to the paramagnetic phase) within 50,kOe. The Co member possesses quite a large canting angle. The Cu member is a magnetic system with low dimensional character and shows slow magnetic relaxation that probably results from the domain dynamics. [source] Comparative Solution Equilibrium Study of the Interactions of Copper(II), Iron(II) and Zinc(II) with Triapine (3-Aminopyridine-2-carbaldehyde Thiosemicarbazone) and Related LigandsEUROPEAN JOURNAL OF INORGANIC CHEMISTRY, Issue 11 2010Éva A. Enyedy Abstract The interactions of CuII, ZnII and FeII with Triapine (3-aminopyridine-2-carbaldehyde thiosemicarbazone), which is currently undergoing phase II clinical trials as a chemotherapeutic antitumour agent, were investigated in a water/DMSO mixture. The proton-dissociation constants of the ligands, the stability constants and the coordination modes of the metal complexes formed were determined by pH-potentiometric, UV/Vis spectrophotometric, EPR, 1H NMR spectroscopic and ESI-MS methods. Two N-terminally dimethylated derivatives of Triapine were also studied. Mono- and bis-ligand complexes in different protonation states were identified. Furthermore, the formation of the dinuclear species [Cu2L3]+ was confirmed for all ligands by EPR spectroscopy and ESI-MS measurements. The results showed that the N-terminally dimethylated ligands are much more potent chelators than Triapine for the divalent metal ions studied. All three ligands formed the least stable complexes with ZnII, whereas the FeII complexes were somewhat more stable than the corresponding CuII species. [source] Isoprenoid biosynthesis in plants , 2C -methyl- d -erythritol-4-phosphate synthase (IspC protein) of Arabidopsis thalianaFEBS JOURNAL, Issue 19 2006Felix Rohdich The ispC gene of Arabidopsis thaliana was expressed in pseudomature form without the putative plastid-targeting sequence in a recombinant Escherichia coli strain. The recombinant protein was purified by affinity chromatography and was shown to catalyze the formation of 2C -methyl- d -erythritol 4-phosphate from 1-deoxy- d -xylulose 5-phosphate at a rate of 5.6 µmol·min,1·mg,1 (kcat 4.4 s,1). The Michaelis constants for 1-deoxy- d -xylulose 5-phosphate and the cosubstrate NADPH are 132 and 30 µm, respectively. The enzyme has an absolute requirement for divalent metal ions, preferably Mn2+ and Mg2+, and is inhibited by fosmidomycin with a Ki of 85 nm. The pH optimum is 8.0. NADH can substitute for NADPH, albeit at a low rate (14% as compared to NADPH). The enzyme catalyzes the reverse reaction at a rate of 2.1 µmol·min -1·mg -1. [source] Comparative metal binding and genomic analysis of the avian (chicken) and mammalian metallothioneinFEBS JOURNAL, Issue 3 2006Laura Villarreal Chicken metallothionein (ckMT) is the paradigm for the study of metallothioneins (MTs) in the Aves class of vertebrates. Available literature data depict ckMT as a one-copy gene, encoding an MT protein highly similar to mammalian MT1. In contrast, the MT system in mammals consists of a four-member family exhibiting functional differentiation. This scenario prompted us to analyse the apparently distinct evolutionary patterns followed by MTs in birds and mammals, at both the functional and structural levels. Thus, in this work, the ckMT metal binding abilities towards Zn(II), Cd(II) and Cu(I) have been thoroughly revisited and then compared with those of the mammalian MT1 and MT4 isoforms, identified as zinc- and copper-thioneins, respectively. Interestingly, a new mechanism of MT dimerization is reported, on the basis of the coordinating capacity of the ckMT C-terminal histidine. Furthermore, an evolutionary study has been performed by means of in silico analyses of avian MT genes and proteins. The joint consideration of the functional and genomic data obtained questions the two features until now defining the avian MT system. Overall, in vivo and in vitro metal-binding results reveal that the Zn(II), Cd(II) and Cu(I) binding abilities of ckMT lay between those of mammalian MT1 and MT4, being closer to those of MT1 for the divalent metal ions but more similar to those of MT4 for Cu(I). This is consistent with a strong functional constraint operating on low-copy number genes that must cope with