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L DeLucas

Publications and source records attributed to L DeLucas.

14 recordsLinked to original sources
PubMed2002

Crystallization and structure analysis of Thermus flavus 5S rRNA helix B.

The crystallization conditions of the synthetic RNA duplex r(GCGGCGU)*r(GCGCCGC), part of the Thermus flavus 5S rRNA domain B, were investigated in detail. The crystallization analysis revealed a relative narrow crystallization zone. Single sequence variations did not enhance the crystal quality, however the crystallization under microgravity provided crystals of higher quality. They belong to the space group P3(1)21 with unit cell dimensions of a = b = 35.0 A and c = 141.2 A. Diffraction data up to 2.6 A were collected and the structure subsequently analysed and refined to an R-value of 22.4 %. The conformation of the two molecules in the asymmetric unit is stabilized by intermolecular hydrogen bonds. The two molecules A and B are perpendicular to each other and interacting head to tail with symmetry related molecules. They form pseudo-continuous infinite helices in the crystal lattice.

Base Sequence↗
PubMed2002

Crystallisation under microgravity of mistletoe lectin I from Viscum album with adenine monophosphate and the crystal structure at 1.9 A resolution.

The crystal structure of the ribosome-inactivating protein (RIP) mistletoe lectin I (ML-I) from Viscum album in complex with adenine has been refined to 1.9 A resolution. High quality crystals of the ML-I complex were obtained by the method of vapour diffusion using the high density protein crystal growth system (HDPCG) on the international space station, mission ISS 6A. Hexagonal crystals were grown during three months under microgravity conditions. Diffraction data to 1.9A were collected applying synchrotron radiation and cryo- techniques. The structure was refined subsequently to analyse the structure of ML-I and particularly the active site conformation, complexed by adenine that mimics the RNA substrate binding.

Adenosine Monophosphate↗
PubMed2001

Stabilization of active-site loops in NH3-dependent NAD+ synthetase from Bacillus subtilis.

The NH(3)-dependent NAD(+) synthetase (NADS) participates in the biosynthesis of nicotinamide adenine dinucleotide (NAD(+)) by transforming nicotinic acid adenine dinucleotide (NaAD) to NAD(+). The structural behavior of the active site, including stabilization of flexible loops 82-87 and 204-225, has been studied by determination of the crystal structures of complexes of NADS with natural substrates and a substrate analog. Both loops are stabilized independently of NaAD and solely from the ATP-binding site. Analysis of the binding contacts suggests that the minor loop 82-87 is stabilized primarily by a hydrogen bond with the adenine base of ATP. Formation of a coordination complex with Mg(2+) in the ATP-binding site may contribute to the stabilization of the major loop 204-225. The major loop has a role in substrate recognition and stabilization, in addition to the protection of the reaction intermediate described previously. A second and novel Mg(2+) position has been observed closer to the NaAD-binding site in the structure crystallized at pH 7.5, where the enzyme is active. This could therefore be the catalytically active Mg(2+).

Amide Synthases↗
PubMed2000

Crystal structure of Dengue virus NS3 protease in complex with a Bowman-Birk inhibitor: implications for flaviviral polyprotein processing and drug design.

Dengue viruses are members of the Flaviviridae and cause dengue fever and the more severe dengue hemorrhagic fever. Although nearly 40 % of the world's population is at risk of dengue infection, there is currently no effective vaccine or chemotherapy for the disease. Processing of the dengue polyprotein into structural and non-structural proteins in a host, which is essential for assembly of infective virions, is carried out by the combined action of host proteases and the trypsin-like, two-component viral NS2B/NS3 serine protease. Although NS2B strongly stimulates the catalytic NS3 protease domain, the latter is fully active against small substrates and possesses detectable activity against larger substrates, making both forms of the enzyme possible targets for drug design. In the crystal structure of a complex of the protease with a Bowman-Birk inhibitor reported here, an Arg residue at the P1 position of the inhibitor is bound in a manner distinctly different from that in other serine proteases of comparable specificity. However, because the regulatory component, NS2B, is not present in the complex, the physiological implications of this observations are currently unclear. The redundant nature of interaction of P1 Arg and Lys residues with Asp129, Tyr150 and Ser163 of the enzyme provides an explanation for the observed behavior of several site-specific mutants of Asp129 in the protease. The strong level of conservation of residues in the protease that interact with the P1 Arg, along with conservation of Arg at P1 of most cleavage sites in other flaviviruses, suggests that observations from this structure are likely to be applicable to many flaviviruses. The structure provides a starting point for design of site-specific mutations to probe the mechanism of catalysis by the catalytic domain, its activation by the regulatory domain and for design of specific inhibitors of enzymatic activity.

