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Heavy Atoms (heavy + atom)
Selected Abstracts2,5-Bis(p -R-arylethynyl)rhodacyclopentadienes Show Intense Fluorescence: Denying the Presence of a Heavy Atom,ANGEWANDTE CHEMIE, Issue 13 2010Andreas Steffen Dr. Schwermetall-Lichtshow: Photophysikalische Untersuchungen ergaben für eine Reihe von Rhodiumkomplexen unerwartet hohe Fluoreszenzquantenausbeuten (,f bis 69,%, ,f,1,3,ns) und ein unerwartet langsames Intersystem-Crossing. Diese neue Verbindungsklasse hinterfragt das gängige Modell zum Verhalten angeregter elektronischer Zustände und zur Rolle von Schweratomen in Intersystem-Crossing-Prozessen. THF=Tetrahydrofuran, Tol=Toluol. [source] Ab initio Thermochemistry Involving Heavy Atoms: An Investigation of the Reactions Hg + IX (X: I, Br, Cl, O)CHEMINFORM, Issue 6 2006Benjamin C. Shepler Abstract For Abstract see ChemInform Abstract in Full Text. [source] The Effect of Heavy Atoms on Photoinduced Electron Injection from Nonthermalized and Thermalized Donor States of MII,Polypyridyl (M=Ru/Os) Complexes to Nanoparticulate TiO2 Surfaces: An Ultrafast Time-Resolved Absorption StudyCHEMISTRY - A EUROPEAN JOURNAL, Issue 2 2010Sandeep Verma Abstract We have synthesized ruthenium(II), and osmium(II),polypyridyl complexes ([M(bpy)2L]2+, in which M=OsII or RuII, bpy=2,2,-bipyridyl, and L=4-(2,2,-bipyridinyl-4-yl)benzene-1,2-diol) and studied the interfacial electron-transfer process on a TiO2 nanoparticle surface using femtosecond transient-absorption spectroscopy. Ruthenium(II)- and osmium(II)-based dyes have a similar molecular structure; nevertheless, we have observed quite different interfacial electron-transfer dynamics (both forward and backward). In the case of the RuII/TiO2 system, single-exponential electron injection takes place from photoexcited nonthermalized metal-to-ligand charge transfer (MLCT) states. However, in the case of the OsII/TiO2 system, electron injection takes place biexponentially from both nonthermalized and thermalized MLCT states (mainly 3MLCT states). Larger spin,orbit coupling for the heavier transition-metal osmium, relative to that of ruthenium, accounts for the more efficient population of the 3MLCT states in the OsII -based dye during the electron-injection process that yields biexponential dynamics. Our results tend to suggest that appropriately designed OsII,polypyridyl dye can be a better sensitizer molecule relative to its RuII analogue not only due to much broader absorption in the visible region of the solar-emission spectrum, but also on account of slower charge recombination. [source] Determination of atenolol by the micelle-stabilized room-temperature phosphorescence methodologyLUMINESCENCE: THE JOURNAL OF BIOLOGICAL AND CHEMICAL LUMINESCENCE, Issue 6 2007Marcela A. Castillo Abstract A micellar-stabilized room-temperature phosphorescence (MS,RTP) method for the determination of atenolol has been developed in micellar solutions of sodium dodecylsulphate (SDS) in the presence of thallium(I) as a heavy atom and sodium sulphite as an oxygen scavenger. The effects of thallium(I) nitrate, SDS and sodium sulphite concentrations on atenolol MS,RTP intensity were studied. Optimized conditions to obtain maximum sensitivity were 0.015 mol/L thallium(I) nitrate, 0.1 mol/L SDS and 0.0075 mol/L sodium sulphite. The maximum phosphorescence signal was completely developed in 10 min and the intensity was measured at ,ex = 272 nm and ,em = 412 nm. The linear range of application obtained was 2.01,16.00 µg/mL. The detection limit estimated from the least-squares regression analysis was 0.86 µg/mL and the relative standard deviation of 10 replicates was 1.7%. The proposed method was applied to the determination of atenolol in a pharmaceutical formulation. The quantitation was carried out by means of standard calibration, standard-additions calibration and Youden calibration. These three experiments were necessary to evaluate the presence of constant and proportional errors due to the matrix. Copyright © 2007 John Wiley & Sons, Ltd. [source] Application of functional group modified substrate in room temperature phosphorescence, II,heavy atom-chelated filter paper for selective determination of , -naphthalene acetic acidLUMINESCENCE: THE JOURNAL OF BIOLOGICAL AND CHEMICAL LUMINESCENCE, Issue 4-5 2005Ruohua Zhu Abstract Heavy atom-chelated