Structural and Functional Studies of a Bothropic Myotoxin Complexed to Rosmarinic Acid: New Insights into Lys49-PLA(2) Inhibition

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

Structural and Interaction Studies of Bacterial Polysaccharides by NMR Spectroscopy

An introduction to bacterial polysaccharides and the methods for structural determination are described in the first two parts of the thesis. In a structural elucidation of bacterial polysaccharides NMR experiments are important as is component analysis. A short description of immunochemical methods such as enzyme immunoassays is included. Two NMR techniques used for interaction studies, trNOE and STD NMR, are also discussed. The third part of the thesis discusses and summarizes the results from the included papers. The structures of the exopolysaccharides produced by two lactic acid bacteria are determined by one- and two dimensional NMR experiments. One is a heteropolysaccharide produced by Streptococcus thermophilus and the other a homopolysaccharide produced by Propionibacterium freudenreichii. The structure of an acidic polysaccharide from a marine bacterium with two serine residues in the repeating unit is also investigated. The structural and immunological relationship between two O-antigenic polysaccharides from Escherichia coli strain 180/C3 and O5 is discussed and investigated. Finally, interaction studies of an octasaccharide derived from the Salmonella enteritidis O-antigen and a bacteriophage are described which were performed with NMR experiments.

Structure to function studies in UDP-glucose dehydrogenases and nitroreductases

The thesis is divided into two parts corresponding to structural studies on two different proteins. The first part concerns the study of two UDP-glucose dehydrogenases (UGDs) from Sphingomonas elodea ATCC 31461 and Burkholderia cepacia IST 408, both involved in exopolysaccharide production. Their relevance arises because some of these bacterial exopolysaccharides are valuable as established biotechnological products, the former case, whilst others are highly problematic, when used by pathogens in biofilm formation over biological surfaces, as the latter case, namely in the human lungs. The goal of these studies is to increase our knowledge regarding UGDs structural properties, which can potentiate either the design of activity enhancers to respond to the increased demand of useful biofilms, or the design of inhibitors of biofilm production, in order to fight invading pathogens present in several infections. The thesis reports the production and crystallisation of both proteins, the determination of initial phases by single-wavelength anomalous dispersion (SAD) in S. elodea crystals using a seleno-methionine isoform, and phasing of B. cepacia crystals by molecular replacement (MR) using the S. elodea model, as well as the refinement, structural analysis and comparison between the several UGDs structures available during this work.(...)

Glutamate receptors as seen by light: spectroscopic studies of structure-function relationships

Ionotropic glutamate receptors are major excitatory receptors in the central nervous system and also have a far reaching influence in other areas of the body. Their modular nature has allowed for the isolation of the ligand-binding domain and for subsequent structural studies using a variety of spectroscopic techniques. This review will discuss the role of specific ligand:protein interactions in mediating activation in the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid subtype of glutamate receptors as established by various spectroscopic investigations of the GluR2 and GluR4 subunits of this receptor. Specifically, this review will provide an introduction to the insight gained from X-ray crystallography and nuclear magnetic resonance investigations and then go on to focus on studies utilizing vibrational spectroscopy and fluorescence resonance energy transfer to study the behavior of the isolated ligand-binding domain in solution and discuss the importance of specific ligand:protein interactions in the mechanism of receptor activation.

The structural effects of divalent cations and insulin on phospholipid model membranes /

It is well accepted that structural studies with model membranes are of considerable value in understanding the structure of biological membranes. Many studies with models of pure phospholipids have been done; but the effects of divalent cations and protein on these models would make these studies more applicable to intact membrane. The present study, performed with above view, is a structural analysis of divalent io~cardio1ipin complexes using the technique of x-ray diffraction. Cardiolipin, precipitated from dilute solution by divalent ionscalcium, magnesium and barium, contains little water and the structure formed is similar to the structure of pure cardiolipin with low water content. The calcium-cardiolipin complex forms a pure hexagonal type II phase that exists from 40 to 400 C. The molar ratio of calcium and cardiolipin in the complex is 1 : 1. Cardiolipin, precipitated with magnesium and barium forms two co-existing phases, lamellar and hexagonal, the relative quantity of the two phases being dependent on temperature. The hexagonal phase type II consisting of water filled channels formed by adding calcium to cardiolipin may have a remarkable permeability property in intact membrane. Pure cardiolipin and insulin at pH 3.0 and 4.0 precipitate but form no organised structure. Lecithin/cardiolipin and insulin precipitated at pH 3.0 give a pure lamellar phase. As the lecithin/cardiolipin molar ratio changes from 93/7 to SO/50, (a) the repeat distance of the lamellar changes from 72.8 X to 68.2 A; (b) the amount of protein bound increases in such a way that cardiolipin/insulin molar ratio in the complex reaches a maximum constant value at lecithin/cardiolipin molar ratio 70/30. A structural model based on these data shows that the molecular arrangement of lipid and protein is a lipid bilayer coated with protein molecules. The lipid-protein interaction is chiefly electrostatic and little, if any, hydrophobic bonding occurs in this particular system. So, the proposed model is essentially the same as Davson-Daniellifs model of biological membrane.

