Decoralin, a novel linear cationic alpha-helical peptide from the venom of the solitary eumenine wasp Oreumenes decoratus

A novel peptide, decoralin, was isolated from the venom of the solitary eumenine wasp Oreumenes decoratus. its sequence, Ser-Leu-Leu-Ser-Leu-Ile-Arg-Lys-Leu-Ile-Thr, was determined by Edman degradation and corroborated by solid-phase synthesis. This sequence has the characteristic features of linear cationic a-helical peptides; rich in hydrophobic and basic amino acids with no disulfide bond, and accordingly, it can be predicted to adopt an amphipathic a-helix secondary structure. In fact, the CD spectra of decoralin in the presence of TFE or SDS showed a high a-helical conformation content. In a biological evaluation, decoralin exhibited a significant broad-spectrum antimicrobial activity, and moderate mast cell degranulation and leishmanicidal activities, but showed virtually no hemolytic activity. A synthetic analog with C-terminal amidation showed a much more potent activity in all the biological assays. (c) 2007 Elsevier B.V. All rights reserved.

Effects of cardiomyopathic mutations on the biochemical and biophysical properties of the human alpha-tropomyosin

Mutations in the protein alpha-tropomyosin (Tm) can cause a disease known as familial hypertrophic cardiomyopathy. In order to understand how such mutations lead to protein dysfunction, three point mutations were introduced into cDNA encoding the human skeletal tropomyosin, and the recombinant Tms were produced at high levels in the yeast Pichia pastoris. Two mutations (A63V and K70T) were located in the N-terminal region of Tm and one (E180G) was located close to the calcium-dependent troponin T binding domain. The functional and structural properties of the mutant Tms were compared to those of the wild type protein. None of the mutations altered the head-to-tail polymerization, although slightly higher actin binding was observed in the mutant Tm K70T, as demonstrated in a cosedimentation assay. The mutations also did not change the cooperativity of the thin filament activation by increasing the concentrations of Ca2+. However, in the absence of troponin, all mutant Tms were less effective than the wild type in regulating the actomyosin subfragment 1 Mg2+ ATPase activity. Circular dichroism spectroscopy revealed no differences in the secondary structure of the Tms. However, the thermally induced unfolding, as monitored by circular dichroism or differential scanning calorimetry, demonstrated that the mutants were less stable than the wild type. These results indicate that the main effect of the mutations is related to the overall stability of Tm as a whole, and that the mutations have only minor effects on the cooperative interactions among proteins that constitute the thin filament.

Water regulation of actinomycin-D binding to DNA: the interplay among drug affinity, DNA long-range conformation, and hydration

Actiaomycin-D (actD) binds to natural DNA at two different classes of binding sites, weak and strong. The affinity for these sites is highly dependent on DNA se(sequence and solution conditions, and the interaction appears to be purely entropic driven Although the entropic character of this reaction has been attributed to the release of water molecules upon drug to DNA complex formation, the mechanism by which hydration regulates actD binding and discrimination between different classes of binding sites on natural DNA is still unknown. In this work, we investigate the role of hydration on this reaction using the osmotic stress method. We skew that the decrease of solution water activity, due to the addition of sucrose, glycerol ethylene glycol, and betaine, favors drug binding to the strong binding sites on DNA by increasing both the apparent binding affinity Delta G, and the number of DNA base pairs apparently occupied by the bound drug n(bp/actD). These binding parameters vary linearly with the logarithm of the molar fraction of water in solution log(X-w), which indicates the contribution of water binding to the energetic of the reaction. It is demonstrated that the hydration change measured upon binding increases proportionally to the apparent size of the binding site n(bp/uctD). This indicates that n(bp/actD) measured from the Scatchard plod is a measure of the size of the DNA molecule changing conformation due to ligand binding. We also find that the contribution of DNA deformation, gauged by n(bp/act) to the total free energy of binding Delta G, is given by Delta G = Delta G(local) + n(bp/actD) x delta G(DNA), where Delta G(local), = -8020 +/- 51 cal/mol of actD bound and delta G(DNa) = -24.1 +/- 1.7cal/mol of base pair at 25 degrees C. We interpret Delta G(local), as the energetic contribution due to the direct interactions of actD with the actual tetranucleotide binding site, and it n(bp/actB) X delta G(DNA) as that due to change inconformation, induced by binding, of it n(bp/actD) DNA base pairs flanking the local site. This interpretation is supported by the agreement found between the value of delta G(DNA) and the torsional free energy change measured independently. We conclude suggesting an allosteric model for ligand binding to DNA, such that the increase in binding affinity is achieved by increasing the relaxation of the unfavorable free energy of binding storage at the local site through a larger number of DNA base pairs. The new aspect on this model is that the size of the complex is not fixed but determined by solutions conditions, such as water activity, which modulate the energetic barrier to change helix conformation. These results may suggest that long-range allosteric transitions of duplex DNA are involved in the inhibition of RNA synthesis by actD, and more generally, in the regulation of transcription. (C) 2000 John Wiley & Sons, Inc.

