Fibrinogen stability under surfactant interaction

Differential scanning calorimetry (DSC), circular dichroism (CD), difference spectroscopy (UV-vis), Raman spectroscopy, and small-angle X-ray scattering (SAXS) measurements have been performed in the present work to provide a quantitatively comprehensive physicochemical description of the complexation between bovine fibrinogen and the sodium perfluorooctanoate, sodium octanoate, and sodium dodecanoate in glycine buffer (pH 8.5). It has been found that sodium octanoate and dodecanoate act as fibrinogen destabilizer. Meanwhile, sodium perfluorooctanoate acts as a structure stabilizer at low molar concentration and as a destabilizer at high molar concentration. Fibrinogen`s secondary structure is affected by all three studied surfactants (decrease in alpha-helix and an increase in beta-sheet content) to a different extent. DSC and UV-vis revealed the existence of intermediate states in the thermal unfolding process of fibrinogen. In addition, SAXS data analysis showed that pure fibrinogen adopts a paired-dimer structure in solution. Such a structure is unaltered by sodium octanoate and perfluoroctanoate. However, interaction of sodium dodecanoate with the fibrinogen affects the protein conformation leading to a complex formation. Taken together, all results evidence that both surfactant hydrophobicity and tail length mediate the fibrinogen stability upon interaction. (C) 2011 Elsevier Inc. All rights reserved.

Cloning, expression, purification and characterization of recombinant glutathione-S-transferase from Xylella fastidiosa

Xylella fastidiosa is an important pathogen bacterium transmitted by xylem-feedings leafhoppers that colonizes the xylem of plants and causes diseases on several important crops including citrus variegated chlorosis (CVC) in orange and lime trees. Glutathione-S-transferases (GST) form a group of multifunctional isoenzymes that catalyzes both glutathione (GSH)-dependent conjugation and reduction reactions involved in the cellular detoxification of xenobiotic and endobiotic compounds. GSTs are the major detoxification enzymes found in the intracellular space and mainly in the cytosol from prokaryotes to mammals, and may be involved in the regulation of stress-activated signals by suppressing apoptosis signal-regulating kinase 1. In this study, we describe the cloning of the glutathione-S-transferase from X. fastidiosa into pET-28a(+) vector, its expression in Escherichia coli, purification and initial structural characterization. The purification of recombinant xfGST (rxfGST) to near homogeneity was achieved using affinity chromatography and size-exclusion chromatography (SEC). SEC demonstrated that rxfGST is a homodimer in solution. The secondary and tertiary structures of recombinant protein were analyzed by circular dichroism and fluorescence spectroscopy, respectively. The enzyme was assayed for activity and the results taken together indicated that rxfGST is a stable molecule, correctly folded, and highly active. Several members of the GST family have been extensively studied. However, xfGST is part of a less-studied subfamily which yet has not been structurally and biochemically characterized. In addition, these studies should provide a useful basis for future studies and biotechnological approaches of rxfGST. (C) 2008 Elsevier Inc. All rights reserved.

Purification, and Biochemical and Biophysical Characterization of Cellobiohydrolase I from Trichoderma harzianum IOC 3844

Because of its elevated cellulolytic activity, the filamentous fungus Trichoderma harzianum has a considerable potential in biomass hydrolysis applications. Trichoderma harzianum cellobiohydrolase I (ThCBHI), an exoglucanase, is an important enzyme in the process of cellulose degradation. Here, we report an easy single-step ion-exchange chromatographic method for purification of ThCBHI and its initial biophysical and biochemical characterization. The ThCBHI produced by induction with microcrystalline cellulose under submerged fermentation was purified on DEAE-Sephadex A-50 media and its identity was confirmed by mass spectrometry. The ThCBHI biochemical characterization showed that the protein has a molecular mass of 66 kDa and pi of 5.23. As confirmed by small-angle X-ray scattering (SAXS), both full-length ThCBHI and its catalytic core domain (CCD) obtained by digestion with papain are monomeric in solution. Secondary structure analysis of ThCBHI by circular dichroism revealed alpha-helices and beta-strands contents in the 28% and 38% range, respectively. The intrinsic fluorescence emission maximum of 337 nm was accounted for as different degrees of exposure of ThCBHI tryptophan residues to water. Moreover, ThCBHI displayed maximum activity at pH 5.0 and temperature of 50 degrees C with specific activities against Avicel and p-nitrophenyl-beta-D-cellobioside of 1.25 U/mg and 1.53 U/mg, respectively.

