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At least 127 records · Page 7Linked to original sources
PubMed1988

A mass spectral study of cyclophosphamide concerning a thermally induced rearrangement reaction.

The electron impact mass spectra of cyclophosphamide (1) are very sensitive towards experimental conditions in view of the kind of sample handling, the type of mass spectrometer used and the temperature of evaporation. The reason for this phenomenon is the elimination of HCl from the molecular ion by a specific 1,5-hydrogen transfer yielding an ion at m/z 224 which is structurally related to the bicyclic compound 4 with its typical fragment ions at m/z 175 and 147. Thermal excitation of the sample increases the intensity of this fragmentation pathway. The fragmentation pattern of 1 and the thermally induced rearrangement reaction has been elucidated by means of isotopic labelling, high-resolution data, metastable ion analysis and some tandem mass spectrometric experiments. Various samples of 1 monohydrate and its commercially available preparations, which are triturates with sodium chloride, differing in the crystal size distribution, showed nearly identical mass spectra on two different magnetic mass spectrometers, provided that the materials were introduced as solids under careful control of the evaporation temperature. The fragmentation via m/z 224 prevails in case of non-crystalline, pre-dissolved samples on one of the instruments used which might be explained by a differing construction of the ion source and the sample cup holder. The conclusions of Mruzek et al. concerning different proportions of stereoisomers in pharmaceutical preparations of 1 lack any analytical evidence.

Chemical Phenomena↗
PubMedDate not supplied

An automated measurement system for characterization of RF and gradient coil parameters.

A fully automated laboratory-based measurement system for characterization of coil system parameters is presented. This method uses an inexpensive personal computer (PC)-controlled stepper motor positioning system in conjunction with a network/spectrum analyzer and an analog-to-digital converter (A/D) board that allows high resolution data acquisition in an unattended manner. A graphical interface was created for complete control of stepper motor movement, measurement, and data acquisition. The system is capable of performing a wide range of measurements that can, either individually or combined, characterize radiofrequency (RF) and gradient coils used in MRI. Measurement methods, theory, and results for conductor and shield current distributions, mutual impedance, and magnetic fields are given. Comparisons with theoretical calculations are included to validate the accuracy and utility of the system.

Calibration↗
PubMed1990

Structure determination and refinement of Bacillus stearothermophilus lactate dehydrogenase.

Structures have been determined of Bacillus stearothermophilus "apo" and holo lactate dehydrogenase. The holo-enzyme had been co-crystallized with the activator fructose 1,6-bisphosphate. The "apo" lactate dehydrogenase structure was solved by use of the known apo-M4 dogfish lactate dehydrogenase molecule as a starting model. Phases were refined and extended from 4 A to 3 A resolution by means of the noncrystallographic molecular 222 symmetry. The R-factor was reduced to 28.7%, using 2.8 A resolution data, in a restrained least-squares refinement in which the molecular symmetry was imposed as a constraint. A low occupancy of coenzyme was found in each of the four subunits of the "apo"-enzyme. Further refinement proceeded with the isomorphous holo-enzyme from Bacillus stearothermophilus. After removing the noncrystallographic constraints, the R-factor dropped from 30.3% to a final value of 26.0% with a 0.019 A and 1.7 degrees r.m.s. deviation from idealized bond lengths and angles, respectively. Two sulfate ions per subunit were included in the final model of the "apo"-form--one at the substrate binding site and one close to the molecular P-axis near the location of the fructose 1,6-bisphosphate activator. The final model of the holo-enzyme incorporated two sulfate ions per subunit, one at the substrate binding site and another close to the R-axis. One nicotinamide adenine dinucleotide coenzyme molecule per subunit and two fructose 1,6-bisphosphate molecules per tetramer were also included. The phosphate positions of fructose 1,6-bisphosphate are close to the sulfate ion near the P-axis in the "apo" model. This structure represents the first reported refined model of an allosteric activated lactate dehydrogenase. The structure of the activated holo-enzyme showed far greater similarity to the ternary complex of dogfish M4 lactate dehydrogenase with nicotinamide adenine dinucleotide and oxamate than to apo-M4 dogfish lactate dehydrogenase. The conformations of nicotinamide adenine dinucleotide and fructose 1,6-bisphosphate were also analyzed.