Studies on the insecticide - nematicide-oxamyl and its quantitative determination by gas chromatographic method

Ox amyl , an insecticide/nematicide with the chemical name; methyl ~'. ~·-dimethyl-~-(methylcarbamoyl)oxy-l-thiooxamimidate, and its major degradation compound; oxime or oximino compound, methyl ~',~'-dimethyl-~-hydroxy-l-thiooxamimidate were studied in this work. NMR and mass spectrometry were utilized in the structural studies. An attempt was made to explain the fragmentation patterns of some major peaks in the mass spectra of oxamyl and oxime. A new gas chromatographic method for the detection and determination of submicrogram levels of intact oxamyl using a electron-capture detector was developed. The principle of this method is to produce a derivative which is highly sensitive to an electron-capture detector. The derivative described is dinitrophenyl methylamine( DNPMA ) • Experimental conditions such as pH , reaction temperature , reaction time, the amount of reagent ( Dinitrofluaro benzene) etc. were thoroughly investigated and optimized. This method was successfully applied to the determination of oxamyl residues in tobacco leaves and soil. Throughout this J9D:oject , thin layer chromatography was also used in the separation:and clean up of oxamyl and oxime samples.

Structural, topographical and magnetic evolution of RF-sputtered Fe-Ni alloy based thin films with thermal annealing

Metglas 2826 MB having a nominal composition of Fe40Ni38Mo4B18 is an excellent soft magnetic material and finds application in sensors and memory heads. However, the thin-film forms of Fe40Ni38Mo4B18 are seldom studied, although they are important in micro-electro-mechanical systems/nano-electromechanical systems devices. The stoichiometry of the film plays a vital role in determining the structural and magnetic properties of Fe40Ni38Mo4B18 thin films: retaining the composition in thin films is a challenge. Thin films of 52 nm thickness were fabricated by RF sputtering technique on silicon substrate from a target of nominal composition of Fe40Ni38Mo4B18. The films were annealed at temperatures of 400 °C and 600 °C. The micro-structural studies of films using glancing x-ray diffractometer (GXRD) and transmission electron microscope (TEM) revealed that pristine films are crystalline with (FeNiMo)23B6 phase. Atomic force microscope (AFM) images were subjected to power spectral density analysis to understand the probable surface evolution mechanism during sputtering and annealing. X-ray photoelectron spectroscopy (XPS) was employed to determine the film composition. The sluggish growth of crystallites with annealing is attributed to the presence of molybdenum in the thin film. The observed changes in magnetic properties were correlated with annealing induced structural, compositional and morphological changes

Ditopic hexaazamacrocycles containing pyridine: synthesis, protonation and complexation studies

Structural studies of metal complexes of five ditopic hexaazamacrocycles containing two pyridine rings ([n] py(2)N(4) n = 18, 20, 22, 24 and 26) have been carried out. The synthesis of macrocycles [22]- to [26]- py(2)N(4) are also reported. The protonation constants of the last three compounds and the stability constants of their complexes with Ni2+, Cu2+, Zn2+, and Pb2+ were determined at 25 degreesC in 0.10 mol dm(-3) KNO3 in aqueous solution. Our results with [22] py(2)N(4) show significant differences from those described previously, while [24] py(2)N(4) has not been studied before and [ 26] py2N4 is a new compound. Mononuclear and dinuclear complexes of the divalent metal ions studied with [ 22]- to [26]- py(2)N(4) were found in solution. The stability constants for the ML complexes of the three ligands follow the Irving - Williams order: NiL2+ < CuL2+ >> ZnL2+ > PbL2+, however for the dinuclear complexes the values for Pb2+ complexes are higher than the corresponding values for the Ni2+ and the Zn2+ complexes. The X-ray single crystal structures of the supramolecular aggregates [Cu-2([20] py(2)N(4))(H2O)(4)][Cu(H2O)(6)](SO4)(3) . 3H(2)O ( 1) and [Cu-2([20] py(2)N(4))(CH3CN)(4)][Ni([20] py(2)N(4))](2)(ClO4)(8) . H2O (2), which are composed of homodinuclear [Cu-2([20] py(2)N(4)])(H2O)(4)](4+) ( 1a) and [Cu-2([20] py(2)N(4)])(CH3CN))(4)](4+) (2a), and mononuclear species, [Cu(H2O)(6)](2+) (1b) and [Ni([20] py(2)N(4))](2+) ( 2b), respectively, assembled by an extensive network of hydrogen bonds, are also reported. In both homodinuclear complexes the copper centres are located at the end of the macrocycle and display distorted square pyramidal coordination environments with the basal plane defined by three consecutive nitrogen donors and one solvent molecule, water in 1a and acetonitrile in 2a. The macrocycle adopts a concertina-type conformation leading to the formation of macrocyclic cavities with the two copper centres separated by intramolecular distances of 5.526(1) and 5.508(7) Angstrom in 1a and 2a, respectively. The mononuclear complex [Ni([20] py(2)N(4)])](2+) displays a distorted octahedral co-ordination environment with the macrocycle wrapping the metal centre in a helical shape. EPR spectroscopy of the copper complexes indicated the presence of mono- and dinuclear species.

