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.

Mg(2+) Ions Bind at the C-Terminal Region of Skeletal Muscle alpha-Tropomyosin

Tropomyosin (Tm) is a dimeric coiled-coil protein that polymerizes through head-to-tail interactions. These polymers bind along actin filaments and play an important role in the regulation of muscle contraction. Analysis of its primary structure shows that Tm is rich in acidic residues, which are clustered along the molecule and may from sites for divalent cation binding. In a previous study, we showed that the Mg(2+)-induced increase in stability of the C-terminal half of Tin is sensitive to imitations near the C-terminus. In the present report, we study the interaction between Mg(2+) and full-length Tin and smaller fragments corresponding to the last 65 and 26 Tin residues. Although the smaller Tin peptide (Tm(259-284(W269))) is flexible and to large extent unstructured, the larger Tm(220-284(W269)) fragments forms a coiled coil in solution whose stability increases significantly in the presence of Mg(2+). NMR analysis shows thin Mg(2+) induces chemical shift perturbations in both Tm(220-284(W269)) and Tm(259-284(W269)) in the vicinity of His276, in which are located several negatively charged residues. (C) 2009 Wiley Periodicals, Inc. Biopolymers 91: 583-590, 2009.

INSECT CHYMOTRYPSINS: CHLOROMETHYL KETONE INACTIVATION AND SUBSTRATE SPECIFICITY RELATIVE TO POSSIBLE COEVOLUTIONAL ADAPTATION OF INSECTS AND PLANTS

Insect digestive chymotrypsins are present in a large variety of insect orders but their substrate specificity still remains unclear. Ewer insect chymotrypsins from 3 different insect orders (Dictyoptera, Coleoptera and two Lepidoptera) were isolated using affinity chromatography. Enzymes presented molecular masses in the range of 20 to 31 kDa and pH optima in the range of 7.5 to 10.0. Kinetic characterization. using different, colorimetric and fluorescent substrates indicated that insect chymotrypsins differ from, bovine chymotrypsin in their primary specificity toward small substrates (like N-benzoyl-L-Tyr p-nitroanilide) rather than on their preference for large substrates (exemplified by Succynil-Ala-Ala-Pro-Phe P-nitroanilide). Chloromethyl ketones (TPCK, N-alpha-tosyl-L-Phe chloromethyl ketone and Z-GGF-CK, N-carbobenzoxy-Gly-Gly-phe-CK) inactivated all chymotrypsins legated. Inactivation rates follow apparent first-order kinetics with variable second order rates (TPCK, 42 to 130 M(-1)s(-1); Z-GGF-CK, 150 to 450 M(-1)s(-1) that may be remarkably low for S. frugiperda chymotrypsin (TPCK, 6 M(-1)s(-1); Z-GGF-CK, 6.1 M(-1) s(-1)). Homology modelling and sequence alignment showed that. in lepidopteran chymotrypsins, differences in the amino acid residues in the neighborhood of the catalytic His 57 may affect its pKa, value. This is Proposed as the cause of the decrease in His 57 reactivity toward chloromethyl ketones. Such amino acid replacement in the active site is proposed. to be an adaptation to the presence of dietary ketones. (C) 2009 Wiley Periodicals, Inc.

Deciphering the role of the electrostatic interactions in the alpha-tropomyosin head-to-tail complex

Skeletal alpha-tropomyosin (Tm) is a dimeric coiled-coil protein that forms linear assemblies under low ionic strength conditions in vitro through head-to-tail interactions. A previously published NMR structure of the Tin head-to-tail complex revealed that it is formed by the insertion of the N-terminal coiled-coil of one molecule into a cleft formed by the separation of the helices at the C-terminus of a second molecule. To evaluate the contribution of charged residues to complex stability, we employed single and double-mutant Tm fragments in which specific charged residues were changed to alanine in head-to-tail binding assays, and the effects of the mutations were analyzed by thermodynamic double-mutant cycles and protein-protein docking. The results show that residues K5, K7, and D280 are essential to the stability of the complex. Though D2, K6, D275, and H276 are exposed to the solvent and do not participate in intermolecular contacts in the NMR structure, they may contribute to head-to-tail complex stability by modulating the stability of the helices at the Tm termini.