Amino Acid Sequence↗
PubMed1990

Crystallographic refinement of human serum retinol binding protein at 2A resolution.

Human serum retinol binding protein (RBP) in complex with retinol has been crystallographically refined to an R-factor of 18.1% with 2A resolution data. The protein topology results in an anti-parallel beta-barrel that encapsulates the retinol ligand. A detailed description of the protein and the binding site is provided. Our structural work has helped to define a family of proteins, many of which are carrier proteins for smaller ligand molecules. We describe the structural basis for the conservation of sequence within the family.

Amino Acid Sequence↗
PubMed1993

Growth and analysis of crystal forms of toxic shock syndrome toxin 1.

Native toxic shock syndrome toxin 1 (TSST-1) purified from Staphylococcus aureus has been crystallized in four different forms. The highest resolution data (2.05 A) was collected from orthorhombic crystals belonging to the space group C222(1). The unit cell dimensions are a = 108.7 A, b = 177.5 A, c = 97.6 A. Rotation function analysis of this form indicates that there is trimer of toxin molecules in the asymmetric unit with a local 3-fold axis parallel to the crystallographic c axis. Crystals of a double mutant of TSST-1 have been grown which has a single molecule in the asymmetric unit and diffract to 1.9 A. The space group is P2(1) with unit cell parameters of a = 44.4 A, b = 34.0 A, c = 55.2 A, beta = 93.0 degrees.

Bacterial Toxins↗
PubMed2001

Toward the three-dimensional structure of the Escherichia coli DNA-binding protein H-NS: A CD and fluorescence study.

The DNA-binding protein H-NS compacts DNA and acts as a specific transcription factor regulating the expression of various bacterial genes. The small abundant protein binds to curved DNA without apparent sequence specificity and the exact nature of its DNA interaction is still unknown. H-NS lacks any common DNA-binding or oligomerization motif and except for a C-terminal fragment of the protein no high resolution structural information is available today. Since the complete structure of H-NS is of considerable interest for understanding its versatile regulatory features, and in lack of high-resolution data for the complete molecule, we have combined circular dichroism (CD) and fluorescence measurements to collect secondary- and higher-order structural information on H-NS. Comparison of CD analyses of wild type H-NS and functional defective mutants allowed assigning secondary structure elements to the N-terminal oligomerization domain of the protein. Moreover, according to fluorescence energy-transfer data we calculate a 45 A distance between the DNA-binding and the oligomerization domain of H-NS.

Bacterial Proteins↗
PubMed1993

Refined X-ray structures of haloalkane dehalogenase at pH 6.2 and pH 8.2 and implications for the reaction mechanism.

The crystal structure of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 has been refined at 1.9 A resolution at two different pH values, the pH of crystallization (pH 6.2) and the pH of optimal activity (pH 8.2), to final R-factors of 16.8% and 16.4%, respectively. Both models show good stereochemical quality. Two non-glycine residues have main-chain torsion angles that are located outside the "allowed" regions in a Ramachandran plot. One of them is the nucleophilic residue Asp124, which, together with the two other active site residues His289 and Asp260, is situated in an internal, predominantly hydrophobic cavity. The other residue, Asn148, helps stabilize the conformations of two of these active-site residues, Asp124 and Asp260. Comparison of the models at pH 6.2 and pH 8.2 revealed one major structural difference. At pH 6.2, a salt-bridge is present between the N epsilon 2 atom of His289 and the O delta 1 atom of Asp124, while at pH 8.2, this salt-bridge is absent, indicating that the N epsilon 2 atom of the histidine residue is mostly deprotonated at the pH of optimum activity. This is in agreement with the putative reaction mechanism in which the O delta 1 atom of Asp124 performs a nucleophilic attack on the substrate, resulting in an intermediate ester. This ester is subsequently cleaved by a hydrolytic water molecule. The high-resolution data sets clearly show the exact position of this water molecule. It is in an ideal position for donating a proton to the N epsilon 2 atom of His289 and subsequently cleaving the covalently bound intermediate ester, releasing the alcohol product. Detailed investigation of both refined models showed a number of unusual structural features. Four out of 11 helices contain an internal proline residue other than in the first turn. Two other alpha-helices have adopted in their central part a 3(10) conformation. A novel four-residue turn between a helix and a strand, the alpha beta 4 turn, is located at the site of the bend in the central eight-stranded beta-sheet of the dehalogenase structure.