differentiating functional limitation. Finally, a second MT gene has been identified in silico in the chicken genome, ckMT2, exhibiting all the features to be considered an active coding region. The results presented here allow a new insight into the metal binding abilities of warm blooded vertebrate MTs and their evolutionary relationships. [source] Intramolecular proton transfer induced by divalent alkali earth metal cation in the gas stateINTERNATIONAL JOURNAL OF QUANTUM CHEMISTRY, Issue 4 2003Hongqi Ai Abstract Interactions between divalent alkali earth metal (DAEM) ions M (MBe, Mg, Ca, Sr, Ba) and the second stable glycine conformer in the gas phase, which can transfer into the ground-state glycine-M2+ (except the glycine,Be2+) among each corresponding isomers when these divalent metal ions are bound, are studied at the hybrid three-parameter B3LYP level with three different basis sets. Proton transfers from the hydroxyl to the amino nitrogen of the glycine without energy barriers have been first observed in the gas phase in these glycine,M2+ systems. The interaction between the glycine and these DAEM ions except beryllium and magnesium ion only create an amino hydrogen pointing to the original hydroxyl due to their weaker interaction relative to those divalent transition metal (DTM) ion-bound glycine derivatives, being obviously different from that between the glycine and DTM ions, in which two amino hydrogens point to the original hydroxyl oxygen when these metal-chelated glycine derivatives are produced. The interaction energy between the glycine and divalent magnesium would be the boundary of one or two amino hydrogens pointing to the hydrogyl oxygen, i.e., the ,170.3 kcal/mol of binding energy is a critical point. Similar intramolecular proton transfer has also been predicted for those DTM ion-chelated glycine systems; however, that in the gas state has not been observed in the monovalent metal ion-coordinated glycine systems. The binding energy between some monovalent TM ion and the glycine is similar to that of the glycine,Ba2+, which has the lowest binding strength among these DAEM,ion chelated glycine complexes. The difference among them only lies in the larger electrostatic and polarized effects in the latter, which favor the stability of the zwitterionic glycine form in the gas phase. According to these observations, we predict that the zwitterionic glycine would exist in the field of two positive charges in the gas phase. © 2003 Wiley Periodicals, Inc. Int J Quantum Chem 94: 205,214, 2003 [source] Eight-Step Synthesis of RoutiennocinADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 4 2008Kenji Matsumoto Abstract Routiennocin is a member of a family of polycyclic pyrrole ether antibiotics that simultaneously uncouple oxidative phosphorylation and inhibit ATPase as a result of selective complexation of divalent metal ions. We describe a concise synthesis of routiennocin with the longest linear sequence of 8 steps. Our synthesis features a unique bi-directional strategy, which entails a sequential ring-opening/cross metathesis of a highly strained cyclopropenone acetal. This approach enables rapid and highly convergent assembly of the fully extended polyketide subunit of this natural product from readily available homoallylic alcohol precursors. [source] Sorption properties of the iminodiacetate ion exchange resin, amberlite IRC-718, toward divalent metal ionsJOURNAL OF APPLIED POLYMER SCIENCE, Issue 2 2008Charef Noureddine Abstract The sorption properties of the commercially available cationic exchange resin, Amberlite IRC-718, that has the iminodiacetic acid functionality, toward the divalent metal-ions, Fe2+, Cu2+, Zn2+, and Ni2+ were investigated by a batch equilibration technique at 25°C as a function of contact time, metal ion concentration, mass of resin used, and pH. Results of the study revealed that the resin exhibited higher capacities and a more pronounced adsorption toward Fe2+ and that the metal-ion uptake follows the order: Fe2+ > Cu2+> Zn2+ >Ni2+. The adsorption and binding capacity of the resin toward the various metal ions investigated are discussed. