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Two Zinc(II) complexes [Zn4(L1)4]·2H2O (1) and [Zn2(L2)2]·2H2O (2) of pyruvaldehydethiosemicarbazone ligands are reported. The complexes were characterized by elemental analysis, IR, NMR, UV-vis spectroscopy and by single-crystal X-ray crystallography. X-ray crystal structure determinations of the complexes show that though Zn : ligand stoichiometry is 1 : 1 in both the complexes, the molecular unit is tetranuclear for 1 and binuclear for 2. Both the complexes show selective sensing of ATP at pH 7.4 (0.01 M HEPES) in CH3CN–H2O (9 : 1) medium in the presence of other anions like AcO−, NO3−, F−, Cl−, H2PO4−, HPO42− and P2O72−. The UV-titration experiments of complexes 1 and 2 with ATP results in binding constants of 2.0(±0.07) × 104 M−1 and 7.1(±0.05) × 103 M−1 respectively. The calculated detection limits of 6.7 μM and 1.7 μM for 1 and 2 respectively suggest that the complexes are sensitive detectors of ATP. High selectivity of the complexes is confirmed by the addition of ATP in presence of an excess of other anions. DFT studies confirm that the ATP complexes are more favorable than those with the other inorganic phosphate anions, in agreement with the experimental results. Phosphatase like activity of both complexes is investigated spectrophotometrically using 4-nitrophenylphosphate (NPP) as a substrate, indicating the complexes possess significant phosphate ester hydrolytic efficiency. The kinetics for the hydrolysis of the substrate NPP was studied by the initial rate method at 25 °C. Michaelis–Menten derived kinetic parameters indicate that rate of hydrolysis of the P–O bond by complex 1 is much greater than that of complex 2, the kcat values being 212(±5) and 38(±2) h−1 respectively. The DNA binding studies of the complexes were investigated using electronic absorption spectroscopy and fluorescence quenching. The absorption spectral titrations of the complexes with DNA indicate that the CT-DNA binding affinity (Kb) of complex 1 (2.10(±0.07) × 106 M−1) is slightly greater than that of 2 (1.11(±0.04) × 106 M−1). From fluorescence spectra the apparent binding constant (Kapp) values were calculated and they are found to be 5.41(±0.01) × 105 M−1 for 1 and 3.93(±0.02) × 105 M−1 for 2. The molecular dynamics simulation demonstrates that the Zn(II) complex 1 is a good intercalator of DNA.

Stimulation of cultured epithelial cells with scatter factor/hepatocyte growth factor (HGF) results in individual cells detaching and assuming a migratory and invasive phenotype. Epithelial scattering recapitulates cancer progression and studies have implicated HGF signaling as a driver of cancer metastasis. Inhibitors of HGF signaling have been proposed to act as anti-cancer agents. We previously screened a small molecule library for compounds that block HGF-induced epithelial scattering. Most hits identified in this screen exhibit anti-mitotic properties. Here we assess the biological mechanism of a compound that blocks HGF-induced scattering with limited anti-mitotic activity. Analogs of this compound have one of two distinct activities: inhibiting either cell migration or cell proliferation with cell cycle arrest in G2/M. Each activity bears unique structure–activity relationships. The mechanism of action of anti-mitotic compounds is by inhibition of microtubule polymerization; these compounds entropically and enthalpically bind tubulin in the colchicine binding site, generating a conformational change in the tubulin dimer.

Cu(II) complexes of eight N,N,O-donor hydrazone ligands, obtained by condensation of pyridine-2-carbaldehyde and 2-acetylpyridine with four aroyl hydrazides, are reported. Reactions of Cu(ClO4)2·6H2O with the pyridine-2-carbaldehyde hydrazones (L1H, L2H, L3H, L4H) in presence of Et3N, in 1:2:2 molar ratio, lead to isolation of mononuclear octahedral Cu(II) complexes of formula [Cu(L)2] (L = L1−, L2−, L3−, L4−). However, for the 2-acetylpyridine hydrazones (L5H, L6H, L7H, L8H) reactions under similar conditions yield dichloro bridged complexes of the type [Cu2(μ-Cl)2(L′)2] (L′ = L5−, L6−, L7−, L8−), where each Cu(II) ion is in a square-pyramidal geometry, the chloride ions coming from the in situ reduction of perchlorate by the 2-acetylpyridine hydrazones. The X-ray crystal structures of [Cu(L1)2] and [Cu2(μ-Cl)2(L5)2] are reported. The spectroscopic and electrochemical behaviors of the complexes are also reported.

