Crystal structure of Staphylococcus aureus exfoliative toxin D-like protein: structural basis for the high specificity of exfoliative toxins

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

Flutter analysis including structural uncertainties

The common practice in industry is to perform flutter analyses considering the generalized stiffness and mass matrices obtained from finite element method (FEM) and aerodynamic generalized force matrices obtained from a panel method, as the doublet lattice method. These analyses are often reperformed if significant differences are found in structural frequencies and damping ratios determined from ground vibration tests compared to FEM. This unavoidable rework can result in a lengthy and costly process of analysis during the aircraft development. In this context, this paper presents an approach to perform flutter analysis including uncertainties in natural frequencies and damping ratios. The main goal is to assure the nominal system’s stability considering these modal parameters varying in a limited range. The aeroelastic system is written as an affine parameter model and the robust stability is verified solving a Lyapunov function through linear matrix inequalities and convex optimization

Structural synaptic elements are differentially regulated in superior temporal cortex of schizophrenia patients

Inaccurate wiring and synaptic pathology appear to be major hallmarks of schizophrenia. A variety of gene products involved in synaptic neurotransmission and receptor signaling are differentially expressed in brains of schizophrenia patients. However, synaptic pathology may also develop by improper expression of intra- and extra-cellular structural elements weakening synaptic stability. Therefore, we have investigated transcription of these elements in the left superior temporal gyrus of 10 schizophrenia patients and 10 healthy controls by genome-wide microarrays (Illumina). Fourteen up-regulated and 22 downregulated genes encoding structural elements were chosen from the lists of differentially regulated genes for further qRT-PCR analysis. Almost all genes confirmed by this method were downregulated. Their gene products belonged to vesicle-associated proteins, that is, synaptotagmin 6 and syntaxin 12, to cytoskeletal proteins, like myosin 6, pleckstrin, or to proteins of the extracellular matrix, such as collagens, or laminin C3. Our results underline the pivotal roles of structural genes that control formation and stabilization of pre- and post-synaptic elements or influence axon guidance in schizophrenia. The glial origin of collagen or laminin highlights the close interrelationship between neurons and glial cells in establishment and maintenance of synaptic strength and plasticity. It is hypothesized that abnormal expression of these and related genes has a major impact on the pathophysiology of schizophrenia.

Preparation, structural characterization and catalytic properties of Co/CeO2 catalysts for the steam reforming of ethanol and hydrogen production

In this paper, Co/CeO2 catalysts, with different cobalt contents were prepared by the polymeric precursor method and were evaluated for the steam reforming of ethanol. The catalysts were characterized by N-2 physisorption (BET method), X-ray diffraction (XRD), UV-visible diffuse reflectance, temperature programmed reduction analysis (TPR) and field emission scanning electron microscopy (FEG-SEM). It was observed that the catalytic behavior could be influenced by the experimental conditions and the nature of the catalyst employed. Physical-chemical characterizations revealed that the cobalt content of the catalyst influences the metal-support interaction which results in distinct catalyst performances. The catalyst with the highest cobalt content showed the best performance among the catalysts tested, exhibiting complete ethanol conversion, hydrogen selectivity close to 66% and good stability at a reaction temperature of 600 degrees C. (c) 2012 Elsevier B.V. All rights reserved.

Structural features of lignin obtained at different alkaline oxidation conditions from sugarcane bagasse

Lignin is a macromolecule frequently obtained as residue during technological processing of biomass. Modifications in chemical structure of lignin generate valuable products, some with particular and unique characteristics. One of the available methods for modification of industrial lignin is oxidation by hydrogen peroxide. In this work, we conducted systematic studies of the oxidation process that were carried out at various pHs and oxidizing agent concentrations. Biophysical, biochemical, structural properties of the oxidized lignin were analyzed by UV spectrophotometry, Fourier transform infrared spectroscopy, scanning electron microscopy and small angle X-ray scattering. Our results reveal that lignin oxidized with 9.1% H(2)O(2) (m/v) at pH 13.3 has the highest fragmentation, oxidation degree and stability. Although this processing condition might be considered quite severe, we have concluded that the stability of the obtained oxidized lignin was greatly increased. Therefore, the identified processing conditions of oxidation may be of practical interest for industrial applications. (C) 2011 Elsevier B.V. All rights reserved.