Amino Acid Sequence↗
PubMed1995

Refined crystal structure of spinach ferredoxin reductase at 1.7 A resolution: oxidized, reduced and 2'-phospho-5'-AMP bound states.

The crystal structure of spinach ferredoxin-NADP(+)-oxidoreductase (FNR), determined by multiple isomorphous replacement at 2.6 A resolution, has been refined at 1.7 A resolution to an R-factor of 17.9%. The structure of FNR bound to the competitive inhibitor 2'-phospho-5'-AMP (P-AMP) has also been refined at 1.7 A to an R-factor of 17.4% and dithionite-reduced/P-AMP-bound FNR has been refined at 2.0 A to an R-factor of 14.9%. The P-AMP-bound structure was used to construct a model for the binding of NADP+. Over 200 solvation sites were included in each structure, and many of the best defined solvation sites stabilize buried turns. A bulk solvent correction obviated the need for a low-resolution data cutoff. An acidic side-chain likely to be responsible for the low pH requirement for crystallization has been identified. Three large networks of the hydrophobic side-chains help define the FNR structure. One of these contains a large cavity far from the active site, which coincides with the lone site of sequence heterogeneity in FNR, and may provide a site for membrane attachment. The reduced structure shows that Ser96 moves toward atom N-5 of FAD and a water molecule moves toward atom N-1 of FAD, while the flavin moiety remains planar. Possible sources of a proton that must be picked up upon reduction are discussed.

Binding Sites↗
PubMed1994

Crystallization and preliminary crystallographic analysis of NADPH:FMN oxidoreductase from Vibrio harveyi.

Crystals of NADPH:FMN oxidoreductase from Vibrio harveyi have been obtained and characterized by X-ray diffraction. This enzyme plays a role in the generation of light in luminescent bacteria by providing reduced FMN to luciferase. Large, high quality crystals were grown using polyethylene glycol 6000 at pH 7.0. They crystallize in the monoclinic space group P2(1) with cell dimensions a = 51.2 A, b = 85.9 A, c = 58.1 A, beta = 109.3 degrees, and diffract to 1.8 A. We expect two molecules per asymmetric unit. High resolution data sets have been recorded and a search is under way for heavy-atom derivatives.

Crystallization↗
PubMed1994

Towards atomic interpretation of F-actin filament three-dimensional reconstructions.

