Expression and purification of human respiratory syncytial virus recombinant fusion protein

The Human Respiratory Syncytial Virus (HRSV) fusion protein (F) was expressed in Escherichia call BL21A using the pET28a vector at 37 degrees C. The protein was purified from the soluble fraction using affinity resin. The structural quality of the recombinant fusion protein and the estimation of its secondary structure were obtained by circular dichroism. Structural models of the fusion protein presented 46% of the helices in agreement with the spectra by circular dichroism analysis. There are only few studies that succeeded in expressing the HRSV fusion protein in bacteria. This is a report on human fusion protein expression in E. call and structure analysis, representing a step forward in the development of fusion protein F inhibitors and the production of antibodies. (c) 2008 Elsevier Inc. All rights reserved.

Refolded dengue virus type 2 NS1 protein expressed in Escherichia coli preserves structural and immunological properties of the native protein

The dengue virus NS1 protein has been shown to be a protective antigen under different experimental conditions but the recombinant protein produced in bacterial expression systems is usually not soluble and loses structural and immunological features of the native viral protein In the present study, experimental conditions leading to purification and refolding of the recombinant dengue virus type 2 (DENV-2) NS1 protein expressed in Escherichia coil are described The refolded recombinant protein was recovered as heat-stable soluble dimers with preserved structural features, as demonstrated by spectroscopic methods In addition, antibodies against epitopes of the NS1 protein expressed in eukaryotic cells recognized the refolded protein expressed in E coli but not the denatured form or the same protein submitted to a different refolding condition Collectively, the results demonstrate that the recombinant NS1 protein preserved important conformation and antigenic determinants of the native virus protein and represents a valuable reagent either for the development of vaccines or for diagnostic methods. (C) 2010 Elsevier B V All rights reserved

Evolutionary dynamics of the immunodominant repeats of the Plasmodium vivax malaria-vaccine candidate circumsporozoite protein (CSP)

The circumsporozoite protein (CSP) of Plasmodium vivax, a major target for malaria vaccine development, has immunodominant B-cell epitopes mapped to central nonapeptide repeat arrays. To determine whether rearrangements of repeat motifs during mitotic DNA replication of parasites create significant CSP diversity under conditions of low effective meiotic recombination rates, we examined csp alleles from sympatric P. vivax isolates systematically sampled from an area of low malaria endemicity in Brazil over a period of 14 months. Nine unique csp types, comprising six different nona peptide repeats, were observed in 45 isolates analyzed. Identical or nearly identical repeats predominated in most arrays, consistent with their recent expansion. We found strong linkage disequilibrium at sites across the chromosome 8 segment flanking the csp locus, consistent with rare meiotic recombination in this region. We conclude that CSP repeat diversity may not be severely constrained by rare meiotic recombination in areas of low malaria endemicity. New repeat variants may be readily created by nonhomologous recombination even when meiotic recombination is rare, with potential implications for CSP-based vaccine development. (C) 2010 Elsevier B.V. All rights reserved.

Structure and Mode of Action of Microplusin, a Copper II-chelating Antimicrobial Peptide from the Cattle Tick Rhipicephalus (Boophilus) microplus

Microplusin, a Rhipicephalus (Boophilus) microplus antimicrobial peptide (AMP) is the first fully characterized member of a new family of cysteine-rich AMPs with histidine-rich regions at the N and C termini. In the tick, microplusin belongs to the arsenal of innate defense molecules active against bacteria and fungi. Here we describe the NMR solution structure of microplusin and demonstrate that the protein binds copper II and iron II. Structured as a single alpha-helical globular domain, microplusin consists of five alpha-helices: alpha 1 (residues Gly-9 to Arg-21), alpha 2 (residues Glu-27 to Asn-40), alpha 3 (residues Arg-44 to Thr-54), alpha 4 (residues Leu-57 to Tyr-64), and alpha 5 (residues Asn-67 to Cys-80). The N and C termini are disordered. This structure is unlike any other AMP structures described to date. We also used NMR spectroscopy to map the copper binding region on microplusin. Finally, using the Gram-positive bacteria Micrococcus luteus as a model, we studied of mode of action of microplusin. Microplusin has a bacteriostatic effect and does not permeabilize the bacterial membrane. Because microplusin binds metals, we tested whether this was related to its antimicrobial activity. We found that the bacteriostatic effect of microplusin was fully reversed by supplementation of culture media with copper II but not iron II. We also demonstrated that microplusin affects M. luteus respiration, a copper-dependent process. Thus, we conclude that the antibacterial effect of microplusin is due to its ability to bind and sequester copper II.