Septin C-Terminal Domain Interactions: Implications for Filament Stability and Assembly

Septins form a conserved family of filament forming GTP binding proteins found in a wide range of eukaryotic cells. They share a common structural architecture consisting of an N-terminal domain, a central GTP binding domain and a C-terminal domain, which is often predicted to adopt a coiled-coil conformation, at least in part. The crystal structure of the human SEPT2/SEPT6/SEPT7 heterocomplex has revealed the importance of the GTP binding domain in filament formation, but surprisingly no electron density was observed for the C-terminal domains and their function remains obscure. The dearth of structural information concerning the C-terminal region has motivated the present study in which the putative C-terminal domains of human SEPT2, SEPT6 and SEPT7 were expressed in E. coli and purified to homogeneity. The thermal stability and secondary structure content of the domains were studied by circular dichroism spectroscopy, and homo- and hetero-interactions were investigated by size exclusion chromatography, chemical cross-linking, analytical ultracentrifugation and surface plasmon resonance. Our results show that SEPT6-C and SEPT7-C are able to form both homo- and heterodimers with a high alpha-helical content in solution. The heterodimer is elongated and considerably more stable than the homodimers, with a K (D) of 15.8 nM. On the other hand, the homodimer SEPT2-C has a much lower affinity, with a K (D) of 4 mu M, and a moderate alpha-helical content. Our findings present the first direct experimental evidence toward better understanding the biophysical properties and coiled-coil pairings of such domains and their potential role in filament assembly and stability.

Low resolution structural characterization of the Hsp70-interacting protein - Hip - from Leishmania braziliensis emphasizes its high asymmetry

The Hsp70 is an essential molecular chaperone in protein metabolism since it acts as a pivot with other molecular chaperone families. Several co-chaperones act as regulators of the Hsp70 action cycle, as for instance Hip (Hsp70-interacting protein). Hip is a tetratricopeptide repeat protein (TPR) that interacts with the ATPase domain in the Hsp70-ADP state, stabilizing it and preventing substrate dissociation. Molecular chaperones from protozoans, which can cause some neglected diseases, are poorly studied in terms of structure and function. Here, we investigated the structural features of Hip from the protozoa Leishmania braziliensis (LbHip), one of the causative agents of the leishmaniasis disease. LbHip was heterologously expressed and purified in the folded state, as attested by circular dichroism and intrinsic fluorescence emission techniques. LbHip forms an elongated dimer, as observed by analytical gel filtration chromatography, analytical ultracentrifugation and small angle X-ray scattering (SAXS). With the SAXS data a low resolution model was reconstructed, which shed light on the structure of this protein, emphasizing its elongated shape and suggesting its domain organization. We also investigated the chemical-induced unfolding behavior of LbHip and two transitions were observed. The first transition was related to the unfolding of the TPR domain of each protomer and the second transition of the dimer dissociation. Altogether. LbHip presents a similar structure to mammalian Hip, despite their low level of conservation, suggesting that this class of eukaryotic protein may use a similar mechanism of action. (C) 2012 Elsevier Inc. All rights reserved.

Interaction between bradykinin potentiating nonapeptide (BPP9a) and beta-cyclodextrin: A structural and thermodynamic study

Herein, we demonstrate the physical and chemical characterizations of the supramolecular complex formed between beta-cyclodextrin (beta CD) and bradykinin potentiating nonapeptide (BPP9a), an endogenous toxin found in Bothrops jararaca. Circular dichroism results indicate a conformational change in the BPP9a secondary structure upon its complexation with beta CD. Nuclear magnetic resonance results, mainly from NOESY experiments, and theoretical calculations showed a favorable interaction between the tryptophan residue of BPP9a and the beta CD cavity. Thermodynamic inclusion parameters were investigated by isothermal titration calorimetry, demonstrating that beta CD/BPP9a complex formation is an exothermic process that results in a reduction in entropy. Additionally, in vitro degradation study of BPP9a against trypsin (37 degrees C, pH 7.2) showed higher stability of peptide in presence of beta CD. This beta CD/BPP9a complex, which presents new chemical properties arising from the peptide inclusion process, may be useful as an antihypertensive drug in oral pharmaceutical formulations. (C) 2011 Elsevier B.V. All rights reserved.

