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E Querol

Publications and source records attributed to E Querol.

At least 55 records · Page 3Linked to original sources
PubMed1996

Cloning, sequencing and expression of the transferrin-binding protein 1 gene from Actinobacillus pleuropneumoniae.

Two outer-membrane proteins are involved in the uptake of iron from transferrin by certain Gram-negative bacteria, transferrin-binding proteins 1 and 2. The gene encoding transferrin-binding protein 1 from a serotype 1 isolate of the Gram-negative pathogen Actinobacillus pleuropneumoniae was cloned, and a fragment encoding 700 amino acids of Tbp1 was expressed in Escherichia coli. We also report here sequencing of the tbpl gene and a comparison of the deduced amino acid sequence with Tbpls from related species. The predicted polypeptide product of tbpl is a 106 kDa protein with a 22-residue signal peptide.

Actinobacillus pleuropneumoniae↗
PubMed1996

On the sensitivity of MD trajectories to changes in water-protein interaction parameters: the potato carboxypeptidase inhibitor in water as a test case for the GROMOS force field.

A critical evaluation is presented of the sensitivity of the results of molecular dynamics simulations of proteins to changes in the parameters describing water-protein and protein-protein van der Waals interactions in the GROMOS force field. The origin of the van der Waals and electrostatic parameters of the GROMOS standard force field is reviewed, and possible weaknesses are discussed. Four alternate sets of van der Waals parameters for the oxygen types of the GROMOS force field that have been suggested by different authors are then tested against the original force field. Six 500 ps molecular dynamics simulations of the potato carboxypeptidase inhibitor (PCI) in solution using the different parameter sets are analyzed and the results compared with the available X-ray and NMR data. It is shown that the behavior of the molecular system is very sensitive to changes in the van der Waals parameters of the oxygens, especially when affecting the interactions between water and aliphatic or aromatic groups. It is also shown that correction of just the repulsive van der Waals parameter of the water oxygen for its interactions with nonpolar groups is sufficient to correct the main deficiency of the original GROMOS parameter set. Nevertheless, the present study suggest that further refinement of the current parameters is still needed for a proper representation of nonbonded interactions.

Algorithms↗
PubMed1996

Glycoprotein E of bovine herpesvirus type 1 is involved in virus transmission by direct cell-to-cell spread.

In order to identify the role of the bovine herpesvirus type 1 (BHV-1) glycoprotein E (gE) in the viral infection cycle, we have constructed a BHV-1 gE deletion mutant strain (BHV-1 gE-). This strain was assayed in vitro by comparing its growth kinetics with the wild type strain used as a host of the deletion. Our results indicate that those conditions which prevent the infection by direct adsorption to the cells (presence of a semi-solid medium or presence of neutralizing antibodies in the medium) selectively inhibit the growth of the gE- strain, suggesting that gE plays a central role in the BHV-1 spread by direct cell-to-cell transmission, a major mechanism of the BHV-1 in vivo virulence.

Animals↗
PubMed1996

Analysis of protein conformational characteristics related to thermostability.

The thermal stability of proteins was studied, 195 single amino acid residue replacements reported elsewhere being analysed for several protein conformational characteristics: type of residue replacement; conservative versus nonconservative substitution; replacement being in a homologous stretch of amino acid residues; change in hydrogen bond, van der Waals and secondary structure propensities; solvent-accessible versus inaccessible replacement; type of secondary structure involved in the substitution; the physico-chemical characteristics to which the thermostability enhancement can be attributed; and the relationship of the replacement site to the folding intermediates of the protein, when known. From the above analyses, some general rules arise which suggest where amino acid substitutions can be made to enhance protein thermostability: substitutions are conservative according to the Dayhoff matrix; mainly occur on conserved stretches of residues; preferentially occur on solvent-accessible residues; maintain or enhance the secondary structure propensity upon substitution; contribute to neutralize the dipole moment of the caps of helices and strands; and tend to increase the number of potential hydrogen bonding or van der Waals contacts or improve hydrophobic packing.