Cys mutants in functional regions of Sticholysin I clarify the participation of these residues in pore formation

Experimental evidence shows that the mechanism of pore formation by actinoporins is a multistep process, involving binding of the water-soluble monomer to the membrane and subsequent oligomerization on the membrane surface, leading to the formation of a functional pore. However, as for other eukaryotic pore-forming toxins, the molecular details of the mechanism of membrane insertion and oligomerization are not clear. In order to obtain further insight with regard to the structure-function relationship in sticholysins, we designed and produced three cysteine mutants of recombinant sticholysin I (rStI) in relevant functional regions for membrane interaction: StI E2C and StI F15C (in the N-terminal region) and StI R52C (in the membrane binding site). The conformational characterization derived from fluorescence and CD spectroscopic studies of StI E2C, StI F15C and StI R52C suggests that replacement of these residues by Cys in rStI did not noticeably change the conformation of the protein. The substitution by Cys of Arg(52) in the phosphocholine-binding site, provoked noticeable changes in rStI permeabilizing activity; however, the substitutions in the N-terminal region (Glu(2), Phe(15)) did not modify the toxin`s permeabilizing ability. The presence of a dimerized population stabilized by a disulfide bond in the StI E2C mutant showed higher pore-forming activity than when the protein is in the monomeric state, suggesting that sticholysins pre-ensembled at the N-terminal region could facilitate pore formation. (C) 2011 Elsevier Ltd. All rights reserved.

Structural classification of proteins through amino acid sequence using interval type-2 fuzzy logic system

This paper introduces a new multi-output interval type-2 fuzzy logic system (MOIT2FLS) that is automatically constructed from unsupervised data clustering method and trained using heuristic genetic algorithm for a protein secondary structure classification. Three structure classes are distinguished including helix, strand (sheet) and coil which correspond to three outputs of the MOIT2FLS. Quantitative properties of amino acids are used to characterize the twenty amino acids rather than the widely used computationally expensive binary encoding scheme. Amino acid sequences are parsed into learnable patterns using a local moving window strategy. Three clustering tasks are performed using the adaptive vector quantization method to derive an equal number of initial rules for each type of secondary structure. Genetic algorithm is applied to optimally adjust parameters of the MOIT2FLS with the purpose of maximizing the Q3 measure. Comprehensive experimental results demonstrate the strong superiority of the proposed approach over the traditional methods including Chou-Fasman method, Garnier-Osguthorpe-Robson method, and artificial neural network models.

Tuning the mechanical and morphological properties of self-assembled peptide hydrogels via control over the gelation mechanism through regulation of ionic strength and the rate of pH change

Hydrogels formed by the self-assembly of peptides are promising biomaterials. The bioactive and biocompatible molecule Fmoc-FRGDF has been shown to be an efficient hydrogelator via a π-β self-assembly mechanism. Herein, we show that the mechanical properties and morphology of Fmoc-FRGDF hydrogels can be effectively and easily manipulated by tuning both the final ionic strength and the rate of pH change. The increase of ionic strength, and consequent increase in rate of gelation and stiffness, does not interfere with the underlying π-β assembly of this Fmoc-protected peptide. However, by tuning the changing rate of the system's pH through the use of glucono-δ-lactone to form a hydrogel, as opposed to the previously reported HCl methodology, the morphology (nano- and microscale) of the scaffold can be manipulated.