Preferential location of lidocaine and etidocaine in lecithin bilayers as determined by EPR, fluorescence and H-2 NMR

We have examined the effect of the uncharged species of lidocaine (LDC) and etidocaine (EDC) on the acyl chain moiety of egg phosphatidylcholine liposomes. Changes in membrane organization caused by both anesthetics were detected through the use of EPR spin labels (5, 7 and 12 doxyl stearic acid methyl ester) or fluorescence probes (4, 6, 10, 16 pyrene-fatty acids). The disturbance caused by the LA was greater when the probes were inserted in more external positions of the acyl chain and decreased towards the hydrophobic core of the membrane. The results indicate a preferential insertion of LDC at the polar interface of the bilayer and in the first half of the acyl chain, for EDC. Additionally, 2 H NMR spectra of multilamellar liposomes composed by acyl chain-perdeutero DMPC and EPC (1:4 mol%) allowed the determination of the segmental order (S-mol) and dynamics (T-1) of the acyl chain region. In accordance to the fluorescence and EPR results, changes in molecular orientation and dynamics are more prominent if the LA preferential location is more superficial, as for LDC while EDC seems to organize the acyl chain region between carbons 2-8, which is indicative of its positioning. We propose that the preferential location of LDC and EDC inside the bilayers creates a ""transient site"", which is related to the anesthetic potency since it could modulate the access of these molecules to their binding site(s) in the voltage-gated sodium channel. (C) 2007 Elsevier B.V. All rights reserved.

The catalytic and other residues essential for the activity of the midgut trehalase from Spodoptera frugiperda

Trehalase (EC 3.2.1.28) hydrolyzes only alpha, alpha`- trehalose and is present in a variety of organisms, but is most important in insects and fungi. Crystallographic data showed that bacterial trehalase has 0312 and E496 as the catalytical residues and three Arg residues in the active site. Those residues have homologous in all family 37 trehalases including Spodoptera frugiperda trehalase (0322, E520, R169, R227, R287). To test the role of these residues, mutants of trehalase were produced. All mutants were at least four orders of magnitude less active than wild type trehalase and no structural difference between these mutants and wild type enzyme were discernible by circular dichroism. D322A and E520 pH-activity profile lacked the alkaline arm and the acid arm, respectively, suggesting that D322 is the acid and E520 the basic catalyst. Azide increases E520A activity three times, confirming its action as the basic catalyst. Taking into account the decrease in activity after substitution for alanine residue, the three arginine residues are as important as the catalytical ones to trehalase activity. This clarifies the previous misidentification of an Arg residue as the acid catalyst. As far as we know, this is the first report on the functional identification residues important for trehalase activity. (C) 2010 Elsevier Ltd. All rights reserved.

Structure, Dynamics, and RNA Interaction Analysis of the Human SBDS Protein

Shwachman-Bodian-Diamond syndrome is an autosomal recessive genetic syndrome with pleiotropic phenotypes, including pancreatic deficiencies, bone marrow dysfunctions with increased risk of myelodysplasia or leukemia, and skeletal abnormalities. This syndrome has been associated with mutations in the SBDS gene, which encodes a conserved protein showing orthologs in Archaea and eukaryotes. The Shwachman-Bodian-Diamond syndrome pleiotropic phenotypes may be an indication of different cell type requirements for a fully functional SBDS protein. RNA-binding activity has been predicted for archaeal and yeast SBDS orthologs, with the latter also being implicated in ribosome biogenesis. However, full-length SBDS orthologs function in a species-specific manner, indicating that the knowledge obtained from model systems may be of limited use in understanding major unresolved issues regarding SBDS function, namely, the effect of mutations in human SBDS on its biochemical function and the specificity of RNA interaction. We determined the solution structure and backbone dynamics of the human SBDS protein and describe its RNA binding site using NMR spectroscopy. Similarly to the crystal structures of Archaea, the overall structure of human SBDS comprises three well-folded domains. However, significant conformational exchange was observed in NMR dynamics experiments for the flexible linker between the N-terminal domain and the central domain, and these experiments also reflect the relative motions of the domains. RNA titrations monitored by heteronuclear correlation experiments and chemical shift mapping analysis identified a classic RNA binding site at the N-terminal FYSH (fungal, Yhr087wp, Shwachman) domain that concentrates most of the mutations described for the human SBDS. (C) 2010 Elsevier Ltd. All rights reserved.