filter paper was synthesized and used as the substrate for room-temperature phosphorescence (RTP). The synthesis conditions for chelated paper were studied. The Pb-chelated filter paper could selectively induce the RTP of , -naphthalene acetic acid (, -NAA). The excitation and emission wavelengths of RTP of , -NAA were 300 nm and 521 nm, respectively. The concentration of , -NAA was linear with the RTP intensity in the range 2 × 10,6,6 × 10,4 mol/L (correlation coefficient, 0.9999). The concentration detection limit was 1.35 × 10,7 mol/L and the absolute detection limit was 0.25 ng/spot. The RSD (n = 10) was 1.7%. The method was applied to the analysis of water and vegetable samples with satisfactory results. Because the heavy atom was directly chelated onto the filter paper, the heavy-atom effect on the RTP of NAA was further increased and the analysis procedure was simple, fast and economical. Copyright © 2005 John Wiley & Sons, Ltd. [source] Selenium incorporation using recombinant techniquesACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2010Helen Walden Using selenomethionine to phase macromolecular structures is common practice in structure determination, along with the use of selenocysteine. Selenium is consequently the most commonly used heavy atom for MAD. In addition to the well established recombinant techniques for the incorporation of selenium in prokaryal expression systems, there have been recent advances in selenium labelling in eukaryal expression, which will be discussed. Tips and things to consider for the purification and crystallization of seleno-labelled proteins are also included. [source] Direct methods and protein crystallography at low resolutionACTA CRYSTALLOGRAPHICA SECTION D, Issue 10 2000Christopher J. Gilmore The tools of modern direct methods are examined and their limitations for solving protein structures discussed. Direct methods need atomic resolution data (1.1,1.2,Å) for structures of around 1000 atoms if no heavy atom is present. For low-resolution data, alternative approaches are necessary and these include maximum entropy, symbolic addition, Sayre's equation, group scattering factors and electron microscopy. [source] Three-dimensional structure of the histidine-containing phosphocarrier protein (HPr) from Enterococcus faecalis in solutionFEBS JOURNAL, Issue 3 2001Till Maurer The histidine-containing phosphocarrier protein (HPr) transfers a phosphate group between components of the prokaryotic phosphoenolpyruvate-dependent phosphotransferase system (PTS), which is finally used to phosphorylate the carbohydrate transported by the PTS through the cell membrane. Recently it has also been found to act as an intermediate in the signaling cascade that regulates transcription of genes related to the carbohydrate-response system. Both functions involve phosphorylation/dephosphorylation reactions, but at different sites. Using multidimensional 1H-NMR spectroscopy and angular space simulated annealing calculations, we determined the structure of HPr from Enterococcus faecalis in aqueous solution using 1469 distance and 44 angle constraints derived from homonuclear NMR data. It has a similar overall fold to that found in HPrs from other organisms. Four , strands, A, B, C, D, encompassing residues 2,7, 32,37, 40,42 and 60,66, form an antiparallel , sheet lying opposite the two antiparallel , helices, a and c (residues 16,26 and 70,83). A short , helix, b, from residues 47,53 is also observed. The pairwise root mean square displacement for the backbone heavy atoms of the mean of the 16 NMR structures to the crystal structure is 0.164 nm. In contrast with the crystalline state, in which a torsion angle strain in the active-center loop has been described [Jia, Z., Vandonselaar, M., Quail, J.W. & Delbaere, L.T.J. (1993) Nature (London) 361, 94,97], in the solution structure, the active-site His15 rests on top of helix a, and the phosphorylation site N,1 of the histidine ring is oriented towards the surface, making it easily accessible to the solvent. Back calculation of the 2D NOESY NMR spectra from both the NMR and X-ray structures shows that the active-center structure derived by X-ray crystallography is not compatible with experimental data recorded in solution. The observed torsional strain must either be a crystallization artefact or represents a conformational