We have recorded dark field images of negatively stained F-actin filaments polymerized with 2 mM MgCl2 and 50 mM KCl with a scanning transmission electron microscope and computed 3-D reconstructions using a helical parameter search to optimize simultaneously the helical repeat length, the radial position of the filament axis, and the helical selection rule. The resulting optimized averaged filament 3-D reconstruction at 2.5 nm resolution is remarkably similar to an atomic model of the F-actin filament. By comparison, several structural features of the reconstruction can be interpreted at the level of distinct secondary structure elements, and predictions made by the atomic model could be verified: for instance, the density connecting the two long-pitch helical strands in our reconstruction co-localizes with an extended beta-hairpin, the "hydrophobic loop" (i.e. residues 262 to 274), which according to the atomic model establishes the major intersubunit contact between the two long-pitch helical strands. The most pronounced structural variations among individual filament 3-D reconstructions were observed in (1) the details of the intersubunit contact pattern between the two long-pitch helical strands, and (2) the exact size and shape of subdomain 2 of the F-actin molecule, which appears rather flexible and easily deformed. In addition, we found that all phenotypes of F-actin filament 3-D reconstructions that arise from small deviations from the optimal helical parameters or from lowering the nominal resolution exhibited stronger intersubunit contacts between than along the two long-pitch helical strands, a structural feature that has been emphasized for a number of F-actin filament 3-D reconstructions in the past. Since this is clearly at variance with the relative strength of the intersubunit contacts as predicted by the atomic model, it may represent an artifactual structural feature arising from low-resolution data or suboptimal helical data processing, and should therefore be interpreted with caution in terms of indicating chemical, mechanical or conformational states of the F-actin filament.

Actins↗
PubMed1995

Crystallographic structure of a PLP-dependent ornithine decarboxylase from Lactobacillus 30a to 3.0 A resolution.

Ornithine decarboxylase from Lactobacillus 30a (L30a OrnDC) is representative of the large, pyridoxal-5'-phosphate-dependent decarboxylases that act on lysine, arginine or ornithine. The crystal structure of the L30a OrnDC has been solved to 3.0 A resolution using MIR phases in combination with density modification (space group P6; a = 195.6 A, c = 97.6 A; dimer of 1460 amino acid residues/asymmetric unit; VM = 3.26 A3/Da). The refined crystallographic R-value was 0.219 (Rfree = 0.268) using 2-fold restraints with a 4 sigma cutoff and 8.0 to 3.0 A resolution data. Six dimers related by C6 symmetry compose the enzymatically active dodecamer (approximately 10(6) Da). Each monomer of L30a OrnDC can be described in terms of five sequential folding domains. The amino-terminal domain, residues 1 to 107, consists of a five-stranded beta-sheet termed the "wing" domain. Two wing domains of each dimer project inward towards the center of the dodecamer and contribute to dodecamer stabilization. The "linker" domain, residues 108 to 160, consists of short alpha-helices separated by a loop that fills in the PLP pocket. The third domain, residues 161 to 413, is an alpha/beta domain containing a seven stranded beta-sheet that resembles the PLP-binding domain of the aspartate aminotransferases. The fourth domain, residues 414 to 569, resembles the "small" domain of the aspartate aminotransferases, but is significantly larger due to insertions. The remaining carboxy-terminal domain, residues 570 to 730, is organized into multiple antiparallel loops and seven alpha-helices that help form a deep channel leading to the PLP-binding site.

Amino Acid Sequence↗
PubMed1997

The 2.6 A structure of antithrombin indicates a conformational change at the heparin binding site.

The crystal structure of a dimeric form of intact antithrombin has been solved to 2.6 A, representing the highest-resolution structure of an active, inhibitory serpin to date. The crystals were grown under microgravity conditions on Space Shuttle mission STS-67. The overall confidence in the structure, determined earlier from lower resolution data, is increased and new insights into the structure-function relationship are gained. Clear and continuous electron density is present for the reactive centre loop region P12 to P14 inserting into the top of the A-beta-sheet. Areas of the extended amino terminus, unique to antithrombin and important in the binding of the glycosaminoglycan heparin, can now be traced further than in the earlier structures. As in the earlier studies, the crystals contain one active and one latent molecule per asymmetric unit. Better definition of the electron density surrounding the D-helix and of the residues implicated in the binding of the heparin pentasaccharide (Arg47, Lys114, Lys125, Arg129) provides an insight into the change of affinity of binding that accompanies the change in conformation. In particular, the observed hydrogen bonding of these residues to the body of the molecule in the latent form explains the mechanism for the release of newly formed antithrombin-protease complexes into the circulation for catabolic removal.