Global Gene Expression Analysis during Sporulation of the Aquatic Fungus Blastocladiella emersonii

The Blastocladiella emersonii life cycle presents a number of drastic biochemical and morphological changes, mainly during two cell differentiation stages: germination and sporulation. To investigate the transcriptional changes taking place during the sporulation phase, which culminates with the production of the zoospores, motile cells responsible for the dispersal of the fungus, microarray experiments were performed. Among the 3,773 distinct genes investigated, a total of 1,207 were classified as differentially expressed, relative to time zero of sporulation, at at least one of the time points analyzed. These results indicate that accurate transcriptional control takes place during sporulation, as well as indicating the necessity for distinct molecular functions throughout this differentiation process. The main functional categories overrepresented among upregulated genes were those involving the microtubule, the cytoskeleton, signal transduction involving Ca(2+), and chromosome organization. On the other hand, protein biosynthesis, central carbon metabolism, and protein degradation were the most represented functional categories among downregulated genes. Gene expression changes were also analyzed in cells sporulating in the presence of subinhibitory concentrations of glucose or tryptophan. Data obtained revealed overexpression of microtubule and cytoskeleton transcripts in the presence of glucose, probably causing the shape and motility problems observed in the zoospores produced under this condition. In contrast, the presence of tryptophan during sporulation led to upregulation of genes involved in oxidative stress, proteolysis, and protein folding. These results indicate that distinct physiological pathways are involved in the inhibition of sporulation due to these two classes of nutrient sources.

Pseudomonas aeruginosa PA14 cupD transcription is activated by the RcsB response regulator, but repressed by its putative cognate sensor RcsC

The opportunistic pathogen Pseudomonas aeruginosa PA14 possesses four fimbrial cup clusters, which may confer the ability to adapt to different environments. cupD lies in the pathogenicity island PAPI-1 next to genes coding for a putative phosphorelay system composed of the hybrid histidine kinase RcsC and the response regulator RcsB. The main focus of this work was the regulation of cupD at the mRNA level. It was found that the HN-S-like protein MvaT does not exert a strong influence on cupD transcript levels, as it does for cupA. cupD transcription is higher in cultures grown at 28 degrees C, which agrees with a cupD mutant presenting attenuated virulence only in a plant model, but not in a mouse model of infection. Whereas an rcsC in-frame deletion mutant presented higher levels of cupD mRNA, rcsB deletion had the opposite effect. Accordingly, overexpression of RcsB increased the levels of cupD transcription, and promoted biofilm formation and the appearance of fimbriae. A single transcription start site was determined for cupD and transcription from this site was induced by RcsB. A motif similar to the enterobacterial RcsB/RcsA-binding site was detected adjacent to the -35 region, suggesting that this could be the RcsB-binding site. Comparison of P. aeruginosa and Escherichia coli Rcs may provide insights into how similar systems can be used by different bacteria to control gene expression and to adapt to various environmental conditions.

Overexpression of Nrp/b (nuclear restrict protein in brain) suppresses the malignant phenotype in the C6/ST1 glioma cell line

Upon searching for glucocorticoid-regulated cDNA sequences associated with the transformed to normal phenotypic reversion of C6/ST1 rat glioma cells, we identified Nrp/b (nuclear restrict protein in brain) as a novel rat gene. Here we report on the identification and functional characterization of the complete sequence encoding the rat NRP/B protein. The cloned cDNA presented a 1767 nucleotides open-reading frame encoding a 589 aminoacids residues sequence containing a BTB/POZ (broad complex Tramtrack bric-a-brac/Pox virus and zinc finger) domain in its N-terminal region and kelch motifs in its C-terminal region. Sequence analysis indicates that the rat Nrp/b displays a high level of identity with the equivalent gene orthologs from other organisms. Among rat tissues, Nrp/b expression is more pronounced in brain tissue. We show that overexpression of the Nrp/b cDNA in C6/ST1 cells suppresses anchorage independence in vitro and tumorigenicity in vivo, altering their malignant nature towards a more benign phenotype. Therefore, Nrp/b may be postulated as a novel tumor suppressorgene, with possible relevance for glioblastoma therapy. (C) 2009 Elsevier Ltd. All rights reserved.

