Purification and preliminary crystallographic analysis of a new Lys49-PLA2 from B-jararacussu

BjVIII is a new myotoxic Lys49-PLA2 isolated from Bothrops jararacussu venom that exhibits atypical effects on human platelet aggregation. To better understand the mode of action of BjVIII, crystallographic studies were initiated. Two crystal forms were obtained, both containing two molecules in the asymmetric unit (ASU). Synchrotron radiation diffraction data were collected to 2.0 angstrom resolution and 1.9 angstrom resolution for crystals belonging to the space group P2(1)2(1)2(1) (a = 48.4 angstrom, b = 65.3 angstrom, c = 84.3 angstrom) and space group P3(1)21 (a = b = 55.7 angstrom, c = 127.9 angstrom), respectively. Refinement is currently in progress and the refined structures are expected to shed light on the unusual platelet aggregation activity observed for BjVIII.

Crystallization and preliminary X-ray crystallographic analysis of the heterodimeric crotoxin complex and the isolated subunits crotapotin and phospholipase A(2)

Crotoxin, a potent neurotoxin from the venom of the South American rattlesnake Crotalus durissus terrificus, exists as a heterodimer formed between a phospholipase A(2) and a catalytically inactive acidic phospholipase A(2) analogue (crotapotin). Large single crystals of the crotoxin complex and of the isolated subunits have been obtained. The crotoxin complex crystal belongs to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 38.2, b = 68.7, c = 84.2 angstrom, and diffracted to 1.75 angstrom resolution. The crystal of the phospholipase A(2) domain belongs to the hexagonal space group P6(1)22 (or its enantiomorph P6(5)22), with unit-cell parameters a = b = 38.7, c = 286.7 angstrom, and diffracted to 2.6 angstrom resolution. The crotapotin crystal diffracted to 2.3 angstrom resolution; however, the highly diffuse diffraction pattern did not permit unambiguous assignment of the unit-cell parameters.

Crystallization and preliminary X-ray crystallographic analysis of chorismate synthase from Mycobacterium tuberculosis

The enzymes of the shikimate pathway are potential targets for the development of new therapies because they are essential for bacteria but absent from mammals. The last step in this pathway is performed by chorismate synthase (CS), which catalyzes the conversion of 5-enolpyruvylshikimate-3-phosphate to chorismate. Optimization of crystallization trials allowed the crystallization of homogeneous recombinant CS from Mycobacterium tuberculosis (MtCS). The crystals of MtCS belong to space group P6(4)22 (or P6(2)22) and diffract to 2.8 Angstrom resolution, with unit-cell parameters a = b = 129.7, c = 156.8 Angstrom. There are two molecules in the asymmetric unit. Molecular-replacement trials were not sucessful. Heavy-atom derivative screening is in progress.

Crystallization and preliminary crystallographic analysis of SMase I, a sphingomyelinase from Loxosceles laeta spider venom

SMase I, a 32 kDa sphingomyelinase found in Loxosceles laeta venom, is responsible for the major pathological effects of spider envenomation. This toxin has been cloned and functionally expressed as a fusion protein containing a 6 x His tag at its N-terminus to yield a 33 kDa protein [Fernandes-Pedrosa et al. (2002), Biochem. Biophys. Res. Commun. 298, 638 - 645]. The recombinant protein possesses all the biological properties ascribed to the whole L. laeta venom, including dermonecrotic and complement-dependent haemolytic activities. Dynamic light-scattering experiments conducted at 291 K demonstrate that the sample possesses a monomodal distribution, with a hydrodynamic radius of 3.57 nm. L. laeta SMase I was crystallized by the hanging-drop vapour-diffusion technique using the sparse-matrix method. Single crystals were obtained using a buffer solution consisting of 0.08 M HEPES and 0.9 M trisodium citrate, which was titrated to pH 7.5 using 0.25 M sodium hydroxide. Complete three-dimensional diffraction data were collected to 1.8 Angstrom at the Laboratorio Nacional de Luz Sincrotron (LNLS, Campinas, Brazil). The crystals belong to the hexagonal system ( space group P6(1) or P6(5)), with unit-cell parameters a = b = 140.6, c = 113.6 Angstrom. A search for heavy-atom derivatives has been initiated and elucidation of the crystal structure is currently in progress.

