Prediction of novel and analogous folds using fragment assembly and fold recognition

A number of new and newly improved methods for predicting protein structure developed by the Jones–University College London group were used to make predictions for the CASP6 experiment. Structures were predicted with a combination of fold recognition methods (mGenTHREADER, nFOLD, and THREADER) and a substantially enhanced version of FRAGFOLD, our fragment assembly method. Attempts at automatic domain parsing were made using DomPred and DomSSEA, which are based on a secondary structure parsing algorithm and additionally for DomPred, a simple local sequence alignment scoring function. Disorder prediction was carried out using a new SVM-based version of DISOPRED. Attempts were also made at domain docking and “microdomain” folding in order to build complete chain models for some targets.

The DISOPRED server for the prediction of protein disorder

Dynamically disordered regions appear to be relatively abundant in eukaryotic proteomes. The DISOPRED server allows users to submit a protein sequence, and returns a probability estimate of each residue in the sequence being disordered. The results are sent in both plain text and graphical formats, and the server can also supply predictions of secondary structure to provide further structural information.

Improvement of the GenTHREADER method for genomic fold recognition

Motivation: In order to enhance genome annotation, the fully automatic fold recognition method GenTHREADER has been improved and benchmarked. The previous version of GenTHREADER consisted of a simple neural network which was trained to combine sequence alignment score, length information and energy potentials derived from threading into a single score representing the relationship between two proteins, as designated by CATH. The improved version incorporates PSI-BLAST searches, which have been jumpstarted with structural alignment profiles from FSSP, and now also makes use of PSIPRED predicted secondary structure and bi-directional scoring in order to calculate the final alignment score. Pairwise potentials and solvation potentials are calculated from the given sequence alignment which are then used as inputs to a multi-layer, feed-forward neural network, along with the alignment score, alignment length and sequence length. The neural network has also been expanded to accommodate the secondary structure element alignment (SSEA) score as an extra input and it is now trained to learn the FSSP Z-score as a measurement of similarity between two proteins. Results: The improvements made to GenTHREADER increase the number of remote homologues that can be detected with a low error rate, implying higher reliability of score, whilst also increasing the quality of the models produced. We find that up to five times as many true positives can be detected with low error rate per query. Total MaxSub score is doubled at low false positive rates using the improved method.

What are the baselines for protein fold recognition?

What constitutes a baseline level of success for protein fold recognition methods? As fold recognition benchmarks are often presented without any thought to the results that might be expected from a purely random set of predictions, an analysis of fold recognition baselines is long overdue. Given varying amounts of basic information about a protein—ranging from the length of the sequence to a knowledge of its secondary structure—to what extent can the fold be determined by intelligent guesswork? Can simple methods that make use of secondary structure information assign folds more accurately than purely random methods and could these methods be used to construct viable hierarchical classifications?

High yield production of a soluble bifidobacterial β-galactosidase (BbgIV) inE. coliDH5α with improved catalytic efficiency for the synthesis of prebiotic galactooligosaccharides

The bifidobacterial β-galactosidase (BbgIV) was produced in E. coli DH5α at 37 and 30 °C in a 5 L bioreactor under varied conditions of dissolved oxygen (dO2) and pH. The yield of soluble BbgIV was significantly (P < 0.05) increased once the dO2 dropped to 0–2% and remained at such low values during the exponential phase. Limited dO2 significantly (P < 0.05) increased the plasmid copy number and decreased the cells growth rate. Consequently, the BbgIV yield increased to its maximum (71–75 mg per g dry cell weight), which represented 20–25% of the total soluble proteins in the cells. In addition, the specific activity and catalytic efficiency of BbgIV were significantly (P < 0.05) enhanced under limited dO2 conditions. This was concomitant with a change in the enzyme secondary structure, suggesting a link between the enzyme structure and function. The knowledge generated from this work is very important for producing BbgIV as a biocatalyst for the development of a cost-effective process for the synthesis of prebiotic galactooligosaccharides from lactose.

Ruminal dry matter and nitrogen degradation in relation to condensed tannin and protein molecular structures in sainfoin (Onobrychis viciifolia) and lucerne (Medicago sativa).