state that exists only to a small extent in solution. [source] Solution structure of the functional domain of Paracoccus denitrificans cytochrome c552 in the reduced stateFEBS JOURNAL, Issue 13 2000Primo, Pristov In order to determine the solution structure of Paracoccus denitrificans cytochrome c552 by NMR, we cloned and isotopically labeled a 10.5-kDa soluble fragment (100 residues) containing the functional domain of the 18.2-kDa membrane-bound protein. Using uniformly 15N-enriched samples of cytochrome c552 in the reduced state, a variety of two-dimensional and three-dimensional heteronuclear double-resonance NMR experiments was employed to achieve complete 1H and 15N assignments. A total of 1893 distance restraints was derived from homonuclear 2D-NOESY and heteronuclear 3D-NOESY spectra; 1486 meaningful restraints were used in the structure calculations. After restrained energy minimization a family of 20 structures was obtained with rmsd values of 0.56 ± 0.10 Å and 1.09 ± 0.09 Å for the backbone and heavy atoms, respectively. The overall topology is similar to that seen in previously reported models of this class of proteins. The global fold consists of two long helices at the N-terminus and C-terminus and three shorter helices surrounding the heme moiety; the helices are connected by well-defined loops. Comparison with the X-ray structure shows some minor differences in the positions of the Trp57 and Phe65 side-chain rings as well as the heme propionate groups. [source] A comparison of a microfocus X-ray source and a conventional sealed tube for crystal structure determinationJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 5 2009Thomas Schulz Experiments are described in which a direct comparison was made between a conventional 2,kW water-cooled sealed-tube X-ray source and a 30,W air-cooled microfocus source with focusing multilayer optics, using the same goniometer, detector, radiation (Mo,K,), crystals and software. The beam characteristics of the two sources were analyzed and the quality of the resulting data sets compared. The Incoatec Microfocus Source (IµS) gave a narrow approximately Gaussian-shaped primary beam profile, whereas the Bruker AXS sealed-tube source, equipped with a graphite monochromator and a monocapillary collimator, had a broader beam with an approximate intensity plateau. Both sources were mounted on the same Bruker D8 goniometer with a SMART APEX II CCD detector and Bruker Kryoflex low-temperature device. Switching between sources simply required changing the software zero setting of the 2, circle and could be performed in a few minutes, so it was possible to use the same crystal for both sources without changing its temperature or orientation. A representative cross section of compounds (organic, organometallic and salt) with and without heavy atoms was investigated. For each compound, two data sets, one from a small and one from a large crystal, were collected using each source. In another experiment, the data quality was compared for crystals of the same compound that had been chosen so that they had dimensions similar to the width of the beam. The data were processed and the structures refined using standard Bruker and SHELX software. The experiments show that the IµS gives superior data for small crystals whereas the diffracted intensities were comparable for the large crystals. Appropriate scaling is particularly important for the IµS data. [source] Completion of crystal structures from powder data: the use of the coordination polyhedraJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 6 2000Angela Altomare Direct methods applied to powder diffraction data often provide well located heavy atoms and unreliable light-atom positions. The completion of the crystal structure is then not always straightforward and may require a considerable amount of user intervention. The heavy-atom connectivity provided by the trial solution may be used to guess the nature of the coordination polyhedra. A Monte Carlo procedure is described which, in the absence of a well defined structural model, is able to locate the light atoms correctly under the restraints of the experimental heavy-atom connectivity model. The correctness of the final model is assessed by criteria based on the agreement between the whole experimental diffraction pattern and the calculated one. The