Chorismate synthase: An attractive target for drug development against orphan diseases

The increase in incidence of infectious diseases worldwide, particularly in developing countries, is worrying. Each year, 14 million people are killed by infectious diseases, mainly HIV/AIDS, respiratory infections, malaria and tuberculosis. Despite the great burden in the poor countries, drug discovery to treat tropical diseases has come to a standstill. There is no interest by the pharmaceutical industry in drug development against the major diseases of the poor countries, since the financial return cannot be guaranteed. This has created an urgent need for new therapeutics to neglected diseases. A possible approach has been the exploitation of the inhibition of unique targets, vital to the pathogen such as the shikimate pathway enzymes, which are present in bacteria, fungi and apicomplexan parasites but are absent in mammals. The chorismate synthase (CS) catalyses the seventh step in this pathway, the conversion of 5-enolpyruvylshikimate-3-phosphate to chorismate. The strict requirement for a reduced flavin mononucleotide and the anti 1,4 elimination are both unusual aspects which make CS reaction unique among flavin-dependent enzymes, representing an important target for the chemotherapeutic agents development. In this review we present the main biochemical features of CS from bacterial and fungal sources and their difference from the apicomplexan CS. The CS mechanisms proposed are discussed and compared with structural data. The CS structures of some organisms are compared and their distinct features analyzed. Some known CS inhibitors are presented and the main characteristics are discussed. The structural and kinetics data reviewed here can be useful for the design of inhibitors. © 2007 Bentham Science Publishers Ltd.

Orphan nuclear receptor NGFI-B forms dimers with nonclassical interface

The orphan receptor nerve growth factor-induced B (NGFI-B) is a member of the nuclear receptor's subfamily 4A (Nr4a). NGFI-B was shown to be capable of binding both as a monomer to an extended half-site containing a single AAAGGTCA motif and also as a homodimer to a widely separated everted repeat, as opposed to a large number of nuclear receptors that recognize and bind specific DNA sequences predominantly as homo- and/or heterodimers. To unveil the structural organization of NGFI-B in solution, we determined the quaternary structure of the NGFI-B LBD by a combination of ab initio procedures from small-angle X-ray scattering (SAXS) data and hydrogen-deuterium exchange followed by mass spectrometry. Here we report that the protein forms dimers in solution with a radius of gyration of 2.9 nm and maximum dimension of 9.0 nm. We also show that the NGFI-B LBD dimer is V-shaped, with the opening angle significantly larger than that of classical dimer's exemplified by estrogen receptor (ER) or retinoid X receptor (RXR). Surprisingly, NGFI-B dimers formation does not occur via the classical nuclear receptor dimerization interface exemplified by ER and RXR, but instead, involves an extended surface area composed of the loop between helices 3 and 4 and C-terminal fraction of the helix 3. Remarkably, the NGFI-B dimer interface is similar to the dimerization interface earlier revealed for glucocorticoid nuclear receptor (GR), which might be relevant to the recognition of cognate DNA response elements by NGFI-B and to antagonism of NGFI-B-dependent transcription exercised by GR in cells. Published by Cold Spring Harbor Laboratory Press. Copyright © 2007 The Protein Society.

Chemical and biological characterization of four new linear cationic α-helical peptides from the venoms of two solitary eumenine wasps