Amino Acid Sequence↗
PubMed1995

Crystal structure of Bacillus licheniformis 1,3-1,4-beta-D-glucan 4-glucanohydrolase at 1.8 A resolution.

The crystal structure of the 1,3-1,4-beta-D-glucan 4-glucanohydrolase from Bacillus licheniformis is solved at a resolution of 1.8 A and refined to R = 16.5%. The protein has a similar beta-sandwich structure as the homologous enzyme from Bacillus macerans and the hybrid H(A16-M). This demonstrates that the jellyroll fold of these proteins is remarkably rigid and only weakly influenced by crystal contacts. The crystal structure permits to extend mechanistic considerations derived for the B. licheniformis enzyme to the entire class of bacterial 1,3-1,4-beta-D-glucan 4-glucanohydrolases.

Bacillus↗
PubMed1995

Properties of a novel glucose-enhanced beta-glucosidase purified from Streptomyces sp. (ATCC 11238).

An inducible intracellular beta-glucosidase (EC 3.2.1.21) from Streptomyces sp. QM-B814 (ATCC 11238) has been purified and characterized. The purified polypeptide is monomeric with a relative molecular mass of 62 kDa by SDS-PAGE and 42 kDa by size-exclusion chromatography; its isoelectric point is 4.2. The difference in the molecular mass values can be attributed to the glycosylated nature of the protein. The purified enzyme has a pH optimum of 6.0-6.5. The temperature optimum for activity is 50 degrees C; at this temperature the enzyme is stable for 1 h. The enzyme hydrolyzes mainly aryl-beta-glucosides but also presents significant activity against beta-linked disaccharides and maltose. The enzyme displays an unusual kinetic behavior and biphasic Lineweaver-Burk and Eadie-Hofstee plots for p-nitrophenyl-beta-D-glucoside and cellobiose were obtained. The enzyme presents beta-glycosyltransferase activity and an exoglycosidase-type action on cellodextrins. It is inhibited by delta-gluconolactone (Ki 0.44 mM) but, remarkably, glucose in the range 25-200 mM enhances the rate of p-nitrophenyl-beta-D-glucoside hydrolysis.

Amino Acids↗
PubMed1995

Prediction and Fourier-transform infrared-spectroscopy estimation of the secondary structure of a recombinant beta-glucosidase from Streptomyces sp. (ATCC 11238).

The secondary structure of a recombinant beta-glucosidase (EC 3.2.1.21) from Streptomyces sp. (ATCC 11238) has been predicted by computer algorithms and also estimated by Fourier-transform IR spectroscopy. From curve fitting of the deconvoluted IR spectra, the most probable distribution of the secondary-structural classes appears to be about 34% alpha-helix, 30% beta-sheet, 25% reverse turns and 11% non-ordered structures. These data showed a good agreement with data from computer prediction (35% alpha-helix, 23% beta-sheet, 31% reverse turns and 11% non-ordered structures).

Algorithms↗
PubMed1995

Induction and preliminary characterization of intracellular beta-glucosidases from a cellulolytic Streptomyces strain.

The cellulolytic actinomycete Streptomyces sp. QM-B814 possess an intracellular beta-glucosidase system which is induced by cellobiose and carboxymethylcellulose. Maximal beta-glucosidase activity was attained 8-10 h after inducer addition to exponential phase growing cultures. The induction is depressed in the presence of glucose. The system is composed of two electrophoretically different beta-glucosidases forms showing relative molecular masses of about 60 and 35 kDa, and pI values in the range 4.2-4.5. Both beta-glucosidases are synthesized de novo. The enzymes share substrate preference and are both inhibited by delta-gluconolactone and p-chloromercuribenzoate. The induction pattern and glucose inhibition are similar for both enzymes.

Cellobiose↗
PubMed1995

Contribution of a disulfide bridge to the stability of 1,3-1,4-beta-D-glucan 4-glucanohydrolase from Bacillus licheniformis.