procedure requires little CPU computing time and has been implemented as a routine of EXPO [Altomare et al. (1999). J. Appl. Cryst.32, 339,340]. The method has proved to be sufficiently robust against the distortion of the coordination polyhedra and has been successfully applied to some test structures. [source] An expeditious procedure for the synthesis of isotopically labelled fatty acids: preparation of 2,2- d2 -nonadecanoic acidJOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, Issue 8 2006Annalisa Guaragna Abstract We describe a new synthetic path for the preparation of isotopically labelled saturated odd-numbered fatty acids by a homologation procedure for the direct conversion of carboxylic acids into their 2,2-dideuterated homologues. This process can be used to obtain both odd- and even-numbered 2,2-dideuterated fatty acids starting from carboxylic acids. Furthermore, the reiteration of this process can also be used for the synthesis of polydeuterated fatty acids in which the heavy atoms are placed on adjacent carbon atoms or differently arranged in the molecule. Copyright © 2006 John Wiley & Sons, Ltd. [source] Syntheses of deuterated jasmonates for mass spectrometry and metabolism studiesJOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, Issue 11 2005Patrycja W. Galka Abstract Jasmonic acid and its metabolites play an essential role in the regulation of plant development and systemic defense responses. Isotopically labeled standards are required to quantify plant hormones for metabolism studies using mass spectrometry. A convenient method for the preparation of deuterated analogs of jasmonates is demonstrated. Modification of commercially available methyl jasmonate by base-catalyzed proton/deuterium exchange or Wittig reaction introduces either two or three heavy atoms into a molecule. Copyright © 2005 John Wiley & Sons, Ltd. [source] Towards the best model for H atoms in experimental charge-density refinementACTA CRYSTALLOGRAPHICA SECTION A, Issue 4 2009Anna A. Hoser The consequences of different treatments of H atoms in experimental charge-density studies are discussed. Geometric and topological parameters obtained after applying four different H-atom models in multipolar refinement on high-resolution X-ray data only were compared with the results obtained for a reference joint high-resolution X-ray/neutron refinement. The geometry and the topological critical point and integrated parameters closest to the reference values were obtained after a mixed refinement (high-order refinement of heavy atoms, low-angle refinement of H atoms and elongation of the X,H distance to the average neutron bond lengths) supplemented by an estimation of the anisotropic thermal motions of H atoms using the SHADE program. Such a procedure works very well even for strong hydrogen bonds. The worst fit to the reference results for both critical point and integrated parameters was obtained when only the standardization to the average neutron X,H distances was applied. The non-H-atom parameters are also systematically influenced by the H-atom modeling. In order to compare topological and integrated properties calculated for H and non-H atoms in multipolar refinement when there are no neutron data, the same treatment of H atoms (ideally the mixed refinement + estimated anisotropic atomic displacement parameters for H atoms) should be applied. [source] NMR solution structure of KP-TerB, a tellurite-resistance protein from Klebsiella pneumoniaePROTEIN SCIENCE, Issue 4 2008Sheng-Kuo Chiang Abstract Klebsiella pneumoniae (KP), a Gram-negative bacterium, is a common cause of hospital-acquired bacterial infections worldwide. Tellurium (Te) compounds, although relatively rare in the environment, have a long history as antimicrobial and therapeutic agents. In bacteria, tellurite (TeO3,2) resistance is conferred by the ter (Ter) operon (terZABCDEF). Here, on the basis of 2593 restraints derived from NMR analysis, we report the NMR structure of TerB protein (151 amino acids) of KP (KP-TerB), which is mainly composed of seven ,-helices and a 310 helix, with helices II to V apparently forming a four-helix bundle. The ensemble of 20 NMR structures was well-defined, with a RMSD of 0.32 ± 0.06 Å for backbone atoms and 1.11 ± 0.07 Å for heavy atoms, respectively. A unique property of the KP-TerB structure is that the