Antithrombin III↗
PubMed1997

The 1.8 A crystal structure of winged bean albumin 1, the major albumin from Psophocarpus tetragonolobus (L.) DC.

Winged bean albumin-1 (WBA) is the main seed albumin of Psophocarpus tetragonolobus, a legume that has excellent potential as a protein-rich food source for humid tropical climates. WBA crystallises in a tetragonal space group and the structure was solved by X-ray crystallography with a combination of multiple isomorphous replacement using four heavy atom derivatives and molecular replacement with a model based on the structure of Erythrina caffra trypsin inhibitor (ETI). Refinement of the structure proceeded to 1.8 A. WBA has a beta-trefoil fold, similar to that found in the STI-Kunitz type trypsin inhibitors. The final structure has an overall R-factor of 19% for 15 to 1.8 A resolution data, all residues in the allowed regions of the Ramachandran plot, and good agreement with ideal geometry. WBA has sequence similarity with the STI-Kunitz trypsin inhibitors, including the apparent conservation of the functional reactive site residue, lysine 64, at the position of the scissile bond (position P1) in the STI-Kunitz type trypsin inhibitors, however, WBA does not inhibit trypsin. The reason for the lack of inhibitory activity against trypsin is clearly evident from the structure. The loop corresponding to the inhibitory loop in the STI-Kunitz trypsin inhibitors does not conform to the canonical conformation of the inhibitory loops of the "small inhibitors". The lysine residue assigned to the P1 position from sequence alignments is instead part of a four amino acid insertion between residues structurally equivalent to residues P1 and P2 of the inhibitors.

Albumins↗
PubMed1998

Polymorphic quaternary organization of the Bacillus subtilis bacteriophage SPP1 replicative helicase (G40 P).

The Bacillus subtilis bacteriophage SPP1 gene 40 product (G40P), which belongs to the DnaB-like family of helicases, is essential for SPP1 genome replication. The active form of the enzyme is the hexamer, capable of DNA unwinding with a 5' to 3' polarity fueled by the hydrolysis of a nucleoside 5'-triphosphate. We have used electron microscopy of negatively stained G40P samples and image processing techniques to study the structural characteristics of the hexameric assemblies of this protein. Our results provide the first low resolution data on a hexameric helicase of a Gram-positive bacterial origin. A novel approach has been adopted to analyze possible symmetry heterogeneities, an unsupervised method based on a neural network self-organizing algorithm, which has led to the detection of different subclasses of G40P views. Two different quaternary states of G40P homohexamers sharing a C3 symmetry organization have been found, as well as a minor class that seems to reflect an alternative C6 symmetry architecture. These forms show general features known for other hexameric helicases, such as the ring-like arrangement of monomers around a central hole. A clear structural handedness has also been detected in some of these forms. An analysis of these quaternary states and a model for the structural organization of G40P are presented.

Bacillus subtilis↗
PubMed1999

Sulphate ions observed in the 2.12 A structure of a new crystal form of S. cerevisiae phosphoglycerate mutase provide insights into understanding the catalytic mechanism.

The structure of a new crystal form of Saccharomyces cerevisiae phosphoglycerate mutase has been solved and refined to 2.12 A with working and free R-factors of 19.7 and 22.9 %, respectively. Higher-resolution data and greater non-crystallographic symmetry have produced a more accurate protein structure than previously. Prominent among the differences from the previous structure is the presence of two sulphate ions within each active site cleft. The separation of the sulphates suggests that they may occupy the same sites as phospho groups of the bisphosphate ligands of the enzyme. Plausible binding modes for 2,3-bisphosphoglycerate and 1, 3-bisphosphoglycerate are thereby suggested. These results support previous conclusions from mutant studies, highlight interesting new targets for mutagenesis and suggest a possible mechanism of enzyme phosphorylation.

2,3-Diphosphoglycerate↗
PubMed2000

The crystal structure of yeast phenylalanine tRNA at 2.0 A resolution: cleavage by Mg(2+) in 15-year old crystals.

