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James C A Bardwell - One of the best experts on this subject based on the ideXlab platform.
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2005, Mutational analysis of the disulfide catalysts DsbA and DsbB
2015Co-Authors: Jacqueline Tan, James C A BardwellAbstract:In prokaryotes, disulfides are generated by the DsbA-DsbB system. DsbB functions to generate disulfides by quinone reduction. These disulfides are passed to the DsbA Protein and then to folding Proteins. To investigate the DsbA-DsbB catalytic system, we performed an in vivo selection for chromosomal DsbA and dsbB mutants. We rediscovered many residues previously shown to be important for the activity of these Proteins. In addition, we obtained one novel DsbA mutant (M153R) and four novel DsbB mutants (L43P, H91Y, R133C, and L146R). We also mutated residues that are highly conserved within the DsbB family in an effort to identify residues important for DsbB function. We found classes of mutants that specifically affect the apparent Km of DsbB for either DsbA or quinones, suggesting that quinone and DsbA may interact with different regions of the DsbB Protein. Our results are consistent with the interpretation that the residues Q33 and Y46 of DsbB interact with DsbA, Q95 and R48 interact with quinones, and that residue M153 of DsbA interacts with DsbB. All of these interactions could be due to direct amino acid interactions or could be indirect through, for instance, their effect on Protein structure. In addition, we find that the DsbB H91Y mutant severely affects the kcat of the reaction between DsbA and DsbB and that the DsbB L43P mutant is inactive, suggesting that both L43 and H91 are important for the activity of DsbB. These experiments help to better define the residues important for the function of these two Protein-folding catalysts
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oxidative Protein folding in bacteria
Molecular Microbiology, 2002Co-Authors: Jeanfrancois Collet, James C A BardwellAbstract:Summary Ten years ago it was thought that disulphide bond for- mation in prokaryotes occurred spontaneously. Now two pathways involved in disulphide bond formation have been well characterized, the oxidative pathway, which is responsible for the formation of disulphides, and the isomerization pathway, which shuffles in- correctly formed disulphides. Disulphide bonds are donated directly to unfolded polypeptides by the DsbA Protein; DsbA is reoxidized by DsbB. DsbB gen- erates disulphides de novo from oxidized quinones. These quinones are reoxidized by the electron trans- port chain, showing that disulphide bond formation is actually driven by electron transport. Disulphide isomerization requires that incorrect disulphides be attacked using a reduced catalyst, followed by the redonation of the disulphide, allowing alterna- tive disulphide pairing. Two isomerases exist in Escherichia coli, DsbC and DsbG. The membrane Protein DsbD maintains these disulphide isomerases in their reduced and thereby active form. DsbD is kept reduced by cytosolic thioredoxin in an NADPH- dependent reaction.
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crystal structure of the DsbA Protein required for disulphide bond formation in vivo
Nature, 1993Co-Authors: Jennifer L Martin, James C A Bardwell, John KuriyanAbstract:Proteins that contain disulphide bonds are often slow to fold in vitro because the oxidation and correct pairing of the cysteine residues is rate limiting. The folding of such Proteins is greatly accelerated in Escherichia coli by DsbA, but the mechanism of this rate enhancement is not well understood. Here we report the crystal structure of oxidized DsbA and show that it resembles closely the ubiquitous redox Protein thioredoxin, despite very low sequence similarity. An important difference, however, is the presence of another domain which forms a cap over the thioredoxin-like active site of DsbA. The redox-active disulphide bond, which is responsible for the oxidation of substrates, is thus at a domain interface and is surrounded by grooves and exposed hydrophobic side chains. These features suggest that DsbA might act by binding to partially folded polypeptide chains before oxidation of cysteine residues.