Structural effect of cationic amphiphiles in diacetylenic photopolymerizable membranes

Liposomes have been an excellent option as drug delivery systems, since they are able of incorporating lipophobic and/or lipophilic drugs, reduce drug side effects, increase drug targeting, and control delivery. Also, in the last years, their use reached the field of gene therapy, as non-viral vectors for DNA delivery. As a strategy to increase system stability, the use of polymerizable phospholipids has been proposed in liposomal formulations. In this work, through differential scanning calorimetry (DSC) and electron spin resonance (ESR) of spin labels incorporated into the bilayers, we structurally characterize liposomes formed by a mixture of the polymerizable lipid diacetylenic phosphatidylcholine 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC8,9PC) and the zwitterionic lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), in a 1:1 molar ratio. It is shown here that the polymerization efficiency of the mixture (c.a. 60%) is much higher than that of pure DC8,9PC bilayers (c.a. 20%). Cationic amphiphiles (CA) were added, in a final molar ratio of 1:1:0.2 (DC8,9PC:DMPC:CA), to make the liposomes possible carriers for genetic material, due to their electrostatic interaction with negatively charged DNA. Three amphiphiles were tested, 1,2-dioleoyl-3-trimetylammonium-propane (DOTAP), stearylamine (SA) and trimetyl (2-miristoyloxietyl) ammonium chloride (MCL), and the systems were studied before and after UV irradiation. Interestingly, the presence of the cationic amphiphiles increased liposomes polymerization. MCL displaying the strongest effect. Considering the different structural effects the three cationic amphiphiles cause in DC8,9PC bilayers, there seem to be a correlation between the degree of DC8,9PC polymerization and the packing of the membrane at the temperature it is irradiated (gel phase). Moreover, at higher temperatures, in the bilayer fluid phase, more polymerized membranes are significantly more rigid. Considering that the structure and stability of liposomes at different temperatures can be crucial for DNA binding and delivery, we expect the study presented here contributes to the production of new carrier systems with potential applications in gene therapy. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Dissecting structure-function-stability relationships of a thermostable GH5-CBM3 cellulase from Bacillus subtilis 168

Cellulases participate in a number of biological events, such as plant cell wall remodelling, nematode parasitism and microbial carbon uptake. Their ability to depolymerize crystalline cellulose is of great biotechnological interest for environmentally compatible production of fuels from lignocellulosic biomass. However, industrial use of cellulases is somewhat limited by both their low catalytic efficiency and stability. In the present study, we conducted a detailed functional and structural characterization of the thermostable BsCe15A (Bacillus subtilis cellulase 5A), which consists of a GH5 (glycoside hydrolase 5) catalytic domain fused to a CBM3 (family 3 carbohydrate-binding module). NMR structural analysis revealed that the Bacillus CBM3 represents a new subfamily, which lacks the classical calcium-binding motif, and variations in NMR frequencies in the presence of cellopentaose showed the importance of polar residues in the carbohydrate interaction. Together with the catalytic domain, the CBM3 forms a large planar surface for cellulose recognition, which conducts the substrate in a proper conformation to the active site and increases enzymatic efficiency. Notably, the manganese ion was demonstrated to have a hyper-stabilizing effect on BsCel5A, and by using deletion constructs and X-ray crystallography we determined that this effect maps to a negatively charged motif located at the opposite face of the catalytic site.

On the temperature stability of extracellular hemoglobin of Glossoscolex paulistus, at different oxidation states: SAXS and DLS studies

Glossoscolex paulistus hemoglobin (HbGp) was studied by dynamic light scattering (DLS) and small angle X-ray scattering (SAXS). DLS melting curves were measured for met-HbGp at different concentrations. SAXS temperature studies were performed for oxy-, cyanomet- and met-HbGp forms, at several pH values. At pH 5.0 and 6.0, the scattering curves are identical from 20 to 60 degrees C, and R-g is 108 angstrom, independent of the oxidation form. At pH 7.0, protein denaturation and aggregation occurs above 55 degrees C and 60 degrees C, for oxy and met-HbGp, respectively. Cyanomet-HbGp, at pH 7.0, is stable up to 60 degrees C. At alkaline pH (8.0-9.0) and higher temperature, an irreversible dissociation process is observed, with a decrease of R-g, D-max and I(0). Analysis by p(r), obtained from GNOM, and OLIGOMER, was used to fit the SAXS experimental scattering curves by a combination of theoretical curves obtained for HbLt fragments from the crystal structure. Our results show clearly the increasing contribution of smaller molecular weight fragments, as a function of increasing pH and temperature, as well as, the order of thermal stabilities: cyanomet-> oxy- > met-HbGp. (C) 2012 Elsevier B.V. All rights reserved.