Limited global diversity of the Plasmodium vivax merozoite surface protein 4 gene

Merozoite surface proteins (MSPs) of the malaria parasites are major candidates for vaccine development targeting asexual blood stages. However, the diverse antigenic repertoire of these antigens that induce strain-specific protective immunity in human is a major challenge for vaccine design and often determines the efficacy of a vaccine. Here we further assessed the genetic diversity of Plasmodium vivax MSP4 (PvMSP4) protein using 195 parasite samples collected mostly from Thailand, Indonesia and Brazil. Overall, PvMSP4 is highly conserved with only eight amino acid substitutions. The majority of the haplotype diversity was restricted to the two short tetrapeptide repeat arrays in exon 1 and 2, respectively. Selection and neutrality tests indicated that exon 1 and the entire coding region of PvMSP4 were under purifying selection. Despite the limited nucleotide polymorphism of PvMSP4, significant genetic differentiation among the three major parasite populations was detected. Moreover, microgeographical heterogeneity was also evident in the parasite populations from different endemic areas of Thailand. (C) 2009 Elsevier B.V. All rights reserved.

The leptospiral antigen Lp49 is a two-domain protein with putative protein binding function

Pathogenic Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. Currently available vaccines have limited effectiveness and therapeutic interventions are complicated by the difficulty in making an early diagnosis of leptospirosis. The genome of Leptospira interrogans was recently sequenced and comparative genomic analysis contributed to the identification of surface antigens, potential candidates for development of new vaccines and serodiagnosis. Lp49 is a membrane-associated protein recognized by antibodies present in sera from early and convalescent phases of leptospirosis patients. Its crystal structure was determined by single-wavelength anomalous diffraction using selenomethionine-labelled crystals and refined at 2.0 angstrom resolution. Lp49 is composed of two domains and belongs to the all-beta-proteins class. The N-terminal domain folds in an immunoglobulin-like beta-sandwich structure, whereas the C-terminal domain presents a seven-bladed beta-propeller fold. Structural analysis of Lp49 indicates putative protein-protein binding sites, suggesting a role in Leptospira-host interaction. This is the first crystal structure of a leptospiral antigen described to date. (C) 2008 Elsevier Inc. All rights reserved.

S100 beta Protein Expression: Gender- and Age-Related Daily Changes

S100 beta is a soluble protein released by glial cells mainly under the activation of the 5-HT1A receptor. It has been reported as a neuro-trophic and -tropic factor that promotes neurite maturation and outgrowth during development. This protein also plays a role in axonal stability and the plasticity underlying long-term potentiation in adult brains. The ability of S100 beta to rapidly regulate neuronal morphology raises the interesting point of whether there are daily rhythm or gender differences in S100 beta level in the brain. To answer this question, the S100 beta expression in adult female and male rats, as well as in adult female CD-21 and S100 beta -/- female mice, were investigated. Scintillation counting and morphometric analysis of the immunoreactivity of S100 beta, showed rhythmic daily expression. The female and male rats showed opposite cycles. Females presented the highest value at the beginning of the rest phase (5:00 h), while in males the maximum value appeared in the beginning of the motor activity period (21:00 h). These results confirm previous S100 beta evaluations in human serum and cerebrospinal fluid reporting the protein`s function as a biomarker for brain damage (Gazzolo et al. in Clin Chem 49:967-970, 2003; Clin Chim Acta 330:131-133, 2003; Pediatr Res 58:1170-1174, 2005), similar behavior was also observed for GFAP in relation to Alzheimer Disease (Fukuyama et al. in Eur Neurol 46:35-38, 2001). The data should be taken into account when considering S100 beta as a biomarker of health condition. In addition, the results raise questions on which structure or condition imposes these rhythms as well as on the physiological meaning of the observed gender differences.

Percolation in a network with long-range connections: Implications for cytoskeletal structure and function

Cell shape, signaling, and integrity depend on cytoskeletal organization. In this study we describe the cytoskeleton as a simple network of filamentary proteins (links) anchored by complex protein structures (nodes). The structure of this network is regulated by a distance-dependent probability of link formation as P = p/d(s), where p regulates the network density and s controls how fast the probability for link formation decays with node distance (d). It was previously shown that the regulation of the link lengths is crucial for the mechanical behavior of the cells. Here we examined the ability of the two-dimensional network to percolate (i.e. to have end-to-end connectivity), and found that the percolation threshold depends strongly on s. The system undergoes a transition around s = 2. The percolation threshold of networks with s < 2 decreases with increasing system size L, while the percolation threshold for networks with s > 2 converges to a finite value. We speculate that s < 2 may represent a condition in which cells can accommodate deformation while still preserving their mechanical integrity. Additionally, we measured the length distribution of F-actin filaments from publicly available images of a variety of cell types. In agreement with model predictions, cells originating from more deformable tissues show longer F-actin cytoskeletal filaments. (C) 2008 Elsevier B.V. All rights reserved.