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2008 [source] Water-soluble anionic conjugated polymers for metal ion sensing: Effect of interchain aggregationJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 19 2009Yi Chen Abstract Three sulfonato-containing fluorene-based anionic water-soluble conjugated polymers, which are specially designed to link fluorene with alternating moieties such as bipyridine (P1), pyridine (P2), and benzene (P3) have been synthesized via the Pd-catalyzed Sonogashira-coupling reaction, respectively. These polymers had good solubility in water and showed different responses for transition metal ions with different valence in aqueous environments: the fluorescence of bipyridine-containing P1 can be completely quenched by addition of all transition metal ions selected and showed a good selectivity for Ni2+; the pyridine-containing P2 had a little response for monovalent and divalent metal ions while showed good quenching with the addition of trivalent metal ions (with a special selectivity for Fe3+); P3 had responses only for the trivalent metal ions within the ionic concentration we studied. After investigation of the UV-vis absorption spectra, PL emission spectra, DLS, and fluorescence lifetime of P1,P3 in aqueous solution when adding transition metal ions, we found that the different spectrum responses of these polymers are attributed to the different coordination ability of the units linked with fluorene in the main chain. The energy or electron-transfer reactions were the main reason for fluorescence quenching of P1 and P2. On the other hand, interchain aggregation caused by trivalent metal ions lead to fluorescence quenching for P3 and also caused partly fluorescence quenching of P1 and P2. These results revealed the origin of ionochromic effects of these polymers and suggested the potential application for these polymers as novel chemosensors with higher sensing sensitivity in aqueous environments. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 5057,5067, 2009 [source] Copper binding to octarepeat peptides of the prion protein monitored by mass spectrometryPROTEIN SCIENCE, Issue 2 2000Randy M. Whittal Abstract Electrospray ionization mass spectrometry (ESI-MS) was used to measure the binding of Cu2+ ions to synthetic peptides corresponding to sections of the sequence of the mature prion protein (PrP). ESI-MS demonstrates that Cu2+ is unique among divalent metal ions in binding to PrP and defines the location of the major Cu2+ binding site as the octarepeat region in the N-terminal domain, containing multiple copies of the repeat ProHisGlyGlyGlyTrpGlyGln. The stoichiometries of the complexes measured directly by ESI-MS are pH dependent: a peptide containing four octarepeats chelates two Cu2+ ions at pH 6 but four at pH 7.4. At the higher pH, the binding of multiple Cu2+ ions occurs with a high degree of cooperativity for peptides C-terminally extended to incorporate a fifth histidine. Dissociation constants for each Cu2+ ion binding to the octarepeat peptides, reported here for the first time, are mostly in the low micromolar range; for the addition of the third and fourth Cu2+ ions to the extended peptides at pH 7.4, KD's are <100 nm. n-terminal acetylation of the peptides caused some reduction in the stoichiometry of binding at both ph's. cu2+ also binds to a peptide corresponding to the extreme N-terminus of PrP that precedes the octarepeats, arguing that this region of the sequence may also make a contribution to the Cu2+ complexation. Although the structure of the four-octarepeat peptide is not affected by pH changes in the absence of Cu2+, as judged by circular dichroism, Cu2+ binding induces a modest change at pH 6 and a major structural perturbation at pH 7.4. It is possible that PrP functions as a Cu2+ transporter by binding Cu2+ ions from the extracellular medium under physiologic conditions and then releasing some or all of this metal upon exposure to acidic pH in endosomes or secondary lysosomes. [source] Metal-free MIRAS phasing: structure of apo-S100A3ACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2002Peer R. E. Mittl S100 proteins are involved in metal-dependent intracellular signalling. Metal-free S100A3, a cysteine-rich Ca2+ - and Zn2+ -binding protein, has been crystallized by vapour diffusion under the strict