Self-Assembly of Poly(4-vynil-N-alkylpyridinium) Bromide onto Silica: Effect of Side-Chain Length on Structural Aspects at a Molecular Level

Self-assembly of poly(4-vynil-N-alkyl)pyridinium bromide with alkyl side chains of 2, 5, 7, 10, or 16 carbons from ethanolic solutions onto flat silica surfaces was studied by means of ellipsometry, atomic force microscopy (AFM), contact angle measurements, and sum-frequency generation (SFG) vibrational spectroscopy in the CH3 and CH2 stretch region. Ab initio quantum-chemical calculations on the N-alkylpyridinium side-group with restricted Hartree-Fock (RHF) method and 6-311G (d,p) basis set were C one to estimate the charge distribution along the pyridinium ring and the alkyl side-chain. SFG results showed that longer side chains promote the disorientation of the alkyl groups at the surface, corroborating with the contact angle values. AFM images revealed film homogeneity, regardless the alkyl side group. However, after 24 h contact with water, ringlike structures appeared on the film surfaces, when the polycation alkyl side chain had 7 or less carbons, and as the alkyl chain increased to 10 or 16 carbons, the films dewetted because the hydrophobic interactions prevailed over the electrostatic interactions between the pyridinium charged groups and the negatively charged SiO2 surface. Under acid conditions (HCl 0.1 mol.L-1), the film mean thickness values decreased up to 50% of original values when the alkyl side chains were ethyl or pentyl groups due to ion-pair disruption, but for longer groups they remained unchanged. Quantum-chemical optimization and Mulliken electron population showed that (i) from C2 to C15 the positive charge at the headgroup (HG) decreased 0.025, while the charge at combined HG + alpha-CH2 increased 0.037; and (ii) for C6 or longer, the alkyl side group presents a tilt in the geometry, moving away from the plane. Such effects summed up over the whole polymer chain give support to suggest that when the side chains are longer than 7 carbons, the hydrophobic interaction decreases film stability and increases acid resistance.

Purification, characterization and structural determination of chitinases produced by Moniliophthora perniciosa

The enzyme chitinase from Moniliophthora perniciosa the causative agent of the witches' broom disease in Theobroma cacao, was partially purified with ammonium sulfate and filtration by Sephacryl S-200 using sodium phosphate as an extraction buffer. Response surface methodology (RSM) was used to determine the optimum pH and temperature conditions. Four different isoenzymes were obtained: ChitMp I, ChitMp II, ChitMp III and ChitMp IV. ChitMp I had an optimum temperature at 44-73ºC and an optimum pH at 7.0-8.4. ChitMp II had an optimum temperature at 45-73ºC and an optimum pH at 7.0-8.4. ChitMp III had an optimum temperature at 54-67ºC and an optimum pH at 7.3-8.8. ChitMp IV had an optimum temperature at 60ºC and an optimum pH at 7.0. For the computational biology, the primary sequence was determined in silico from the database of the Genome/Proteome Project of M. perniciosa, yielding a sequence with 564 bp and 188 amino acids that was used for the three-dimensional design in a comparative modeling methodology. The generated models were submitted to validation using Procheck 3.0 and ANOLEA. The model proposed for the chitinase was subjected to a dynamic analysis over a 1 ns interval, resulting in a model with 91.7% of the residues occupying favorable places on the Ramachandran plot and an RMS of 2.68.

The Effect of Preservative Interventions on the Chemical- Physical and Structural Characteristics of Panel Painting