Structural characteristics of silica sonogels prepared with additions of isopropyl alcohol

Wet silica gels with similar to 1.4 x 10(-3) mol SiO2/cm(3) and similar to 92 vol% liquid phase were obtained from sonohydrolysis of tetraethoxysilane (TEOS) with different additions of isopropyl alcohol ( IPA). The IPA/TEOS molar ratio R was changed from 0 to 4. Aerogels were obtained by supercritical CO2 extraction. The samples were analyzed by small-angle X-ray scattering (SAXS) and nitrogen adsorption. The wet gels exhibit mass fractal structure with fractal dimension increasing from D similar to 2.10 to D similar to 2.22, characteristic length xi decreasing from similar to 9.5 to similar to 6.9 nm, as R increases from 0 to 4, and an estimated characteristic length for the primary silica particles lower than similar to 0.3 nm. The supercritical process apparently eliminates a fraction of the porosity, increasing the mass fractal dimension and shortening the fractality domain in the mesopore region. The fundamental role of isopropyl alcohol on the structure of the resulting aerogels is to decrease the porosity and the pore mean size as R changes from pure TEOS to R = 4. A secondary structure appearing in the micropore region of the aerogels can be described as a mass/surface fractal structure, with correlated mass fractal dimension D-m similar to 2.7 and surface fractal dimension D-s similar to 2.3, as inferred from SAXS and nitrogen adsorption data.

Anoplin, a novel antimicrobial peptide from the venom of the solitary wasp Anoplius samariensis

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

Modulation of the Pharmacological Activities of Secretory Phospholipase A2 from Crotalus durissus cascavella Induced by Naringin

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

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.

Morfo-anatomia do eixo vegetativo aéreo de Achyrocline alata (Kunth) DC. (Asteraceae)

Achyrocline alata occurs in a dense and low vegetation in Mato Grosso do Sul State, Brazil and it has been studied under physical-chemical and yield aspects, being scarce anatomical data. The present work has as objective the study of the aerial vegetative axis. Stem is cylindrical and hairy, having five wings, which are arisen from their leaves. The blade shows the presence of uniseriate epidermis, which is covered by the cuticle of variable thickness; and non-glandular and glandular trichomes. The non-glandular trichomes are uniseriate and multicellular and have their apex cell in whip form, while the glandular trichomes are multicellular, uniseriate or biseriate. The mesophyll is dorsiventral with uni-stratified palisade parenchyma in most of the cases and lacunal parenchyma formed by three to four layers of irregular cells. Only one collateral vascular bundle occurs in the midvein. Stem in transversal section is covered by epidermis with trichomes similar to leaves; the cortex is constituted by a discontinuous area of angular collenchyma, which is followed by chlorophyll parenchyma. Vascular cylinder that is delimited by pericycle shows vascular bundles of collateral type. In the secondary structure, the periderm is originated from epidermal and subepidermal tissues. In vascular region, the fascicular cambium differs into secondary xylem and phloem, while interfascicular cambium produces sclerenchymatous tissue.

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.

Immunosensor based on immobilization of antigenic peptide NS5A-1 from HCV and silk fibroin in nanostructured films

The peptide NS5A-1 (PPLLESWKDPDYVPPWHG), derived from hepatitis C virus (HCV) NS5A protein, was immobilized into layer-by-layer (LbL) silk fibroin (SF) films. Deposition was monitored by UV-vis absorption measurements at each bilayer deposited. The interaction SF/peptide film induced secondary structure in NS5A-1 as indicated by fluorescence and circular dichroism (CD) measurements. Voltammetric sensor (SF/NS5A-1) properties were observed when the composite film was tested in the presence of anti-HCV. The peptide-silk fibroin interaction studied here showed new architectures for immunosensors based on antigenic peptides and SF as a suitable immobilization matrix. © 2013 American Chemical Society.

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.

Estudo por dinâmica molecular da estabilidade conformacional de dímeros do peptídeo Eumenine mastoparan-AF em água e mistura TFE-água

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

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)