Bacillus 1,3-1,4-beta-glucanases possess a highly conserved disulfide bridge connecting a beta-strand with a solvent-exposed loop lying on top of the extended binding site cleft. The contribution of the disulfide bond and of both individual cysteines (Cys61 and Cys90) in the Bacillus licheniformis enzyme to stability and activity has been evaluated by protein engineering methods. Reduction of the disulfide bond has no effect on kinetic parameters, has only a minor effect on the activity-temperature profile at high temperatures, and destabilizes the protein by less than 0.7 kcal/mol as measured by equilibrium urea denaturation at 37 degrees C. Replacing either of the Cys residues with Ala destabilizes the protein and lowers the specific activity. C90A retains 70% of wild-type (wt) activity (in terms of Vmax), whereas C61A and the double mutant C61A-C90A have 10% of wt Vmax. A larger change in free energy of unfolding is seen by equilibrium urea denaturation for the C61A mutation (loop residue, 3.2 kcal/mol relative to reduced wt) as compared with the C90A mutation (beta-strand residue, 1.8 kcal/mol relative to reduced wt), while the double mutant C61A-C90A is approximately 0.8 kcal/mol less stable than the single C61A mutant. The effects on stability are interpreted as a result of the change in hydrophobic packing that occurs upon removal of the sulfur atoms in the Cys to Ala mutations.

Bacillus↗
PubMed1994

Mapping, cloning and sequencing of a glycoprotein-encoding gene from bovine herpesvirus type 1 homologous to the gE gene from HSV-1.

In order to map and identify the glycoprotein-encoding gene from bovine herpesvirus type 1 (BHV-1), homologous to the gE glycoprotein from herpes simplex virus type 1 (HSV-1), a region of the unique short sequence from the BHV-1 genome has been sequenced. The sequenced region contains an ORF coding for a polypeptide of 575 amino acids (aa). The aa sequence presents substantial similarity to that of the glycoprotein gE from HSV-1 and to homologous proteins of related viruses such as pseudorabies virus, equine herpesvirus type 1 and varicella zoster virus. The aa sequence presents additional characteristics compatible with the structure of a viral glycoprotein: signal peptide, putative glycosylation sites and a long C-terminal transmembrane alpha-helix.

Amino Acid Sequence↗
PubMed1994

The disulfide folding pathway of potato carboxypeptidase inhibitor.

Potato carboxypeptidase inhibitor (PCI) contains 39 amino acids and three disulfides. Reduced and denatured PCI refolds spontaneously in vitro to regain its native structure. The folding pathway of a recombinant form of this protein has been elucidated by structural analysis and stop/go folding experiments of both acid and iodoacetate-trapped intermediates. The results reveal that folding of PCI proceeds through an initial stage of nonspecific disulfide formation (packing), followed by disulfide reshuffling (consolidation) of partially packed intermediates to acquire the native structure. The process of nonspecific packing involves a sequential flow of fully reduced PCI through 1- and 2-disulfide intermediates and leads to the formation of scrambled 3-disulfide species. All three classes of intermediates are highly heterogeneous. Disulfide reshuffling occurs at the final stage which refines and consolidates the scrambled species to acquire the native conformation. The efficiencies of disulfide formation and disulfide reshuffling can be selectively regulated by redox potential. Disulfide formation is promoted by cystine or oxidized glutathione, whereas disulfide reshuffling requires free thiols, such as cysteine, reduced glutathione, or beta-mercaptoethanol. Consolidation of scrambled species to form the native PCI represents the major rate-limiting step. When folding of PCI was carried out in the presence of cystine (2 mM) alone, more than 98% of the intermediates accumulate as the scrambled species after 1 min of folding. Furthermore, denaturant (5 M GdmCl or 8 M urea) mainly disrupts the final stage of PCI folding and exerts no apparent influence on the early stage of nonspecific packing. The compositions of 1- and 2-disulfide intermediates, according to their high performance liquid chromatography patterns, remain indistinguishable regardless of whether folding is performed in the absence or presence of denaturant.