In bacteria, stalled ribosomes are recycled by a hybrid transfer–messenger RNA (tmRNA). Like tRNA, tmRNA is aminoacylated with alanine and is delivered to the ribosome by EF-Tu, where it reacts with the growing polypeptide chain. tmRNA entry into stalled ribosomes poses a challenge to our understanding of ribosome function because it occurs in the absence of a codon–anticodon interaction. Instead, tmRNA entry is licensed by the binding of its protein partner, SmpB, to the ribosomal decoding center. We analyzed a series of SmpB mutants and found that its C-terminal tail is essential for tmRNA accommodation but not for EF-Tu activation. We obtained evidence that the tail likely functions as a helix on the ribosome to promote accommodation and identified key residues in the tail essential for this step. In addition, our mutational analysis points to a role for the conserved K131GKK tail residues in trans-translation after peptidyl transfer to tmRNA, presumably EF-G-mediated translocation or translation of the tmRNA template. Surprisingly, analysis of A1492, A1493, and G530 mutants reveals that while these ribosomal nucleotides are essential for normal tRNA selection, they play little to no role in peptidyl transfer to tmRNA. These studies clarify how SmpB interacts with the ribosomal decoding center to license tmRNA entry into stalled ribosomes.

The crystal structures of four new diphosphinated chromium Fischer amino carbenes with the compositions fac-[(P-P)(CO)3Cr=C(NR2′)(R)] (R = Me, NR2′ = pyrrolidino, P-P = dppe, 1; R = Me, NR2′ = pyrrolidino, P-P = dppp, 2; R = Me, R′ = Me, P-P = dppe, 3; R = Me, R′ = Me, P-P = dppp, 4) have been determined at 243 K. Compound 1 crystallizes in the monoclinic system, space group P21 /n with a = 12.1597(11) A, b = 20.1556(17) A, c = 14.0557(12) A, β = 114.163(3)°, V = 3143.0(5) A3, and Z = 4. Compound 2 crystallizes in the triclinic system, space group P − 1 with a = 7.4424(3) A, b = 10.8830(5) A, c = 20.6040(9) A, α = 100.9880(10)°, β = 91.7650(10)°, γ = 97.6610(10)°, V = 1620.90(12) A3, and Z = 2. Compound 3 crystallizes in the monoclinic system as a mono-solvate of d5-pyridine, space group P21 /c with a = 11.485(2) A, b = 22.825(5) A, c = 14.092(3) A, β = 108.53(3)°, V = 3502.7(12) A3, and Z = 4. Compound 4 crystallizes in the orthorhombic system, space group P2 1 2 1 2 1 with a = 8.359(3) A, b = 15.364(6) A, c = 23.784(9) A, V = 3055(2) A3, and Z = 4. Steric repulsions with the diphosphine ligand favor a conformation with the amino moiety directed away from the diphosphine backbone in 1–4.

The synthesis and molecular structure of the novel 1-[ferrocenyl(hydroxy)methyl]-1,7-dicarba-closo-dodecaborane (1) is described. Compound 1 was synthesized from reaction of m-carborane and ferrocene carboxaldehyde using n-butyllithium (n-BuLi) or tetrabutylammonium fluoride (TBAF) in THF in 45% and 36% yield, respectively. Compound 1 consists of a ferrocene molecule tethered to m-carborane through a methylhydroxy bridge. The crystal structure of 1 was determined by single crystal X-ray diffraction analysis. Crystal data: 1 [Fe(C5H5)(C5H4-CH2O-1,7-C2B10H12)], formula weight = 359.17, crystallized in orthorhombic system, space group Pna2 1 with a=19.698(4) A, b=10.709(2) A, c=8.520(2) A, and V=1797.3(7) A3 and Z=4. Refined to R 1=0.043 for 4124 observed reflections with I/σ>2σ(I). The compound was crystallized as racemic twins in a ratio of 73(2)/27(2). The unsubstituted Cp ring was disordered and modeled as two conformations in a 53(3)/47(3) ratio. Intermolecular hydrogen bonding was observed from the hydrogen of the meta-carbon on the carborane cluster towards the hydroxyl oxygen.