positively and negatively charged clusters are formed by the N-terminal positively and C-terminal negatively charged residues, respectively. To the best of our knowledge, the protein sequence and structures of KP-TerB are unique. [source] Synthesis and NMR solution structure of an ,-helical hairpin stapled with two disulfide bridgesPROTEIN SCIENCE, Issue 5 2000Philippe Barthe Abstract Helical coiled-coils and bundles are some of the most common structural motifs found in proteins. Design and synthesis of ,-helical motifs may provide interesting scaffolds that can be useful as host structures to display functional sites, thus allowing the engineering of novel functional miniproteins. We have synthesized a 38-amino acid peptide, ,2p8, encompassing the ,-helical hairpin present in the structure of p8MTCP1, as an ,-helical scaffold particularly promising for its stability and permissiveness of sequence mutations. The three-dimensional structure of this peptide has been solved using homonuclear two-dimensional NMR techniques at 600 MHz. After sequence specific assignment, a total of 285 distance and 29 dihedral restraints were collected. The solution structure of ,2p8 is presented as a set of 30 DIANA structures, further refined by restrained molecular dynamics, using simulated annealing protocol with the AMBER force field. The RMSD values for the backbone and all heavy atoms are 0.65 ± 0.25 and 1.51 ± 0.21 Å, respectively. Excised from its protein context, the ,-hairpin keeps its native structure: an ,-helical coiled-coil, similar to that found in superhelical structures, with two helices spanning residues 4-16 and 25,36, and linked by a short loop. This motif is stabilized by two interhelical disulfide bridges and several hydrophobic interactions at the helix interface, leaving most of its solvent-exposed surface available for mutation. This ,-helical hairpin, easily amenable to synthetic chemistry and biological expression system, may represent a stable and versatile scaffold to display new functional sites and peptide libraries. [source] X-ray atomic orbital analysis.ACTA CRYSTALLOGRAPHICA SECTION A, Issue 4 2008The scattering unit of X-ray crystal structure analysis is changed from atoms to the subshell electrons by X-ray atomic orbital analysis (XAO). All the atoms in the unit cell are divided into groups of subshell electrons in the XAO analysis. Each subshell is treated as an independent pseudo-atom, which enables the atomic orbitals (AO's) and the electron population of each AO expressed as a linear combination of s/p/d/f orbitals in each subshell to be determined. When the environmental condition of the sample is varied, the electron transfer among the AO's in the crystal can be traced with XAO. It is applicable mainly to analyses of the electron-density distribution in ionic solids including those with a nonstoichiometric structure. The expansion coefficients of each AO are calculated with the perturbation theory putting a point charge on each atom in the unit cell. This automatically makes the perturbation potential have the point-group symmetry of the atom in the crystal field. Then the coefficients of each AO are refined to fit to the observed structure factors keeping the orthonormal relationships among the AO's. Complex basis functions with , or , spin as well as real ones are employed for heavy atoms and the relationships among the coefficients for the AO's of an electron in the crystal fields of the 32 point-group symmetries are derived for p, d and f orbitals. The AO's thus derived can be applicable to an anti-symmetrized multi-electron system, although X-ray diffraction cannot specify the atomic terms occupied when the crystal symmetry permits the atom to have many terms. [source] Structure determination of the minimal complex between Tfb5 and Tfb2, two subunits of the yeast transcription/DNA-repair factor TFIIH: a retrospective studyACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2010Denis E. Kainov Tfb5 interacts with the Tfb2 subunit of the general transcription factor TFIIH to ensure efficient nucleotide-excision repair in eukaryotes. The crystal structure of the complex between Tfb5 and the C-terminal region of Tfb2 (Tfb2C) from Saccharomyces cerevisiae has recently been reported. Here, the structure-determination process is described as a case study. Although crystals were