Protein lethality investigated in terms of long range dynamical interactions

The relationship between network structure/dynamics and biological function constitutes a fundamental issue in systems biology. However, despite many related investigations, the correspondence between structure and biological functions is not yet fully understood. A related subject that has deserved particular attention recently concerns how essentiality is related to the structure and dynamics of protein interactions. In the current work, protein essentiality is investigated in terms of long range influences in protein-protein interaction networks by considering simulated dynamical aspects. This analysis is performed with respect to outward activations, an approach which models the propagation of interactions between proteins by considering self-avoiding random walks. The obtained results are compared to protein local connectivity. Both the connectivity and the outward activations were found to be strongly related to protein essentiality.

Characterisation, immunolocalisation and antifungal activity of a lipid transfer protein from chili pepper (Capsicum annuum) seeds with novel alpha-amylase inhibitory properties

Lipid transfer proteins (LTPs) were thus named because they facilitate the transfer of lipids between membranes in vitro. This study was triggered by the characterization of a 9-kDa LTP from Capsicum annuum seeds that we call Ca-LTP(1). Ca-LTP(1) was repurified, and in the last chromatographic purification step, propanol was used as the solvent in place of acetonitrile to maintain the protein`s biological activity. Bidimensional electrophoresis of the 9-kDa band, which corresponds to the purified Ca-LTP(1), showed the presence of three isoforms with isoelectric points (pIs) of 6.0, 8.5 and 9.5. Circular dichroism (CD) analysis suggested a predominance of alpha-helices, as expected for the structure of an LTP family member. LTPs immunorelated to Ca-LTP(1) from C. annuum were also detected by western blotting in exudates released from C. annuum seeds and also in other Capsicum species. The tissue and subcellular localization of Ca-LTP(1) indicated that it was mainly localized within dense vesicles. In addition, isolated Ca-LTP(1) exhibited antifungal activity against Colletotrichum lindemunthianum, and especially against Candida tropicalis, causing several morphological changes to the cells including the formation of pseudohyphae. Ca-LTP(1) also caused the yeast plasma membrane to be permeable to the dye SYTOX green, as verified by fluorescence microscopy. We also found that Ca-LTP(1) is able to inhibit mammalian alpha-amylase activity in vitro.

Crystallographic Structure of Phosphofructokinase-2 from Escherichia coli in Complex with Two ATP Molecules. Implications for Substrate Inhibition

Phosphofructokinase-1 and -2 (Pfk-1 and Pfk-2, respectively) from Escherichia coli belong to different homologous superfamilies. However, in spite of the lack of a common ancestor, they share the ability to catalyze the same reaction and are inhibited by the substrate MgATP. Pfk-2, an ATP-dependent 6-phosphofructokinase member of the ribokinase-like superfamily, is a homodimer of 66 kDa subunits whose oligomerization state is necessary for catalysis and stability. The presence of MgATP favors the tetrameric form of the enzyme. In this work, we describe the structure of Pfk-2 in its inhibited tetrameric form, with each subunit bound to two ATP molecules and two Mg ions. The present structure indicates that substrate inhibition occurs due to the sequential binding of two MgATP molecules per subunit, the first at the usual site occupied by the nucleotide in homologous enzymes and the second at the allosteric site, making a number of direct and Mg-mediated interactions with the first. Two configurations are observed for the second MgATP, one of which involves interactions with Tyr23 from the adjacent subunit in the dimer and the other making an unusual non-Watson-Crick base pairing with the adenine in the substrate ATP. The oligomeric state observed in the crystal is tetrameric, and some of the structural elements involved in the binding of the Substrate and allosteric ATPs are also participating in the dimer-dimer interface. This structure also provides the grounds to compare analogous features of the nonhomologous phosphofructokinases from E. coli. (C) 2008 Elsevier Ltd. All rights reserved.

Only subtle protein conformational adaptations are required for ligand binding to thyroid hormone receptors: Simulations using a novel multipoint steered molecular dynamics approach

Thyroid hormone receptors (TR) are hormone-dependent transcription regulators that play a major role in human health, development, and metabolic functions. The thyroid hormone resistance syndrome, diabetes, obesity, and some types of cancer are just a few examples of important diseases that are related to TR malfunctioning, particularly impaired hormone binding. Ligand binding to and dissociation from the receptor ultimately control gene transcription and, thus, detailed knowledge of binding and release mechanisms are fundamental for the comprehension of the receptor`s biological function and development of pharmaceuticals. In this work, we present the first computational study of ligand entry into the ligand binding domain (LBD) of a nuclear receptor. We report molecular dynamics simulations of ligand binding to TRs using a generalization of the steered molecular dynamics technique designed to perform single-molecule pulling simulations along arbitrarily nonlinear driving pathways. We show that only gentle protein movements and conformational adaptations are required for ligand entry into the LBDs and that the magnitude of the forces applied to assist ligand binding are of the order of the forces involved in ligand dissociation. Our simulations suggest an alternative view for the mechanisms ligand binding and dissociation of ligands from nuclear receptors in which ligands can simply diffuse through the protein surface to reach proper positioning within the binding pocket. The proposed picture indicates that the large-amplitude protein motions suggested by the apo- and holo-RXR alpha crystallographic structures are not required, reconciling conformational changes of LBDs required for ligand entry with other nuclear receptors apo-structures that resemble the ligand-bound LBDs.