The synthesis, spectroscopic studies, computational analysis, and crystal structure of (6S)-2,6-diphenylcyclohex-2-enone oxime are described. The oxime crystallizes in the monoclinic space group P2l/c with a = 11.5269(11) A, b = 6.6724(6) A, c = 19.0105(18) A, β = 100.863(3)°, V = 1435.9(2) A3, and Z = 4. Semi-empirical (AM1), ab initio (MP2/6-31G*), and density functional theory (B3LYP/6-31G*) calculations suggest that there are two low-energy conformations available for the oxime. Significant differences were observed between the dihedral angles in the conformers. Good agreement was found between the crystal data and one of the conformers from the calculations. The absence of certain splitting patterns in the 1H NMR spectrum of the title compound is in contrast to what would be expected on the basis of the dihedral angles and suggests that rapid interconversion is possible in solution. Analysis of the crystal packing suggests that one conformer is favored in the solid state, stabilized by packing interactions, and interconversion is prohibited due to a blocking effect.

Ca(2+) is essential for in vitro activity of Erwinia chrysanthemi pectate lyase C (PelC). Crystallographic analyses of 11 PelC-Ca(2+) complexes, formed at pH 4.5, 9.5, and 11.2 under varying Ca(2+) concentrations, have been solved and refined at a resolution of 2.2 A. The Ca(2+) site represents a new motif for Ca(2+), consisting primarily of beta-turns and beta-strands. The principal differences between PelC and the PelC-Ca(2+) structures at all pH values are the side-chain conformations of Asp-129 and Glu-166 as well as the occupancies of four water molecules. According to calculations of pK(a) values, the presence of Ca(2+) and associated structural changes lower the pK(a) of Arg-218, the amino acid responsible for proton abstraction during catalysis. The Ca(2+) affinity for PelC is weak, as the K(d) was estimated to be 0.132 (+/-0.004) mm at pH 9.5, 1.09 (+/-0.29) mm at pH 11.2, and 5.84 (+/-0.41) mm at pH 4.5 from x-ray diffraction studies and 0.133 (+/-0.045) mm at pH 9.5 from intrinsic tryptophan fluorescence measurements. Given the pH dependence of Ca(2+) affinity, PelC activity at pH 4.5 has been reexamined. At saturating Ca(2+) concentrations, PelC activity increases 10-fold at pH 4.5 but is less than 1% of maximal activity at pH 9.5. Taken together, the studies suggest that the primary Ca(2+) ion in PelC has multiple functions.

Cytochrome c′ isolated from Rhodobacter sphaeroides strain R26 (RSCP) crystallizes as a dimer of two identical 14-kDa subunits, in trigonal space group P31, with cell parameters a, b = 48.10 A, c = 115.80 A. The crystal structure of RSCP has been solved by molecular replacement using cytochrome c′ from Rhodobacter capsulatus (PDB ID: 1CPQ) as a search model. To ensure effective phase bias removal, the RSCP model was iteratively built into maps generated by a modified wARP procedure, Shake&wARP. The 1.8 A model (PDB ID: 1GQA) has been refined to an R = 0.204 and freeR = 0.254. Each subunit consists of four antiparallel α-helices, with the pentacoordinate heme covalently bound to a C–X–Y–C–H motif near the C-terminus. F14, located on helix A, blocks direct access to what would be the sixth “distal” ligand binding site of the heme. The dimer subunits form a flattened “X” shape, intermediate between the Type 1 and Type 2 cytochromes c′. The presence of the aromatic F14 and a deep channel between helices B and C places RSCP into Group 1 cytochromes c′. Clear electron density has revealed that the amino acid sequences for the cytochrome c′ from strains R26 and 2.4.1 are identical.

In vitro activity of the aminoglycoside 6′- N -acetyltransferase type Ib [AAC(6′)-Ib] was inhibited by ZnCl 2 with a 50% inhibitory concentration (IC 50 ) of 15 μM. Growth of Acinetobacter baumannii or Escherichia coli harboring aac ( 6 ′)- Ib in cultures containing 8 μg/ml amikacin was significantly inhibited by the addition of 2 μM Zn 2+ in complex with the ionophore pyrithione (ZnPT).

The three-dimensional structure of a complex between the pectate lyase C (PelC) R218K mutant and a plant cell wall fragment has been determined by x-ray diffraction techniques to a resolution of 2.2 A and refined to a crystallographic R factor of 18.6%. The oligosaccharide substrate, alpha-D-GalpA-([1-->4]-alpha-D-GalpA)3-(1-->4)-D-GalpA , is composed of five galacturonopyranose units (D-GalpA) linked by alpha-(1-->4) glycosidic bonds. PelC is secreted by the plant pathogen Erwinia chrysanthemi and degrades the pectate component of plant cell walls in soft rot diseases. The substrate has been trapped in crystals by using the inactive R218K mutant. Four of the five saccharide units of the substrate are well ordered and represent an atomic view of the pectate component in plant cell walls. The conformation of the pectate fragment is a mix of 21 and 31 right-handed helices. The substrate binds in a cleft, interacting primarily with positively charged groups: either lysine or arginine amino acids on PelC or the four Ca2+ ions found in the complex. The observed protein-oligosaccharide interactions provide a functional explanation for many of the invariant and conserved amino acids in the pectate lyase family of proteins. Because the R218K PelC-galacturonopentaose complex represents an intermediate in the reaction pathway, the structure also reveals important details regarding the enzymatic mechanism. Notably, the results suggest that an arginine, which is invariant in the pectate lyase superfamily, is the amino acid that initiates proton abstraction during the beta elimination cleavage of polygalacturonic acid.