Carboxypeptidases↗
PubMed1994

C-tail valine is a key residue for stabilization of complex between potato inhibitor and carboxypeptidase A.

Although the carboxypeptidase A-potato carboxypeptidase inhibitor (CPA-PCI) complex is a well known example of protein-protein interaction, little was known about the basis of its thermodynamic stability. Site-directed mutagenesis has been used to identify key residues in the PCI tail and estimate the contribution of their chemical groups to the binding to CPA. Two deletion mutants were created, one lacking the C-terminal residue of the tail (Gly39) and another one lacking the two C-terminal residues (Val38, Gly39). The last mutant had an inhibition constant for CPA 10(4)-fold higher than that of wild-type PCI, indicating that Val38 is a key residue. The interactions of Val38 with CPA residues contribute 5.4-5.7 kcal mol-1 to the overall stability of the CPA-PCI complex (11.9-12.1 kcal mol-1). A series of PCI point mutants at valine 38 were created, and their inhibition constant for CPA was measured. Two of these mutants with smaller side chains, V38G and V38A, allowed us to estimate that the contribution of the three side chain aliphatic groups of valine 38 to the overall stability of the complex is 3.4-4 kcal mol-1. Another two mutants with larger side chains, V38L and V38I, were constructed, the first being a significantly worse inhibitor than the wild type. These results suggest that only aliphatic groups in positions beta and gamma of residue 38 in PCI (but not those in delta) can establish van der Waals interactions with atoms of the active center of CPA and participate in binding. The energetic contribution of each methyl/methylene group in those positions can be estimated as 1-1.5 kcal mol-1. Our hypothesis is supported by computer simulation analysis.

Carboxypeptidases↗
PubMed1994

A beta-glucosidase gene (bgl3) from Streptomyces sp. strain QM-B814. Molecular cloning, nucleotide sequence, purification and characterization of the encoded enzyme, a new member of family 1 glycosyl hydrolases.

A beta-glucosidase gene (bgl3) from Streptomyces sp. QM-B814 (American Type Culture Collection 11238) has been cloned by functional complementation of a beta-glucosidase-negative mutant of Streptomyces lividans. An open-reading frame of 1440 nucleotides encoding a polypeptide of 479 amino acids was found by sequencing. The encoded protein (Bgl3) shows extensive similarity (over 45% identity) with beta-glycosidases from family-1 glycosyl hydrolases. The cloned enzyme, purified following ammonium sulphate precipitation and two chromatographic steps, is monomeric with molecular mass 52.6 kDa, as determined by mass spectrometry, and an isoelectric point of pI 4.4. The enzyme appears to be a beta-glucosidase with broad substrate specificity, is active on cellooligomers, and performs transglycosylation reactions. The estimated apparent Km values for p-nitrophenyl-beta-D-glucopyranoside and cellobiose are 0.27 mM and 7.9 mM, respectively. The Ki values for glucose and delta-gluconolactone, using p-nitrophenyl-beta-D-glucopyranoside as a substrate, are 65 mM and 0.08 mM, respectively. The purified enzyme has a pH optimum of pH 6.5 and the temperature optimum for activity is 50 degrees C.

Amino Acid Sequence↗
PubMed1994

Identification of active site carboxylic residues in Bacillus licheniformis 1,3-1,4-beta-D-glucan 4-glucanohydrolase by site-directed mutagenesis.