Sainfoin is a temperate legume that contains condensed tannins (CT), i.e. polyphenols that are able to bind proteins and thus reduce protein degradation in the rumen. A reduction in protein degradation in the rumen can lead to a subsequent increase in amino acid flow to the small intestine. The effects of CT in the rumen and the intestine differ according to the amount and structure of CT and the nature of the protein molecular structure. The objective of the present study was to investigate the degradability in the rumen of three CT-containing sainfoin varieties and CT-free lucerne in relation to CT content and structure (mean degree of polymerization, proportion of prodelphinidins and cis-flavanol units) and protein structure (amide I and II bands, ratio of amide I-to-amide II, α-helix, β-sheet, ratio of α-helix-to-β-sheet). Protein molecular structures were identified using Fourier transform/infrared-attenuated total reflectance (FT/IR-ATR) spectroscopy. The in situ degradability of three sainfoin varieties (Ambra, Esparcette and Villahoz) was studied in 2008, during the first growth cycle at two harvest dates (P1 and P2, i.e. 5 May and 2 June, respectively) and at one date (P3) during the second growth cycle (2 June) and these were compared with a tannin-free legume, lucerne (Aubigny). Loss of dry matter (DMDeg) and nitrogen (NDeg) in polyester bags suspended in the rumen was measured using rumen-fistulated cows. The NDeg of lucerne compared with sainfoin was 0·80 v. 0·77 at P1, 0·78 v. 0·65 at P2 and 0·79 v. 0·70 at P3, respectively. NDeg was related to the rapidly disappearing fraction (‘a’) fraction (r=0·76), the rate of degradation (‘c’) (r=0·92), to the content (r=−0·81) and structure of CT. However, the relationship between NDeg and the slowly disappearing fraction (‘b’) was weak. There was a significant effect of date and species×date, for NDeg and ‘a’ fraction. The secondary protein structure varied with harvest date (species×date) and was correlated with the fraction ‘b’. Both tannin and protein structures influenced the NDeg degradation. CT content and structure were correlated to the ‘a’ fraction and to the ‘c’. Features of the protein molecular secondary structure were correlated to the ‘b’ fraction.

Interactions between lipid-free apolipoprotein-AI and a lipopeptide incorporating the RGDS cell adhesion motif

The interaction of a designed bioactive lipopeptide C16-GGGRGDS, comprising a hexadecyl lipid chain attached to a functional heptapeptide, with the lipid-free apoliprotein, Apo-AI, is examined. This apolipoprotein is a major component of high density lipoprotein and it is involved in lipid metabolism and may serve as a biomarker for cardiovascular disease and Alzheimers’ disease. We find via isothermal titration calorimetry that binding between the lipopeptide and Apo-AI occurs up to a saturation condition, just above equimolar for a 10.7 μM concentration of Apo-AI. A similar value is obtained from circular dichroism spectroscopy, which probes the reduction in α-helical secondary structure of Apo-AI upon addition of C16-GGGRGDS. Electron microscopy images show a persistence of fibrillar structures due to self-assembly of C16-GGGRGDS in mixtures with Apo-AI above the saturation binding condition. A small fraction of spheroidal or possibly “nanodisc” structures was observed. Small-angle X-ray scattering (SAXS) data for Apo-AI can be fitted using a published crystal structure of the Apo-AI dimer. The SAXS data for the lipopeptide/ Apo-AI mixtures above the saturation binding conditions can be fitted to the contribution from fibrillar structures coexisting with flat discs corresponding to Apo-AI/lipopeptide aggregates.

Studies on Anopheles (Kerteszia) homunculus Komp (Diptera: Culicidae)

The present findings suggest that Anopheles (Kerteszia) homunculus may comprise more than one species. The rDNA ITS2 sequence data corroborate the presence of An. homunculus l.s. in Mata Atlantica, southern Brazil, and suggest that specimens from Trinidad may belong to an unnamed morphologically similar species. There is a need for additional studies to establish the geographical distribution of An. homunculus l.s. in continental South America and in Trinidad, especially in southern Mata Atlantica, Brazil.