New dioxadiaza- and trioxadiaza-macrocycles containing one dibenzofuran unit with two amino pendant arms: synthesis, protonation and complexation studies

New dioxadiaza- and trioxadiaza-macrocycles containing one rigid dibenzofuran unit (DBF) and N-(2-aminoethyl) pendant arms were synthesized, N,N'-bis(2-aminoethyl)-[17]( DBF) N2O2 (L-1) and N,N'-bis(2-aminoethyl)-[22](DBF)N2O3 (L-2), respectively. The binding properties of both macrocycles to metal ions and structural studies of their metal complexes were carried out. The protonation constants of both compounds and the stability constants of their complexes with Co2+, Ni2+, Cu2+, Zn2+, Cd2+, and Pb2+ were determined at 298.2 K, in aqueous solutions, and at ionic strength 0.10 mol dm(-3) in KNO3. Mononuclear complexes with both ligands were formed, and dinuclear complexes were only found for L-2. The thermodynamic binding affinities of the metal complexes of L-2 are lower than those of L-1 as expected, but the Pb2+ complexes of both macrocycles exhibit close stability constant values. On the other hand, the binding affinities of Cd2+ and Pb2+ for L-1 are very high, when compared to those of Co2+, Ni2+ and Zn2+. These interesting properties were explained by the presence of the rigid DBF moiety in the backbone of the macrocycle and to the special match between the macrocyclic cavity size and the studied larger metal ions. To elucidate the adopted structures of complexes in solution, the nickel(II) and copper( II) complexes with both ligands were further studied by UV-vis-MR spectroscopy in DMSO-H2O 1 : 1 (v/v) solution. The copper(II) complexes were also studied by EPR spectroscopy in the same mixture of solvents. The crystal structure of the copper complex of L-1 was also determined. The copper(II) displays an octahedral geometry, the four nitrogen atoms forming the equatorial plane and two oxygen atoms, one from the DBF unit and the other one from the ether oxygen, in axial positions. One of the ether oxygens of the macrocycle is out of the coordination sphere. Our results led us to suggest that this geometry is also adopted by the Co2+ to Zn2+ complexes, and only the larger Cd2+ and Pb2+ manage to form complexes with the involvement of all the oxygen atoms of the macrocyclic backbone.

Structural basis of nuclear import of flap endonuclease 1 (FEN1)

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

Structural Basis of Importin-alpha-Mediated Nuclear Transport for Ku70 and Ku80

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

Biochemical and structural investigations of Bothropstoxin-II, a myotoxic Asp49 phospholipase A(2) from Bothrops jararacussu venom

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

Production, purification, crystallization and preliminary X-ray diffraction studies of the nucleoside diphosphate kinase b from Leishmania major

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

Self-assembly of cyclometallated Pd(II) compounds directed by C-H center dot center dot center dot pi interactions: A structural evidence for metalloaromaticity in a cyclopalladated ring

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Structural analysis of Canavalia maritima and Canavalia gladiata lectins complexed with different dimannosides: New insights into the understanding of the structure-biological activity relationship in legume lectins

Plant lectins, especially those purified from species of the Legummosae family, represent the best studied group of carbohydrate-binding proteins. The legume lectins from Diocleinae subtribe are highly similar proteins that present significant differences in the potency/ efficacy of their biological activities. The structural studies of the interactions between lectins and sugars may clarify the origin of the distinct biological activities observed in this high similar class of proteins. In this way, this work presents a crystallographic study of the ConM and CGL (agglutinins from Canavalia maritima and Canavalia gladiata, respectively) in the following complexes: ConM/ CGL:Man(alpha 1-2)Man(alpha 1-0)Me, ConM/CGL:Man(alpha 1-O)Man(alpha 1-O)Me and ConM/CGL:Man(alpha 1-4)Man(alpha 1-O)Me, which crystallized in different conditions and space group from the native proteins.The structures were solved by molecular replacement, presenting satisfactory values for R-factor and R-factor. Comparisons between ConM, CGL and ConA (Canavalia ensiformis lectin) binding mode with the dimannosides in subject, presented different interactions patterns, which may account for a structural explanation of the distincts biological properties observed in the lectins of Diocleinae subtribe. (C) 2007 Elsevier B.V. All rights reserved.