obtained readily, it was not possible to determine experimental phases from a first crystal form (Tfb2412,513,Tfb52,72) that diffracted to 2.6,Å resolution. Shortening of the Tfb2C from its N-terminus was decisive and modified the crystal packing, leading to a second crystal form (Tfb2435,513,Tfb52,72). These crystals diffracted to 1.7,Å resolution with excellent mosaicity and allowed structure determination by conventional approaches using heavy atoms. The refined structure from the second crystal form was used to solve the structure of the first crystal form by molecular replacement. Comparison of the two structures revealed that the N-terminal region of Tfb2C and (to a lesser extent) the C-terminal region of Tfb5 contributed to the crystal packing. A detailed analysis illustrates how variation in domain boundaries influences crystal packing and quality. [source] The magic triangle goes MAD: experimental phasing with a bromine derivativeACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2010Tobias Beck Experimental phasing is an essential technique for the solution of macromolecular structures. Since many heavy-atom ion soaks suffer from nonspecific binding, a novel class of compounds has been developed that combines heavy atoms with functional groups for binding to proteins. The phasing tool 5-amino-2,4,6-tribromoisophthalic acid (B3C) contains three functional groups (two carboxylate groups and one amino group) that interact with proteins via hydrogen bonds. Three Br atoms suitable for anomalous dispersion phasing are arranged in an equilateral triangle and are thus readily identified in the heavy-atom substructure. B3C was incorporated into proteinase K and a multiwavelength anomalous dispersion (MAD) experiment at the Br,K edge was successfully carried out. Radiation damage to the bromine,carbon bond was investigated. A comparison with the phasing tool I3C that contains three I atoms for single-wavelength anomalous dispersion (SAD) phasing was also carried out. [source] Experimental phasing with SHELXC/D/E: combining chain tracing with density modificationACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2010George M. Sheldrick The programs SHELXC, SHELXD and SHELXE are designed to provide simple, robust and efficient experimental phasing of macromolecules by the SAD, MAD, SIR, SIRAS and RIP methods and are particularly suitable for use in automated structure-solution pipelines. This paper gives a general account of experimental phasing using these programs and describes the extension of iterative density modification in SHELXE by the inclusion of automated protein main-chain tracing. This gives a good indication as to whether the structure has been solved and enables interpretable maps to be obtained from poorer starting phases. The autotracing algorithm starts with the location of possible seven-residue ,-helices and common tripeptides. After extension of these fragments in both directions, various criteria are used to decide whether to accept or reject the resulting poly-Ala traces. Noncrystallographic symmetry (NCS) is applied to the traced fragments, not to the density. Further features are the use of a `no-go' map to prevent the traces from passing through heavy atoms or symmetry elements and a splicing technique to combine the best parts of traces (including those generated by NCS) that partly overlap. [source] The effect of heavy atoms on the conformation of the active-site polypeptide loop in human ABO(H) blood-group glycosyltransferase BACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2007James A. Letts The human ABO(H) blood-group antigens are oligosaccharide structures that are expressed on erythrocyte and other cell surfaces. The terminal carbohydrate residue differs between the blood types and determines the immune reactivity of this antigen. Individuals with blood type A have a terminal N -acetylgalactosamine residue and those with blood type B have a terminal galactose residue. The attachment of these terminal carbohydrates are catalyzed by two different glycosyltransferases: an ,(1,3)N -acetylgalactosaminyltransferase (GTA) and an ,(1,3)galactosyltransferase (GTB) for blood types A and B, respectively. GTA and GTB are homologous enzymes that differ in only four of 354 amino-acid residues (Arg/Gly176, Gly/Ser235, Leu/Met266 and Gly/Ala268 in GTA and