Four novel peptides were isolated from the venoms of the solitary eumenine wasps Eumenes rubrofemoratus and Eumenes fraterculus. Their sequences were determined by MALDI-TOF/TOF (matrix assisted laser desorption/ionization time-of-flight mass spectrometry) analysis, Edman degradation and solid-phase synthesis. Two of them, eumenitin-R (LNLKGLIKKVASLLN) and eumenitin-F (LNLKGLFKKVASLLT), are highly homologous to eumenitin, an antimicrobial peptide from a solitary eumenine wasp, whereas the other two, EMP-ER (FDIMGLIKKVAGAL-NH 2) and EMP-EF (FDVMGIIKKIAGAL-NH 2), are similar to eumenine mastoparan-AF (EMP-AF), a mast cell degranulating peptide from a solitary eumenine wasp. These sequences have the characteristic features of linear cationic cytolytic peptides; rich in hydrophobic and basic amino acids with no disulfide bond, and accordingly, they can be predicted to adopt an amphipathic α-helix secondary structure. In fact, the CD (circular dichroism) spectra of these peptides showed significant α-helical conformation content in the presence of TFE (trifluoroethanol), SDS (sodium dodecylsulfate) and asolectin vesicles. In the biological evaluation, all the peptides exhibited a significant broad-spectrum antimicrobial activity, and moderate mast cell degranulation and leishmanicidal activities, but showed virtually no hemolytic activity. © 2011 Elsevier Ltd.

Dynamics and Conformational Studies of TOAC Spin Labeled Analogues of Ctx(Ile21)-Ha Peptide from Hypsiboas albopunctatus

Antimicrobial peptides (AMPs) isolated from several organisms have been receiving much attention due to some specific features that allow them to interact with, bind to, and disrupt cell membranes. The aim of this paper was to study the interactions between a membrane mimetic and the cationic AMP Ctx(Ile21)-Ha as well as analogues containing the paramagnetic amino acid 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) incorporated at residue positions n = 0, 2, and 13. Circular dichroism studies showed that the peptides, except for [TOAC13]Ctx(Ile21)-Ha, are unstructured in aqueous solution but acquire different amounts of α-helical secondary structure in the presence of trifluorethanol and lysophosphocholine micelles. Fluorescence experiments indicated that all peptides were able to interact with LPC micelles. In addition, Ctx(Ile21)-Ha and [TOAC13]Ctx(Ile21)-Ha peptides presented similar water accessibility for the Trp residue located near the N-terminal sequence. Electron spin resonance experiments showed two spectral components for [TOAC0]Ctx(Ile21)-Ha, which are most likely due to two membrane-bound peptide conformations. In contrast, TOAC2 and TOAC13 derivatives presented a single spectral component corresponding to a strong immobilization of the probe. Thus, our findings allowed the description of the peptide topology in the membrane mimetic, where the N-terminal region is in dynamic equilibrium between an ordered, membrane-bound conformation and a disordered, mobile conformation; position 2 is most likely situated in the lipid polar head group region, and residue 13 is fully inserted into the hydrophobic core of the membrane. © 2013 Vicente et al.

The characterization of a thermostable and cambialistic superoxide dismutase from thermus filiformis

The superoxide dismutase (TfSOD) gene from the extremely thermophilic bacterium Thermus filiformis was cloned and expressed at high levels in mesophilic host. The purified enzyme displayed approximately 25 kDa band in the SDS-PAGE, which was further confirmed as TfSOD by mass spectrometry. The TfSOD was characterized as a cambialistic enzyme once it had enzymatic activity with either manganese or iron as cofactor. TfSOD showed thermostability at 65, 70 and 80°C. The amount of enzyme required to inhibit 50% of pyrogallol autoxidation was 0·41, 0·56 and 13·73 mg at 65, 70 and 80°C, respectively. According to the circular dichroism (CD) spectra data, the secondary structure was progressively lost after increasing the temperature above 70°C. The 3-dimensional model of TfSOD with the predicted cofactor binding corroborated with functional and CD analysis. © 2013 The Society for Applied Microbiology.

Modelagem molecular da interação entre a proteína de fusão do vírus sincicial respiratório humano e inibidores da ação viral. -

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Isolation and biochemical characterization of a gamma-type phospholipase A2 inhibitor from Crotalus durissus collilineatus snake serum

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Investigation of DMSO-Induced Conformational Transitions in Human Serum Albumin Using Two-Dimensional Raman Optical Activity Spectroscopy

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