Oxindole-Schiff base copper(II) complexes interactions with human serum albumin: Spectroscopic, oxidative damage, and computational studies

CD and EPR were used to characterize interactions of oxindole-Schiff base copper(II) complexes with human serum albumin (HSA). These imine ligands form very stable complexes with copper, and can efficiently compete for this metal ion towards the specific N-terminal binding site of the protein, consisting of the amino acid sequence Asp-Ala-His. Relative stability constants for the corresponding complexes were estimated from CD data, using the protein as competitive ligand, with values of log K(CuL) in the range 15.7-18.1, very close to that of [Cu(HSA)] itself, with log K(CuHSA) 16.2. Some of the complexes are also able to interfere in the a-helix structure of the protein, while others seem not to affect it. EPR spectra corroborate those results, indicating at least two different metal species in solution, depending on the imine ligand. Oxidative damage to the protein after incubation with these copper(II) complexes, particularly in the presence of hydrogen peroxide, was monitored by carbonyl groups formation, and was observed to be more severe when conformational features of the protein were modified. Complementary EPR spin-trapping data indicated significant formation of hydroxyl and carbon centered radicals, consistent with an oxidative mechanism. Theoretical calculations at density functional theory (DFT) level were employed to evaluate Cu(II)-L binding energies, L -> Cu(II) donation, and Cu(II) -> L back-donation, by considering the Schiff bases and the N-terminal site of HSA as ligands. These results complement previous studies on cytotoxicity, nuclease and pro-apoptotic properties of this kind of copper(II) complexes, providing additional information about their possibilities of transport and disposition in blood plasma. (C) 2009 Elsevier Inc. All rights reserved.

Stoichiometry and thermodynamics of the interaction between the C-terminus of human 90 kDa heat shock protein Hsp90 and the mitochondrial translocase of outer membrane Tom70

A large majority of the 1000-1500 proteins in the mitochondria are encoded by the nuclear genome, and therefore, they are translated in the cytosol in the form and contain signals to enable the import of proteins into the organelle. The TOM complex is the major translocase of the outer membrane responsible for preprotein translocation. It consists of a general import pore complex and two membrane import receptors, Tom20 and Tom70. Tom70 contains a characteristic TPR domain, which is a docking site for the Hsp70 and Hsp90 chaperones. These chaperones are involved in protecting cytosolic preproteins from aggregation and then in delivering them to the TOM complex. Although highly significant, many aspects of the interaction between Tom70 and Hsp90 are still uncertain. Thus, we used biophysical tools to study the interaction between the C-terminal domain of Hsp90 (C-Hsp90), which contains the EEVD motif that binds to TPR domains, and the cytosolic fragment of Tom70. The results indicate a stoichiometry of binding of one monomer of Tom70 per dimer of C-Hsp90 with a K(D) of 360 30 nM, and the stoichiometry and thermodynamic parameters obtained suggested that Tom70 presents a different mechanism of interaction with Hsp90 when compared with other TPR proteins investigated. (C) 2011 Elsevier Inc. All rights reserved.

Docking and molecular dynamics simulation of quinone compounds with trypanocidal activity

In this work, two different docking programs were used, AutoDock and FlexX, which use different types of scoring functions and searching methods. The docking poses of all quinone compounds studied stayed in the same region in the trypanothione reductase. This region is a hydrophobic pocket near to Phe396, Pro398 and Leu399 amino acid residues. The compounds studied displays a higher affinity in trypanothione reductase (TR) than glutathione reductase (GR), since only two out of 28 quinone compounds presented more favorable docking energy in the site of human enzyme. The interaction of quinone compounds with the TR enzyme is in agreement with other studies, which showed different binding sites from the ones formed by cysteines 52 and 58. To verify the results obtained by docking, we carried out a molecular dynamics simulation with the compounds that presented the highest and lowest docking energies. The results showed that the root mean square deviation (RMSD) between the initial and final pose were very small. In addition, the hydrogen bond pattern was conserved along the simulation. In the parasite enzyme, the amino acid residues Leu399, Met400 and Lys402 are replaced in the human enzyme by Met406, Tyr407 and Ala409, respectively. In view of the fact that Leu399 is an amino acid of the Z site, this difference could be explored to design selective inhibitors of TR.

Caracterização estrutural e avaliação das atividades farmacológicas da fucana B extraída da alga Dictyota menstrualis