Catalytic Domain↗
PubMed2000

Structure of the nucleotide-binding domain of Plasmodium falciparum rab6 in the GDP-bound form.

Rab proteins are small Ras-like GTPases which play important roles in regulating intracellular vesicle trafficking. The nucleotide-binding domain of Rab6 from the malaria parasite Plasmodium falciparum was crystallized with GDP bound to the active site. The MAD phasing technique was used to determine the crystal structure to 2.3 A resolution. Comparisons of the structure of GDP-bound PfRab6 with the recently determined structures of Rab3A in complex with either a GTP analog or with GTP and Rabphillin present structural evidence supporting the traditional model for the molecular GTP/GDP switch in Rab proteins. PfRab6 residues homologous to those distinguishing human Rab6 isoforms, which differ in binding to Rabkinesin-6 in human cells, are located next to the recognized complementarity-determining region (CDR) and constitute a conceptual broadening of that domain. Despite significant observable differences in Golgi ultrastructure, the Rab6 core structure and switch mechanism appear highly conserved when compared with murine Rab3a structures. A significant difference between the PfRab6 and higher eukaryotic Rabs may be the lack of CDR features that allow binding interactions with Rabkinesin-type effectors.

Amino Acid Sequence↗
PubMed2000

Structure of human alpha-thrombin complexed with RWJ-51438 at 1.7 A: unusual perturbation of the 60A-60I insertion loop.

The three-dimensional structure of the ternary complex consisting of human alpha-thrombin, hirugen and the active-site inhibitor RWJ-51438 has been determined at 1.7 A resolution. The crystals of the complex belong to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 62.98, b = 117.52, c = 47.99 A. The refined R and R(free) values are 0.196 and 0.232, respectively. The ketone carbonyl group of the inhibitor is covalently linked to the hydroxyl O atom of Ser195, forming a tetrahedral intermediate hemiketal structure; the benzothiazole ring N atom of RWJ-51438 forms a hydrogen bond with His57. Surprisingly, the carboxylate substituent on the benzothiazole group forms salt bridges with Lys60F NZ and the NZ of the symmetry-related residues Lys236 and Lys240, which introduces steric effects that perturb the 60A-60I insertion loop, especially at residues Trp60D and Phe60H.

Antithrombins↗
PubMed1999

Crystallization, characterization and measurement of MAD data on crystals of dengue virus NS3 serine protease complexed with mung-bean Bowman-Birk inhibitor.

Crystallization and preliminary characterization of the essential dengue virus NS3 serine protease complexed with a Bowman-Birk-type inhibitor from mung beans are reported. As the structure proved resistant to solution by molecular replacement and multiple isomorphous replacement methods, multi-wavelength anomalous diffraction data at the LIII edge of a holmium derivative have been measured. Promising Bijvoet and dispersive signals which are largely consistent with expected values have been extracted from the data. The structure, when determined, will provide a structural basis for the design, synthesis and evaluation of inhibitors of the protease for chemotherapy of dengue infections.

Crystallization↗
PubMed1997

Purification, crystallization and preliminary X-ray diffraction studies of recombinant calcium-binding domain of the small subunit of porcine calpain.

The calcium-binding domain of the small subunit of porcine calpain (domain VI) has been expressed in Escherichia coli, purified, and crystallized in the presence of Ca(2+). Two crystal forms have been obtained by the vapor-diffusion method using PEG 6000 as the precipitant. Crystal form I, belonging to trigonal space group P3(1)21 (or P3(2)21) with cell dimensions a = b = 79.8, c = 57.08 A, alpha = beta = 90.0 and gamma, = 120.0 degrees diffracted to 2.8 A. The second crystal form diffracts to 1.8 A and belongs to monoclinic space group P2(1) with cell dimensions a = 50.1, b = 79.7, c = 57.1 A and beta = 91.2 degrees.