This work studies the impact of two traditional Romanian treatments, Red Petroleum and Propolis, in terms of real efficiency and consequence on the wooden artifacts. The application of these solutions is still a widely adopted and popular technique in preservative conservation but the impact of these solutions is not well known. It is important to know the effect of treatments on chemical-physical and structural characteristics of the artifacts, not only for understanding the influence on present conditions but also for foreseeing the future behavior. These treatments with Romanian traditional products are compared with a commercial antifungal product, Biotin R, which is utilized as reference to control the effectiveness of Red Petroleum and Propolis. Red Petroleum and Propolis are not active against mould while Biotin R is very active. Mould attack is mostly concentrated in the painted layer, where the tempera, containing glue and egg, enhance nutrition availability for moulds. Biotin R, even if is not a real insecticide but a fungicide, was the most active product against insect attack of the three products, followed by Red Petroleum, Propolis and untreated reference. As for colour, it did not change so much after the application of Red Petroleum and Biotin R and the colour difference was almost not perceptible. On the contrary, Propolis affected the colour a lot. During the exposure at different RH, the colour changes significantly at 100% RH at equilibrium and this is mainly due to the mould attack. Red Petroleum penetrates deeply into wood, while Propolis does not penetrate and remains only on the surface. However, Red Petroleum does not interact chemically with wood substance and it is easy volatilized in oven-dry condition. On the contrary Propolis interacts chemically with wood substance and hardly volatilized, even in oven-dry condition and consequently Propolis remains where it penetrated, mostly on the surface. Treatment by immersion has impact on wood physical parameters while treatment by brushing does not have significant impact. Especially Red Petroleum has an apparent impact on moisture content (MC) due to the penetration of solution, while Propolis does not penetrate so much and remains only on surface therefore Propolis does not have so much impact as Red Petroleum. However, if the weight of the solution penetrated in wood is eliminated, there is not significant difference in MC between treated and untreated samples. Considering physical parameters, dimensional stability is an important parameter. The variation of wood moisture content causes shrinkages/swelling of the wood that polychrome layer can only partially follow. The dimension of wooden supports varied under different moisture conditioning; the painted layer cannot completely follow this deformation, and consequently a degradation and deterioration caused by detachment, occurs. That detachment affects the polychrome stratification of the panel painting and eventually the connections between the different layer compositions of the panel painting.

Structural and functional analysis of centromeric chromatin

Animal neocentromeres are defined as ectopic centromeres that have formed in non-centromeric locations and avoid some of the features, like the DNA satellite sequence, that normally characterize canonical centromeres. Despite this, they are stable functional centromeres inherited through generations. The only existence of neocentromeres provide convincing evidence that centromere specification is determined by epigenetic rather than sequence-specific mechanisms. For all this reasons, we used them as simplified models to investigate the molecular mechanisms that underlay the formation and the maintenance of functional centromeres. We collected human cell lines carrying neocentromeres in different positions. To investigate the region involved in the process at the DNA sequence level we applied a recent technology that integrates Chromatin Immuno-Precipitation and DNA microarrays (ChIP-on-chip) using rabbit polyclonal antibodies directed against CENP-A or CENP-C human centromeric proteins. These DNA binding-proteins are required for kinetochore function and are exclusively targeted to functional centromeres. Thus, the immunoprecipitation of DNA bound by these proteins allows the isolation of centromeric sequences, including those of the neocentromeres. Neocentromeres arise even in protein-coding genes region. We further analyzed if the increased scaffold attachment sites and the corresponding tighter chromatin of the region involved in the neocentromerization process still were permissive or not to transcription of within encoded genes. Centromere repositioning is a phenomenon in which a neocentromere arisen without altering the gene order, followed by the inactivation of the canonical centromere, becomes fixed in population. It is a process of chromosome rearrangement fundamental in evolution, at the bases of speciation. The repeat-free region where the neocentromere initially forms, progressively acquires extended arrays of satellite tandem repeats that may contribute to its functional stability. In this view our attention focalized to the repositioned horse ECA11 centromere. ChIP-on-chip analysis was used to define the region involved and SNPs studies, mapping within the region involved into neocentromerization, were carried on. We have been able to describe the structural polymorphism of the chromosome 11 centromeric domain of Caballus population. That polymorphism was seen even between homologues chromosome of the same cells. That discovery was the first described ever. Genomic plasticity had a fundamental role in evolution. Centromeres are not static packaged region of genomes. The key question that fascinates biologists is to understand how that centromere plasticity could be combined to the stability and maintenance of centromeric function. Starting from the epigenetic point of view that underlies centromere formation, we decided to analyze the RNA content of centromeric chromatin. RNA, as well as secondary chemically modifications that involve both histones and DNA, represents a good candidate to guide somehow the centromere formation and maintenance. Many observations suggest that transcription of centromeric DNA or of other non-coding RNAs could affect centromere formation. To date has been no thorough investigation addressing the identity of the chromatin-associated RNAs (CARs) on a global scale. This prompted us to develop techniques to identify CARs in a genome-wide approach using high-throughput genomic platforms. The future goal of this study will be to focalize the attention on what strictly happens specifically inside centromere chromatin.