Seaweeds are a major source of biologically active compounds . In the extracellular matrix of these organisms are sulfated polysaccharides that functions as structural components preventing it against dehydration. The fraction 0.9 (FucB) rich in sulfated fucans obtained from brown seaweed Dictyota menstrualis was chemical characterized and evaluated for pharmacological activity by testing anticoagulant activity, stimulatory action on the synthesis of an antithrombotic heparan sulfate, antioxidant activity and its effects in cell proliferation. The main components were FucB carbohydrates (49.80 ± 0.10 %) and sulfate (42.30 ± 0.015 %), with phenolic compounds ( 3.86 ± 0.016 %) and low protein contamination ( 0.58 ± 0.001 % ) . FucB showed polydisperse profile and analysis of signals in the infrared at 1262, 1074 and 930 cm -1 and 840 assigned to S = O bonds sulfate esters , CO bond presence of 3,6- anhydrogalactose , β -D- galactose non- sulfated sulfate and the axial position of fucose C4 , respectively. FucB exhibited moderate anticoagulant activity , the polysaccharides prolonged time (aPTT ) 200 ug ( > 90s ) partial thromboplastin FucB no effect on prothrombin time (PT), which corresponds to the extrinsic pathway of coagulation was observed. This stimulation promoted fraction of about 3.6 times the synthesis of heparan sulfate (HS) by endothelial cells of the rabbit aorta ( RAEC ) in culture compared with cells not treated with FucB . This has also been shown to compete for the binding site with heparin. The rich fraction sulfated fucans exhibited strong antioxidant activity assays on total antioxidant (109.7 and 89.5 % compared with BHT and ascorbic acid standards ) , reducing power ( 71 % compared to ascorbic acid ) and ferric chelation ( 71 , comparing with 5 % ascorbic acid). The fraction of algae showed cytostatic activity on the RAEC cells revealed that the increase of the synthesis of heparan sulfate is not related to proliferation. FucB showed antiproliferative action on cell lines modified as Hela and Hep G2 by MTT assay . These results suggest that FucB Dictyota menstrualis have anticoagulant , antithrombotic , antioxidant potential as well as a possible antitumor action, promoting the stimulation of the synthesis of antithrombotic HS by endothelial cells and is useful in the prevention of thrombosis, also due to its inhibitory action on species reactive oxygen ( ROS ) in some in vitro systems , being involved in promoting a hypercoagulable state

Modelagem molecular na caracterização eletrônica de oligopeptídeos e na descrição quântica da interação fármaco-receptor

In this dissertation, the theoretical principles governing the molecular modeling were applied for electronic characterization of oligopeptide α3 and its variants (5Q, 7Q)-α3, as well as in the quantum description of the interaction of the aminoglycoside hygromycin B and the 30S subunit of bacterial ribosome. In the first study, the linear and neutral dipeptides which make up the mentioned oligopeptides were modeled and then optimized for a structure of lower potential energy and appropriate dihedral angles. In this case, three subsequent geometric optimization processes, based on classical Newtonian theory, the semi-empirical and density functional theory (DFT), explore the energy landscape of each dipeptide during the search of ideal minimum energy structures. Finally, great conformers were described about its electrostatic potential, ionization energy (amino acids), and frontier molecular orbitals and hopping term. From the hopping terms described in this study, it was possible in subsequent studies to characterize the charge transport propertie of these peptides models. It envisioned a new biosensor technology capable of diagnosing amyloid diseases, related to an accumulation of misshapen proteins, based on the conductivity displayed by proteins of the patient. In a second step of this dissertation, a study carried out by quantum molecular modeling of the interaction energy of an antibiotic ribosomal aminoglicosídico on your receiver. It is known that the hygromycin B (hygB) is an aminoglycoside antibiotic that affects ribosomal translocation by direct interaction with the small subunit of the bacterial ribosome (30S), specifically with nucleotides in helix 44 of the 16S ribosomal RNA (16S rRNA). Due to strong electrostatic character of this connection, it was proposed an energetic investigation of the binding mechanism of this complex using different values of dielectric constants (ε = 0, 4, 10, 20 and 40), which have been widely used to study the electrostatic properties of biomolecules. For this, increasing radii centered on the hygB centroid were measured from the 30S-hygB crystal structure (1HNZ.pdb), and only the individual interaction energy of each enclosed nucleotide was determined for quantum calculations using molecular fractionation with conjugate caps (MFCC) strategy. It was noticed that the dielectric constants underestimated the energies of individual interactions, allowing the convergence state is achieved quickly. But only for ε = 40, the total binding energy of drug-receptor interaction is stabilized at r = 18A, which provided an appropriate binding pocket because it encompassed the main residues that interact more strongly with the hygB - C1403, C1404, G1405, A1493, G1494, U1495, U1498 and C1496. Thus, the dielectric constant ≈ 40 is ideal for the treatment of systems with many electrical charges. By comparing the individual binding energies of 16S rRNA nucleotides with the experimental tests that determine the minimum inhibitory concentration (MIC) of hygB, it is believed that those residues with high binding values generated bacterial resistance to the drug when mutated. With the same reasoning, since those with low interaction energy do not influence effectively the affinity of the hygB in its binding site, there is no loss of effectiveness if they were replaced.