SBTX, a new toxic protein distinct from soyatoxin and other toxic soybean [Glycine max] proteins, and its inhibitory effect on Cercospora sojina growth

SBTX, a novel toxin from soybean, was purified by ammonium sulfate fractionation followed by chromatographic steps DEAE-Cellulose, CM-Sepharose and Superdex 200 HR fast-protein liquid chromatography (FPLC). Lethality of SBTX to mice (LD50 5.6 mg/kg) was used as parameter in the purification steps. SBTX is a 44-kDa basic glycoprotein composed of two polypeptide chains (27 and 17 kDa) linked by a disulfide bond. The N-terminal sequences of the 44 and 27 kDa chains were identical (ADPTFGFTPLGLSEKANLQIMKAYD), differing from that of 17 kDa (PNPKVFFDMTIGGQSAGRIVMEEYA). SBTX contains high levels of Glx, Ala, Asx, Gly and Lys and showed maximum absorption at 280 nm, epsilon(1 cm) (1%) of 6.3, and fluorescence emission in the 290-450nm range upon excitation at 280nm. The secondary structure content was 35% alpha-helix, 13% beta-strand and beta-sheet, 27% beta-turn, 25% unordered, and 1% aromatic residues. Immunological assays showed that SBTX was related to other toxic proteins, such as soyatoxin and canatoxin, and cross-reacted weekly with soybean trypsin inhibitor and agglutinin, but it was devoid of protease-inhibitory and hemagglutinating activities. The inhibitory effect of SBTX on growth of Cercospora sojina, fungus causing frogeye leaf spot in soybeans, was observed at 50 mu g/ml, concentration 112 times lesser than that found to be lethal to mice. This effect on phytopathogenic fungus is a potential attribute for the development of transgenic plants with enhanced resistance to pathogens. (c) 2007 Elsevier Ltd. All rights reserved.

Structural modeling and mutational analysis of yeast eukaryotic translation initiation factor 5A reveal new critical residues and reinforce its involvement in protein synthesis

Eukaryotic translation initiation factor 5A (eIF5A) is a protein that is highly conserved and essential for cell viability. This factor is the only protein known to contain the unique and essential amino acid residue hypusine. This work focused on the structural and functional characterization of Saccharomyces cerevisiae eIF5A. The tertiary structure of yeast eIF5A was modeled based on the structure of its Leishmania mexicana homologue and this model was used to predict the structural localization of new site-directed and randomly generated mutations. Most of the 40 new mutants exhibited phenotypes that resulted from eIF-5A protein-folding defects. Our data provided evidence that the C-terminal alpha-helix present in yeast eIF5A is an essential structural element, whereas the eIF5A N-terminal 10 amino acid extension not present in archaeal eIF5A homologs, is not. Moreover, the mutants containing substitutions at or in the vicinity of the hypusine modification site displayed nonviable or temperature-sensitive phenotypes and were defective in hypusine modification. Interestingly, two of the temperature-sensitive strains produced stable mutant eIF5A proteins - eIF5A(K56A) and eIF5A(Q22H,L93F)- and showed defects in protein synthesis at the restrictive temperature. Our data revealed important structural features of eIF5A that are required for its vital role in cell viability and underscored an essential function of eIF5A in the translation step of gene expression.

gamma-zein secondary structure in solution by circular dichroism

The proline-rich N-terminal domain of gamma-zein has been reported in relevant process, which include its ability to cross the cell membranes. Evidences indicate that synthetic hexapeptide (PPPVHL), naturally found in N-terminal portion of gamma-zein, can adopt the polyproline II (PPII) conformation in aqueous solution. The secondary structure of gamma-zein in maize protein bodies had been analyzed by solid state Fourier transform infrared and nuclear magnetic resonance spectroscopies. However, it was not possible to measure PPII content in physiological environment since the beta-sheet and PPII signals overlap in both solid state techniques. Here, the secondary structure of gamma-zein has been analyzed by circular dichroism in SDS aqueous solution with and without ditiothreitol (DTT), and in 60% of 2-propanol and water with DTT The results show that gamma-zein has high helical content in all solutions. The PPII conformation was present at about 7% only in water/DTT solution. (c) 2007 Wiley Periodicals, Inc.