Journal Article↗
PubMed1997

Crystallization and preliminary cryogenic X-ray diffraction analyses of protein L-isoaspartyl O-methyltransferase from human fetal brain.

Recombinant human fetal brain protein L-isoaspartyl O-methyltransferase, EC 2.1.1.77, was crystallized in PEG 8000 with adenosine homocysteine by a macroseeding technique. The space group was P2(1) with a = 47.4 A, b = 53.9 A, c = 48.7 A and beta = 116.4 degrees for cryofrozen crystals at 90 K. The crystals diffracted to 2.1 A and have one molecule per asymmetric unit.

Brain Chemistry↗
PubMed1984

Crystallization and preliminary x-ray investigation of a protein neurotoxin from the sea anemone Anthopleura xanthogrammica.

Neurotoxic protein AX-I isolated from the sea anemone Anthopleura xanthogrammica has been crystallized. Two crystal forms were obtained. One form is tetragonal, space group P41 or P43 , with cell dimensions a = b = 40.74(2) A and c = 34.13(3) A. The second crystal form is hexagonal, space group P622 , with cell dimensions a = b = 72.23(2) A and c = 39.16(2)A. Density measurements indicate that the tetragonal crystals contain one toxin molecule/asymmetric unit. Both crystal forms diffract to high resolution.

Animals↗
PubMed1979

Ferrocyanide staining of transferrin and ferritin-conjugated antibody to transferrin.

To evaluate the ultrastructural distribution of transferrin on the surface of L1210 ascites tumor cells, we used ferrocyanide to stain ferric iron (Prussian blue reaction) in transferrin, as well as in ferritin conjugated to antibody that was immunologically attached to the transferrin. Small deposits averaging 5 nm in diameter identified transferrin iron, whereas large cuboidal deposits averaging 50 nm in diameter stained ferritin conjugated-antibody that was bound to both transferrin and apotransferrin on the cell surface. The ability of transferrin to deliver iron to ascites tumor cells was confirmed by kinetic studies of transferrin labeled with 59Fe and 125I. These preliminary results are consistent with release of transferrin iron at the cell surface and demonstrate additional uses for ferrocyanide in ultrastructural cytochemical techniques.

Animals↗
PubMed1975

Calcium binding to D-glucuronate residues: crystal structure of a hydrated calcium bromide salt of D-glucuronic acid.

Three-dimensional X-ray diffraction data were used to determine the crystal structure of alpha-D-glucuronate CaBr times 3H20, a model system for investigating the factors involved in the binding of calcium ions to D-glucuronate residues of oligo-and poly-saccharides. Crystals of the salt are monoclinic, space group P21, having a = 6.410 (1), b = 10.784 (2), c = 8.879 (1) A, betta = 92.07 (1)degrees, and Z = 2. Instensity data for 1082 reflections were measured with an automated diffractometer. A trial structure, obtained by the heavy-atom method, was refined by least squares to R = 0.025. The absolute configuration was confirmed by anomalous-dispersion effects. An outstanding feature of the crystal packing is the interaction of D-glucuronate anions with calcium ions. The calcium ion is coordinated to three symmetry-related D-glucuronate anions and to two water molecules. The D-glucuronate anion binds calcium cations through three chelation sites: one that involves a carboxyl-oxygen atom combined with O-5; one that includes the second carboxyl-oxygen atom acting in concert with O-4, and one composed of the O-1-O-2 pair of hydroxyl groups.

Calcium↗
PubMedDate not supplied

Expression of the recombinant human immunoglobulin J chain in Escherichia coli.

Selective transport of polymeric (p) immunoglobulins (Ig) of IgA and IgM isotypes into external secretions by pIg receptor-mediated mechanism depends on the incorporation of joining (J) chain into the polymers. Until now, availability of a free J chain for immunological and biophysical studies has been limited to preparations of denatured J chain forms with moderate yield. Here we report that a recombinant J chain (rJ) can be over-expressed as a soluble fusion protein with thioredoxin using a modified vector pET32 in Escherichia coli. An intact J chain was released by digestion with IgA1 protease from Neisseria gonorrhoeae and isolated in a good yield with immunological and biochemical properties similar to those of J chain obtained by chemical cleavage from pIgA.

Amino Acid Sequence↗