exclusion of oxygen and in the absence of divalent metal ions. Metal binding induces large conformational changes, rendering the apo-S100A3 crystals very sensitive to various metal compounds. Therefore, the structure was solved by MIRAS phasing using potassium iodide and xenon derivatives. Iodide replaces a water molecule at the surface of the S100A3 protein, whereas xenon binds in a hydrophobic cavity at the dimer interface. Despite significant non-isomorphism, the combination of both derivatives was sufficient for structure determination. The overall apo-S100A3 structure resembles the structures of metal-free S100B and S100A6 solution structures. In contrast to the NMR structures, the EF-hand loops are well ordered in the apo-S100A3 crystal structure. In the N-terminal pseudo-EF-hand loop a water molecule occupies the position of the Ca2+ ion. The C-terminal canonical EF-hand loop shows an extended conformation and a different helix arrangement to other S100/metal complex crystal structures. [source] Structure of the endonuclease IV homologue from Thermotoga maritima in the presence of active-site divalent metal ionsACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2010Stephen J. Tomanicek The most frequent lesion in DNA is at apurinic/apyrimidinic (AP) sites resulting from DNA-base losses. These AP-site lesions can stall DNA replication and lead to genome instability if left unrepaired. The AP endonucleases are an important class of enzymes that are involved in the repair of AP-site intermediates during damage-general DNA base-excision repair pathways. These enzymes hydrolytically cleave the 5,-phosphodiester bond at an AP site to generate a free 3,-hydroxyl group and a 5,-terminal sugar phosphate using their AP nuclease activity. Specifically, Thermotoga maritima endonuclease IV is a member of the second conserved AP endonuclease family that includes Escherichia coli endonuclease IV, which is the archetype of the AP endonuclease superfamily. In order to more fully characterize the AP endonuclease family of enzymes, two X-ray crystal structures of the T. maritima endonuclease IV homologue were determined in the presence of divalent metal ions bound in the active-site region. These structures of the T. maritima endonuclease IV homologue further revealed the use of the TIM-barrel fold and the trinuclear metal binding site as important highly conserved structural elements that are involved in DNA-binding and AP-site repair processes in the AP endonuclease superfamily. [source] The RNA-Cleaving Bipartite DNAzyme Is a Distinctive MetalloenzymeCHEMBIOCHEM, Issue 1 2006Anat R. Feldman Dr. Abstract Much interest has focused on the mechanisms of the five naturally occurring self-cleaving ribozymes, which, in spite of catalyzing the same reaction, adopt divergent strategies. These ribozymes, with the exception of the recently described glmS ribozyme, do not absolutely require divalent metal ions for their catalytic chemistries in vitro. A mechanistic investigation of an in vitro-selected, RNA-cleaving DNA enzyme, the bipartite, which catalyzes the same chemistry as the five natural self-cleaving ribozymes, found a mechanism of significant complexity. The DNAzyme showed a bell-shaped pH profile. A dissection of metal usage indicated the involvement of two catalytically relevant magnesium ions for optimal activity. The DNAzyme was able to utilize manganese(II) as well as magnesium; however, with manganese it appeared to function complexed to either one or two of those cations. Titration with hexaamminecobalt(III) chloride inhibited the activity of the bipartite; this suggests that it is a metalloenzyme that utilizes metal hydroxide as a general base for activation of its nucleophile. Overall, the bipartite DNAzyme appeared to be kinetically distinct not only from the self-cleaving ribozymes but also from other in vitro-selected, RNA-cleaving deoxyribozymes, such as the 8,17, 10,23, and 614. [source] Potential for Using Histidine Tags in Purification of Proteins at Large ScaleCHEMICAL ENGINEERING & TECHNOLOGY (CET), Issue 11 2005V. Gaberc-Porekar Abstract Attachment of oligo-histidine tag (His-tag) to the protein N- or C-terminus is a good example of early and successful protein engineering to