AAC(6')-Ib is an important aminoglycoside resistance enzyme to target with enzymatic inhibitors. An in silico screening approach was used to identify potential inhibitors from the ChemBridge library. Several compounds were identified, of which two of them, 4-[(2-{[1-(3-methylphenyl)-4,6-dioxo-2-thioxotetrahydro-5(2H)-pyrimidinylidene]methyl}phenoxy)methyl]benzoic acid and 2-{5-[(4,6-dioxo-1,3-diphenyl-2-thioxotetrahydro-5(2H)-pyrimidinylidene)methyl]-2-furyl}benzoic acid, showed micromolar activity in inhibiting acetylation of kanamycin A. These compounds are predicted to bind the aminoglycoside binding site of AAC(6')-Ib and exhibited competitive inhibition against kanamycin A.

The title compound, C25H16OS2, (I), was synthesized via the alkali-catalysed condensation of 3,3′-thenil and 1,3-di­phenyl-2-propanone in absolute ethanol. Both 3-thienyl rings in (I) exhibit an 18.89% flip disorder. Thienyl-ring flip disorders are often observed with mol­ecules having unsubstituted terminal 3-thienyl rings [Crundwell et al. (2002). Acta Cryst. E58, o668–o670.

RC3/neurogranin is a forebrain-enriched, postnatal-onset, thyroid hormone-dependent, protein kinase C substrate of dendritic spines that interacts with calmodulin. These characteristics suggest a prominent role within the Ca(2+)-mediated second messenger cascades associated with neonatal synaptogenesis and adult neural plasticity. To understand the molecular interactions between RC3 and calmodulin, we characterized recombinant RC3 and four sequence variants: Ser-36-->Ala, Ser-36-->Asp, Ser-36-->Lys, and Phe-37-->Trp. Interactions between CaM and variant Phe-37-->Trp can be monitored by fluorescence spectroscopy, allowing us to determine, by competitive assays, the relative affinities of the wild-type and variant proteins for calmodulin. The effects of salt and Ca2+ on the rank order of these affinities permit partial dissection of hydrophobic, ionic, and structural components of the RC3-CaM interaction and suggest that it is bimodal. We demonstrate that RC3 binds preferentially to CaM when Ca2+ is absent and that the addition of a negative charge to residue 36 is sufficient to disrupt all detectable RC3-CaM interactions. We propose a model wherein a Ca(2+)-"sensitive," bimodal interaction between RC3 and CaM regulates the transduction of postsynaptic Ca2+ fluxes into physiological responses through the modulation of Ca2+/CaM availability.

RC3/Neurogranin is a postnatal-onset, forebrain-specific, thyroid hormone-regulated, protein kinase C (PKC) substrate that binds calmodulin (CaM) and accumulates in dendritic spines. We bacterially expressed and purified RC3 and, for comparison, GAP-43/neuromodulin to near homogeneity using relatively simple procedures. We then raised antisera against recombinant RC3 that does not crossreact with GAP-43 and is suitable for immunohistochemical analysis of brain slices. We also constructed over 30 RC3 sequence variants by PCR-mediated, site-directed mutagenesis, and purified four of these to near homogeneity. The elution profiles displayed by RC3 and sequence variants during purification on CaM-Sepharose columns suggest that two different affinity forms of the RC3.CaM complex coexist when Ca2+ is absent and that GAP-43.CaM interactions are far more sensitive to salt than those that occur between recombinant RC3 and CaM. Variant proteins in which serine 36 was changed failed to serve as a substrate for PKC, implicating this as the target residue.