We have re-determined the crystal structure of yeast tRNA(Phe) to 2. 0 A resolution using 15 year old crystals. The accuracy of the new structure, due both to higher resolution data and formerly unavailable refinement methods, consolidates the previous structural information, but also reveals novel details. In particular, the water structure around the tightly bound Mg(2+) is now clearly resolved, and hence provides more accurate information on the geometry of the magnesium-binding sites and the role of water molecules in coordinating the metal ions to the tRNA. We have assigned a total of ten magnesium ions and identified a partly conserved geometry for high-affinity Mg(2+ )binding. In the electron density map there is also clear density for a spermine molecule binding in the major groove of the TPsiC arm and also contacting a symmetry-related tRNA molecule. Interestingly, we have also found that two specific regions of the tRNA in the crystals are partially cleaved. The sites of hydrolysis are within the D and anticodon loops in the vicinity of Mg(2+).

Base Sequence↗
PubMed2000

Structural basis for isotype selectivity of the human retinoic acid nuclear receptor.

The human retinoic acid receptor (hRAR) belongs to the family of nuclear receptors that regulate transcription in a ligand-dependent way. The isotypes RARalpha,beta and gamma are distinct pharmacological targets for retinoids that are involved in the treatment of various skin diseases and cancers, in particular breast cancer and acute promyelocytic leukemia. Therefore, synthetic retinoids have been developed aiming at isotype selectivity and reduced side-effects. We report the crystal structures of three complexes of the hRARgamma ligand-binding domain (LBD) bound to agonist retinoids that possess selectivity either for RARgamma (BMS184394) or for RARbeta/gamma (CD564), or that are potent for all RAR-isotypes (panagonist BMS181156). The high resolution data (1.3-1. 5 A) provide a description at the atomic level of the ligand pocket revealing the molecular determinants for the different degrees of ligand selectivity. The comparison of the complexes of the chemically closely related retinoids BMS184394 and CD564 shows that the side-chain of Met272 adopts different conformations depending on the presence of a hydrogen bond between its sulfur atom and the ligand. This accounts for their different isotype selectivity. On the other hand, the difference between the pan- and the RARbeta, gamma-selective agonist is probably due to a steric discrimination at the level of the 2-naphthoic acid moiety of CD564. Based on this study, we propose a model for a complex with the RARgamma-specific agonist CD666 that shows the possible applications for structure-based drug design of RAR isotype-selective retinoids.

Binding Sites↗
PubMed2000

Crystal structure of N-acyl-D-glucosamine 2-epimerase from porcine kidney at 2.0 A resolution.

The X-ray crystallographic structure of N-acyl-d-glucosamine 2-epimerase (AGE) from porcine kidney, which has been identified to be a renin-binding protein (RnBP), was determined by the multiple isomorphous replacement method and refined at 2.0 A resolution with a final R-factor of 16.9 % for 15 to 2.0 A resolution data. The refined structure of AGE comprised 804 amino acid residues (one dimer) and 145 water molecules. The dimer of AGE had an asymmetric unit with approximate dimensions 46 Ax48 Ax96 A. The AGE monomer is composed of an alpha(6)/alpha(6)-barrel, the structure of which is found in glucoamylase and cellulase. One side of the AGE alpha(6)/alpha(6)-barrel structure comprises long loops containing five short beta-sheets, and contributes to the formation of a deep cleft shaped like a funnel. The putative active-site pocket and a possible binding site for the substrate N-acetyl-d-glucosamine (GlcNAc) were found in the cleft. The other side of the alpha(6)/alpha(6)-barrel comprises short loops and contributes to the dimer formation. At the dimer interface, which is composed of the short loops and alpha-helices of the subunits, five strong ion-pair interactions were observed, which play a major role in the dimer assembly. This completely ruled out the previously accepted hypothesis that the formation of the RnBP homodimer and RnBP-renin heterodimer requires the leucine zipper motif present in RnBP.

Animals↗