Crystallization and preliminary X-ray crystallographic analysis of importin-alpha from Neurospora crassa

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Estudos conformocionais de diversas proteínas em solução utilizando a técnica de espalhamento de raios-X à baixo ângulo-saxs

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Quantitative phase analysis from X-ray diffraction in Y-TZP dental ceramics: A critical evaluation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Structural Basis for Interaction of Inhibitors with Cyclin-Dependent Kinase 2

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Synthesis, structure, and electronic and EPR spectra of copper(II) complexes containing the [CuBr4]2- anion and triphenylarsine oxide

The preparation and characterization of (Ph3AsOH)2[CuBr4] and [Cu(Ph3AsO)4][CuBr4] are reported (Ph3AsO = triphenylarsine oxide). Crystallographic analysis of the monoclinic crystals of (Ph3AsOH)2[CuBr4] (space group C2/c, a = 17.569 (3) Å, b = 13.090 (2) Å, c = 16.933 (2) Å, and β = 105.64 (2)°, R = 0.055 and Rw = 0.057) revealed the presence of compressed [CuBr4]2- tetrahedra of C2 symmetry with Cu-Br distances of 2.340 (1) and 2.437 (1) Å and trans-Br-Cu-Br angles of 139.2 (1) and 122.4 (1)°. The oxonium cations hydrogen bond to the bromine atoms involved in the longer Cu-Br bonds and the smaller trans-Br-Cu-Br angle. Single-crystal electronic and EPR spectra are interpreted in terms of the observed [CuBr4]2- geometry. Analysis of the electronic and EPR spectra of [Cu(Ph3AsO)4][CuBr4] led to the postulation of the presence of planar [Cu(Ph3AsO)4]2+ cations and distorted tetrahedral [CuBr4]2- anions. © 1992 American Chemical Society.

Avaliação de cálculos urinários em per-operatórios por imagem digital e técnicas físico-químicas e analíticas

Pós-graduação em Engenharia Mecânica - FEG

Complexos de zinco(II), de európio(III) e heterobimetálico com os ácidos tiofeno 2 e 3-carboxílico. Síntese, caracterização e propriedades luminescentes

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Crystallization and preliminary X-ray analysis of S-III-SPIII, a phospholipase A2 isolated from the venom of Bothrops jararacussu.

Large single crystals have been obtained of S-III-SPIII, a phospholipase A(2) from the venom of Bothrops jararacussu. The crystals belong to the orthorhombic system space group C222, and diffract X-rays to a resolution of 1.9 Angstrom. Preliminary analysis reveals the presence of one molecule in the crystallographic asymmetric unit. The crystal structure is currently being determined using molecular replacement techniques.

Crystallographic portrayal of different conformational states of a Lys49 phospholipase A2 homologue: Insights into structural determinants for myotoxicity and dimeric configuration

Catalytically inactive phospholipase A2 (PLA2) homologues play key roles in the pathogenesis induced by snake envenomation, causing extensive tissue damage via a mechanism still unknown. Although, the amino acid residues directly involved in catalysis are conserved, the substitution of Asp49 by Arg/Lys/Gln or Ser prevents the binding of the essential calcium ion and hence these proteins are incapable of hydrolyzing phospholipids. In this work, the crystal structure of a Lys49-PLA2 homologue from Bothrops brazili (MTX-II) was solved in two conformational states: (a) native, with Lys49 singly coordinated by the backbone oxygen atom of Val31 and (b) complexed with tetraethylene glycol (TTEG). Interestingly, the TTEG molecule was observed in two different coordination cages depending on the orientation of the nominal calcium-binding loop and of the residue Lys49. These structural observations indicate a direct role for the residue Lys49 in the functioning of a catalytically inactive PLA2 homologue suggesting a contribution of the active site-like region in the expression of pharmacological effects such as myotoxicity and edema formation. Despite the several crystal structures of Lys49-PLA2 homologues already determined, their biological assembly remains controversial with two possible conformations. The extended dimer with the hydrophobic channel exposed to the solvent and the compact dimer in which the active site-like region is occluded by the dimeric interface. In the MTX-II crystal packing analysis was found only the extended dimer as a possible stable quaternary arrangement. © 2012 Elsevier B.V.

Crystallization and preliminary X-ray diffraction analysis of a myotoxic phospholipase A(2) homologue from Bothrops neuwiedi pauloensis venom

Crystals of a myotoxic phospholipase A(2) from Bothrops neuwiedi pauloensis have been obtained. They diffracted at 2.5 Angstrom resolution using a synchrotron radiation source and belong to space group P3(1)21. Preliminary analysis shows that there are two molecules in the asymmetric unit. (C) 1999 Elsevier B.V. B.V. All rights reserved.

Crystallization and preliminary X-ray diffraction analysis of an acidic phospholipase A(2) complexed with p-bromophenacyl bromide and alpha-tocopherol inhibitors at 1.9-and 1.45-A resolution

An acidic phospholipase A(2) (PLA(2)) isolated from Bothrops jararacussu snake venom was crystallized with two inhibitors: alpha-tocopherol (vitamin E) and p-bromophenacyl bromide (BPB). The crystals diffracted at 1.45- and 1.85-Angstrom resolution, respectively, for the complexes with alpha-tocopherol and p-bromophenacyl bromide. The crystals are not isomorphous with those of the native protein, suggesting the inhibitors binding was successful and changes in the quaternary structure may have occurred. (C) 2004 Elsevier B.V. All rights reserved.