Three new dinitrosyl iron complexes LFe(NO)(2) (L = 2,2'-bipyridine (bipy) (1), 2,2',2''-terpyridine (terpy) (2) and 1,10-phenathroline (phen) (3)) were synthesized by the reaction of Fe(NO)(2)(CO)(2) with corresponding ligands in tetrahydrofuran. Complexes 1-3 were studied using IR, UV-vis, MS, NMR, and electrochemical techniques. Complexes 1 and 2 were also characterized using single crystal X-ray diffraction analysis. IR spectra of complexes 1-3 display two strong characteristic NO stretching frequencies (nu(NO)) in the region reflecting donor properties of the ligands. Cyclic voltammetry studies show two quasi-reversible one-electron reductions for all complexes. Electrochemical investigations using different concentrations show that an irreversible one-electron reduction at -1.85 V for complex 2 and -1.80 V for complex 3 are from solvated species. Single-crystal X-ray structural analysis reveals that complex 1 crystallizes in the triclinic P1 space group and the asymmetric unit consists of one Fe(NO)(2)(bipy) molecule with the two NO groups located on two sides of Fe(bipy) plane. Complex 2 crystallizes in monoclinic P21/n space group, and the asymmetric unit contains one Fe(NO)(2)(terpy) molecule, in which the NO groups are located on two sides of the plane consisted of Fe and two coordinated pyridyl rings, but almost parallel to the uncoordinated pyridyl ring. The crystal packings of both complexes 1 and 2 show intermolecular H-bonding and strong pi-pi stacking interactions.

Aziridines were formed by copper-catalyzed intramolecular nitrene addition to alkenes. The carbamate group was used as the tether between the alkene and the nitrene. Subsequent nucleophilic attack of the aziridine was accomplished using RSH, R2NH, N3-, or ROH as the nucleophile. This addition was found to be regio- and stereoselective. This methodology has been used to demonstrate its utility in the regio- and stereoselective synthesis of a 1,2-diamino-3-hydroxycyclohexane. This substitution pattern is found in natural products such as Tamiflu.

Pectate lyases are depolymerizing enzymes that degrade plant cell walls, causing tissue maceration and death. The laboratory has been engaged in structural studies of several members of the pectate lyase superfamily. Most effort has been focused on Erwinia chrysanthemi pectate lyase C (PelC). There is now an accumulation of data that supports a detailed description of the enzymatic mechanism. The data include structural studies of native PelC, PelC-Ca2+ complexes, and an inactive PelC mutant complexed with an oligogalacturonic acid fragment. The structural studies have been supported by amino acid sequence alignments, site-specific mutagenesis experiments, kinetic studies at various pHs, and deuterium labeling studies. Together, the data implicate an invariant arginine as the amino acid that initiates proton abstraction in the β-elimination cleavage of pectate. The data suggest several potential sources of the proton donated to the scissile glycosidic bond to complete the reaction, but do not provide sufficient evidence for any particular one. An overview of the research and its implications for other members of the pectate lyase superfamily are presented.

The structure and function of Erwinia chrysanthemi pectate lysase C, a plant virulence factor, is reviewed to illustrate one mechanism of pathogenesis at the molecular level. Current investigative topics are discussed in this paper.

Two neuronal protein kinase C substrates, RC3/neurogranin and GAP-43/neuromodulin, preferentially bind to calmodulin (CaM) when Ca2+ is absent. We examine RC3.CaM and GAP-43.CaM interactions by circular dichroism spectroscopy using purified, recombinant RC3 and GAP-43, sequence variants of RC3 displaying qualitative and quantitative differences in CaM binding affinities, and overlapping peptides that cumulatively span the entire amino acid sequence of RC3. We conclude that CaM stabilizes a basic, amphiphilic alpha-helix within RC3 and GAP-43 under physiological salt concentrations only when Ca2+ is absent. This provides structural confirmation for two binding modes and suggests that CaM regulates the biological activities of RC3 and GAP-43 through an allosteric, Ca(2+)-sensitive mechanism that can be uncoupled by protein kinase C-mediated phosphorylation. More generally, our observations imply an alternative allosteric regulatory role for the Ca(2+)-free form of CaM.

In the title compound, C16H10N2S2, (I), which was synthesized via the reaction of 2,2′-thenil and 1,2-phenyl­enedi­amine, the thienyl rings display ideal geometries, with no evidence of ring-flip disorders often found with unsubstituted terminal 2-thienyl rings [Zheng, Wang, Liu, Carducci, Peyghambarian & Jabbourb (2002). Acta Cryst. C58, m50–m52].

A novel cyclic tetra-nuclear dinitrosyl iron complex [Fe(NO)2(Im-H)]4 was isolated and characterized by X-ray crystallography, and in donor solvents this fragments into 17 e− monomeric units that give EPR spectra analogous to the g = 2.03 species seen in mammalian biology.