Active site residues of 1,3-1,4-beta-D-glucan 4-glucanohydrolase (EC 3.2.1.73) from Bacillus licheniformis have been identified by site-directed mutagenesis. Previous work revealed that Glu-134 was essential for enzymatic activity, and it was proposed as the catalytic nucleophile by affinity labeling of the highly homologous Bacillus amyloliquefaciens enzyme. To search for the general acid catalyst, the Asp and Glu residues conserved among the Bacillus isozymes have been mutated to Asn and Gln, respectively. Out of the 14 positions studied, only the E138Q mutation yielded an inactive enzyme, whereas the E134Q and D136N mutants retained less than 0.5% of the wild type activity. Based on the three-dimensional structure of a hybrid B. amyloliquefaciens-Bacillus macerans 1,3-1,4-beta-D-glucan 4-glucanohydrolase, Glu-134, Asp-136, and Glu-138 are the only carboxylic acid residues that are properly located into the active site cleft to participate in catalysis. Glu-138 appears as the most likely candidate to function as the general acid catalyst, while Asp-136 may affect the pK alpha of the catalytic residues.

Amino Acid Sequence↗
PubMed1994

An Excel spreadsheet computer program combining algorithms for prediction of protein structural characteristics.

A program running on personal computers (either Apple Macintosh or PC, using Excel worksheets) for the prediction of some protein structural characteristics is reported. The program runs according to the Chou and Fasman algorithm, with some modifications, for secondary structure prediction. The program also incorporates several complementary analyses for secondary structure prediction to help the user in the decision-making process: rules for amino acid preferences in the N-cap and C-cap of alpha-helices; prediction of the protein structural class and search of sequential motifs related to secondary structure. Additional algorithms performed by the program are: prediction of domain boundaries, prediction of loops, prediction of the state of cysteines (reduced or in disulfide bridge), hydropathy profiles according to Kyte and Doolittle, Hoop and Woods, and flexibility plot according to Karplus and Schulz.

Algorithms↗
PubMed1992

Essential catalytic role of Glu134 in endo-beta-1,3-1,4-D-glucan 4-glucanohydrolase from B. licheniformis as determined by site-directed mutagenesis.

Site-directed mutagenesis experiments designed to identify the active site of Bacillus licheniformis endo-beta-1,3-1,4-D-glucan 4-glucanohydrolase (beta-glucanase) have been performed. Putative catalytic residues were chosen on the basis of sequence similarity analysis to viral and eukaryotic lysozymes. Four mutant enzymes were expressed and purified from recombinant E. coli and their kinetics analysed with barley beta-glucan. Replacement of Glu134 by Gln produced a mutant (E134Q) that retains less than 0.3% of the wild-type activity. The other mutants, D133N, E160Q and D179N, are active but show different kinetic parameters relative to wild-type indicative of their participation in substrate binding and transition-state complex stabilization. Glu134 is essential for activity; it is comprised in a region of high sequence similarity to the active site of T4 lysozyme and matches the position of the general acid catalyst. These results strongly support a lysozyme-like mechanism for this family of Bacillus beta-glucan hydrolases with Glu134 being the essential acid catalyst.

Amino Acid Sequence↗
PubMed1992

Expression of a synthetic gene encoding potato carboxypeptidase inhibitor using a bacterial secretion vector.

A synthetic gene encoding the 39-amino-acid (aa) potato carboxypeptidase inhibitor IIa (PCI-IIa) has been constructed and expressed using the secretion vector, pIN-III-ompA-3, fused in frame to the OmpA signal peptide-encoding sequence. Recombinant Escherichia coli secreted a PCI with 10 additional aa at the N terminus (rePCI + 10). These extra aa were removed by site-directed mutagenesis giving a PCI with no additional aa (rePCI), as shown by fast atom bombardment mass spectrometry (M(r) 4295). The two forms of rePCI were found almost exclusively in the culture medium, not in the periplasmic space, as would be expected from OmpA signal peptide fusions. Both rePCI + 10 and rePCI are biologically active and react strongly with serum raised against PCI from potato. A method for the purification of rePCI to homogeneity has been developed. The purified rePCI shows a Ki for carboxypeptidase A within the range of the natural PCI-IIa (1.5-2.7 nM). These results indicate that both rePCI + 10 and rePCI are properly folded and that their three disulfide bridges are correctly formed. Together with previous reports, our results show that fusion to a secretion signal peptide is an effective way of producing small proteins containing disulfide bridges in a biologically active form.

Amino Acid Sequence↗