design a unique and generalized purification scheme for virtually any protein. Thus relatively strong and specific binding of His-tagged protein is achieved on an Immobilized Metal-Ion Affinity Chromatography (IMAC) matrix. Most popular hexa-histidine tag and recently also deca-histidine tag are used in combination with three chelating molecules: iminodiacetic acid (IDA), nitrilotriacetic acid (NTA), and carboxymethyl aspartic acid (CM-Asp), covalently attached to the chromatographic matrix. The following combinations with divalent metal ions are preferentially used: (Cu, Zn, Ni, Co)-IDA, Ni-NTA, and Co-CM-Asp. At large scale, regarding cost and product purity, a decisive step is precise and efficient cleavage of His-tag by the cleavage enzyme. Two-step IMAC followed by a polishing step appears to be a minimum but still realistic as an approach to generic technology also for more demanding products. Possible drawbacks in using His-tags and IMAC, such as leaching of metal ions, inefficient cleavage, and batch-to-batch reproducibility must be carefully evaluated before transferred to large scale. Although a great majority of reports refer to small laboratory scale isolations for research purposes it appears there is much higher potential for more extensive use of His-tags and IMAC at large scale than currently documented. [source] In Situ Synthesis of Trisubstituted Methanol Ligands and Their Potential as One-Pot Generators of Cubane-like Metal ComplexesCHEMISTRY - A EUROPEAN JOURNAL, Issue 27 2006Brendan F. Abrahams Dr. Abstract Two different one pot routes to a variety of metal cubane compounds are reported; one route is based on an in situ benzilic acid type rearrangement and the other involves in situ nucleophilic attack at a ketone. Diketosuccinic acid in basic solution in the presence of certain divalent metal ions undergoes a benzilic acid type rearrangement to generate the carbon oxyanion, C(CO2,)3O,, which serves as a cubane-forming bridging ligand in a series of octanuclear complexes of composition [M8{C(CO2)3O}4](H2O)12 (M=Mg, Mn, Fe, Co, Ni, Zn). At the heart of each of these highly symmetrical aggregates is an M4O4 cubane core, each oxygen component of which is provided by the alkoxo centre of a C(CO2,)3O, ligand. Reaction of 2,2,-pyridil, (2-C5H4N)COCO(2-C5H4N), and calcium nitrate in basic alcoholic solution, which proceeds by a similar benzilic acid type rearrangement, gives the cubane compounds, [Ca4L4(NO3)4] in which L=(2-C5H4N)2C(COOR)O, (R=Me or Et). Nucleophilic attack by bisulfite ion at the carbonyl carbon atom of 2,2,-dipyridyl ketone in the presence of certain divalent metals generates the electrically neutral complexes, [{(C5H4N)2SO3C(OH)}2M] (M=Mn, Fe, Co, Ni, Zn and Cd). Cubane-like complexes [M4{(C5H4N)2SO3C(O)}4] (M=Zn, Mn) can be obtained directly from 2,2,-dipyridyl ketone in one-pot reaction systems (sealed tube, 120,°C) if a base as weak as acetate ion is present to deprotonate the OH group of the initial [(C5H4N)2SO3C(OH)], bisulfite addition compound; the [(C5H4N)2SO3C(O)]2, ligand in this case plays the same cubane-forming role as the ligands C(COO,)3O, and (2-C5H4N)2C(COOR)O, above. When excess sodium sulfite is used in similar one-pot reaction mixtures, the monoanionic complexes, [M3Na{(C5H4N)2SO3C(O)}4], (M=Zn, Mn, Co) with an M3NaO4 cubane core, are formed directly from 2,2,-dipyridyl ketone. [source] 2,5-Diphenyl-3,4-bis(2-pyridyl)cyclopenta-2,4-dien-1-one as a Redox-Active Chelating LigandCHEMISTRY - A EUROPEAN JOURNAL, Issue 22 2004Ulrich Siemeling Prof. Abstract 2,5-Diphenyl-3,4-bis(2-pyridyl)cyclopenta-2,4-dien-1-one (1), a close relative of tetraphenylcyclopentadienone, is a new ligand platform for use in redox switches and sensors. Compound 1 acts as a molecular electrochemical sensor towards a range of divalent metal ions and exhibits favourable two-wave behaviour. It forms chelates of the type [(1)MX2], whose stability is enhanced by five orders of magnitude upon one-electron reduction. The bite angle of 1 is close to 90° in these complexes. The attachment of the 14-valence-electron Cp*Co fragment to the cyclopentadienone , system reduces the bite angle and thus modulates the binding characteristics of 1. [source] | |||||||||||||||||||||||||