GTB, respectively). Diffraction-quality crystals of GTA and GTB have previously been grown from as little as 10,mg,ml,1 stock solutions in the presence of Hg, while diffraction-quality crystals of the native enzymes require much higher concentrations of protein. The structure of a single mutant C209A has been determined in the presence and absence of heavy atoms and reveals that when mercury is complexed with Cys209 it forces a significant level of disorder in a polypeptide loop (amino acids 179,195) that is known to cover the active site of the enzyme. The observation that the more highly disordered structure is more amenable to crystallization is surprising and the derivative provides insight into the mobility of this polypeptide loop compared with homologous enzymes. [source] How to get the magic triangle and the MAD triangle into your protein crystalACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 10 2009Tobias Beck The magic triangle 5-amino-2,4,6-triiodoisophthalic acid (I3C) and the MAD triangle 5-amino-2,4,6-tribromoisophthalic acid (B3C) are two representatives of a novel class of compounds that combine heavy atoms for experimental phasing with functional groups for protein interactions. These compounds are readily available and provide easy access to experimental phasing. The preparation of stock solutions and the incorporation of the compounds into protein crystals are discussed. As an example of incorporation via cocrystallization, the incorporation of B3C into bovine trypsin, resulting in a single site with high occupancy, is described. [source] Expression, purification, crystallization and preliminary X-ray diffraction analysis of the TonB-dependent haem outer membrane transporter ShuA from Shigella dysenteriaeACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2009Karl Brillet As part of efforts towards understanding the crystallization of membrane proteins and membrane transport across the outer membrane of Gram-negative bacteria, the TonB-dependent haem outer membrane transporter ShuA of Shigella dysenteriae bound to heavy atoms was crystallized in several crystallization conditions using detergents. The insertion of a His6 tag into an extracellular loop of ShuA, instead of downstream of the Escherichia coli peptide signal, allowed efficient targeting to the outer membrane and the rapid preparation of crystallizable protein. Crystals diffracting X-rays beyond 3.5,Å resolution were obtained by co-crystallizing ShuA with useful heavy atoms for phasing (Eu, Tb, Pb) by the MAD method at the synchrotron, and the SAD or SIRAS method at the Cu wavelength. The authors collected X-ray diffraction data at 2.3,Å resolution using one crystal of ShuA-Pb, and at 3.2,Å resolution at an energy remote from the Pb,M absorption edges for phasing on PROXIMA-1 at SOLEIL. [source] Solution Structure of , -Am2766: A Highly Hydrophobic , -Conotoxin from Conus amadis That Inhibits Inactivation of Neuronal Voltage-Gated Sodium ChannelsCHEMISTRY & BIODIVERSITY, Issue 4 2005Siddhartha The three-dimensional (3D) NMR solution structure (MeOH) of the highly hydrophobic , -conotoxin , -Am2766 from the molluscivorous snail Conus amadis has been determined. Fifteen converged structures were obtained on the basis of 262 distance constraints, 25 torsion-angle constraints, and ten constraints based on disulfide linkages and H-bonds. The root-mean-square deviations (rmsd) about the averaged coordinates of the backbone (N, C,, C) and (all) heavy atoms were 0.62±0.20 and 1.12±0.23,Å, respectively. The structures determined are of good stereochemical quality, as evidenced by the high percentage (100%) of backbone dihedral angles that occupy favorable and additionally allowed regions of the Ramachandran map. The structure of , -Am2766 consists of a triple-stranded antiparallel , -sheet, and of four turns. The three disulfides form the classical ,inhibitory cysteine knot' motif. So far, only one tertiary structure of a , -conotoxin has been reported; thus, the tertiary structure of , -Am2766 is the second such example. Another Conus peptide, Am2735 from C. amadis, has also been purified and sequenced. Am2735 shares 96% sequence identity with , -Am2766. Unlike , -Am2766, Am2735 does not inhibit the fast inactivation of Na+ currents in rat brain Nav1.2 Na+ channels at concentrations up to 200,nM. [source] | ||||||||||||||||||||||