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.

Putative outer membrane proteins of Leptospira interrogans stimulate human umbilical vein endothelial cells (HUVECS) and express during infection

Cell adhesion molecules (CAMs) are surface receptors present in eukaryotic cells that mediate cell-cell or cell-extracellular matrix interactions. Vascular endothelium stimulation in vitro that lead to the upregulation of CAMs was reported for the pathogenic spirochaetes, including rLIC10365 of Leptospira interrogans. In this study, we report the cloning of LIC10507, LIC10508, LIC10509 genes of L interrogans using Escherichia coli as a host system. The rational for selecting these sequences is due to their location in L. interrogans serovar Copenhageni genome that has a potential involvement in pathogenesis. The genes encode for predicted lipoproteins with no assigned functions. The purified recombinant proteins were capable to promote the upregulation of intercellular adhesion molecule 1 (ICAM-1) and E-selectin on monolayers of human umbilical vein endothelial cells (HUVECS). In addition, the coding sequences are expressed in the renal tubules of animal during bacterial experimental infection. The proteins are probably located at the outer membrane of the bacteria since they are detected in detergent-phase of L interrogans Triton X-114 extract. Altogether our data suggest a possible involvement of these proteins during bacterial infection and provide new insights into the role of this region in the pathogenesis of Leptospira. (C) 2008 Elsevier Ltd. All rights reserved.

Fibrinogen stability under surfactant interaction

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

Sugarcane Phosphoribosyl Pyrophosphate Synthetase: Molecular Characterization of a Phosphate-independent PRS

Phosphoribosyl pyrophosphate synthetase (PRS-EC:2.7.6.1) is an important enzyme present in several metabolic pathways, thus forming a complex family of isoenzymes. However, plant PRS enzymes have not been extensively investigated. In this study, a sugarcane prs gene has been characterized from the Sugar Cane Expressed Sequence Tag Genome Project. This gene contains a 984-bp open reading frame encoding a 328-amino acid protein. The predicted amino acid sequence has 77% and 78% amino acid sequence identity to Arabidopsis thaliana and Spinacia oleracea PRS4, respectively. The assignment of sugarcane PRS as a phosphate-independent PRS isoenzyme (Class II PRS) is verified following enzyme assay and phylogenetic reconstruction of PRS homologues. To gain further insight into the structural framework of the phosphate independence of sugarcane PRS, a molecular model is described. This model reveals the formation of two conserved domains elucidating the structural features involved in sugarcane PRS phosphate independence. The recombinant PRS retains secondary structure elements and a quaternary arrangement consistent with known PRS homologues, based on circular dichroism measurements.

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

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

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

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

On the Denaturation Mechanisms of the Ligand Binding Domain of Thyroid Hormone Receptors

The ligand binding domain (LBD) of nuclear hormone receptors adopts a very compact, mostly alpha-helical structure that binds specific ligands with very high affinity. We use circular dichroism spectroscopy and high-temperature molecular dynamics Simulations to investigate unfolding of the LBDs of thyroid hormone receptors (TRs). A molecular description of the denaturation mechanisms is obtained by molecular dynamics Simulations of the TR alpha and TR beta LBDs in the absence and in the presence of the natural ligand Triac. The Simulations Show that the thermal unfolding of the LBD starts with the loss of native contacts and secondary Structure elements, while the Structure remains essentially compact, resembling a molten globule state. This differs From most protein denaturation simulations reported to date and suggests that the folding mechanism may start with the hydrophobic collapse of the TR LBDs. Our results reveal that the stabilities of the LBDs of the TR alpha and TR beta Subtypes are affected to different degrees by the binding of the isoform selective ligand Triac and that ligand binding confers protection against thermal denaturation and unfolding in a subtype specific manner. Our Simulations indicate two mechanisms by which the ligand stabilizes the LBD: (1) by enhancing the interactions between H8 and H 11, and the interaction of the region between H I and the Omega-loop with the core of the LBD, and (2) by shielding the hydrophobic H6 from hydration.