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identification of a Protein required for disulfide bond formation in vivo
Cell, 1991Co-Authors: James C A Bardwell, Karen Mcgovern, Jon BeckwithAbstract:Abstract We describe a mutation ( DsbA ) that renders Escherichia coli severely defective in disulfide bond formation. In DsbA mutant cells, pulse-labeled β-lactamase, alkaline phosphatase, and OmpA are secreted but largely lack disulfide bonds. These disulfideless Proteins may represent in vivo folding intermediates, since they are protease sensitive and chase slowly into stable oxidized forms. The DsbA gene codes for a 21,000 M r periplasmic Protein containing the sequence cys-pro-his-cys, which resembles the active sites of certain disulfide oxidoreductases. The purified DsbA Protein is capable of reducing the disulfide bonds of insulin, an activity that it shares with these disulfide oxidoreductases. Our results suggest that disulfide bond formation is facilitated by DsbA in vivo.
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identification of a Protein required for disulfide bond formation in vivo
Cell, 1991Co-Authors: James C A Bardwell, Karen Mcgovern, Jon BeckwithAbstract:We describe a mutation (DsbA) that renders Escherichia coli severely defective in disulfide bond formation. In DsbA mutant cells, pulse-labeled beta-lactamase, alkaline phosphatase, and OmpA are secreted but largely lack disulfide bonds. These disulfideless Proteins may represent in vivo folding intermediates, since they are protease sensitive and chase slowly into stable oxidized forms. The DsbA gene codes for a 21,000 Mr periplasmic Protein containing the sequence cys-pro-his-cys, which resembles the active sites of certain disulfide oxidoreductases. The purified DsbA Protein is capable of reducing the disulfide bonds of insulin, an activity that it shares with these disulfide oxidoreductases. Our results suggest that disulfide bond formation is facilitated by DsbA in vivo.
Jon Beckwith - One of the best experts on this subject based on the ideXlab platform.
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the DsbA signal sequence directs efficient cotranslational export of passenger Proteins to the escherichia coli periplasm via the signal recognition particle pathway
Journal of Bacteriology, 2003Co-Authors: Clark Schierle, Carol A Kumamoto, Mehmet Berkmen, Damon Huber, Dana Boyd, Jon BeckwithAbstract:The Escherichia coli cytoplasmic Protein thioredoxin 1 can be efficiently exported to the periplasmic space by the signal sequence of the DsbA Protein (DsbAss) but not by the signal sequence of alkaline phosphatase (PhoA) or maltose binding Protein (MBP). Using mutations of the signal recognition particle (SRP) pathway, we found that DsbAss directs thioredoxin 1 to the SRP export pathway. When DsbAss is fused to MBP, MBP also is directed to the SRP pathway. We show directly that the DsbAss-promoted export of MBP is largely cotranslational, in contrast to the mode of MBP export when the native signal sequence is utilized. However, both the export of thioredoxin 1 by DsbAss and the export of DsbA itself are quite sensitive to even the slight inhibition of SecA. These results suggest that SecA may be essential for both the slow posttranslational pathway and the SRP-dependent cotranslational pathway. Finally, probably because of its rapid folding in the cytoplasm, thioredoxin provides, along with gene fusion approaches, a sensitive assay system for signal sequences that utilize the SRP pathway.
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identification of a Protein required for disulfide bond formation in vivo
Cell, 1991Co-Authors: James C A Bardwell, Karen Mcgovern, Jon BeckwithAbstract:Abstract We describe a mutation ( DsbA ) that renders Escherichia coli severely defective in disulfide bond formation. In DsbA mutant cells, pulse-labeled β-lactamase, alkaline phosphatase, and OmpA are secreted but largely lack disulfide bonds. These disulfideless Proteins may represent in vivo folding intermediates, since they are protease sensitive and chase slowly into stable oxidized forms. The DsbA gene codes for a 21,000 M r periplasmic Protein containing the sequence cys-pro-his-cys, which resembles the active sites of certain disulfide oxidoreductases. The purified DsbA Protein is capable of reducing the disulfide bonds of insulin, an activity that it shares with these disulfide oxidoreductases. Our results suggest that disulfide bond formation is facilitated by DsbA in vivo.
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identification of a Protein required for disulfide bond formation in vivo
Cell, 1991Co-Authors: James C A Bardwell, Karen Mcgovern, Jon BeckwithAbstract:We describe a mutation (DsbA) that renders Escherichia coli severely defective in disulfide bond formation. In DsbA mutant cells, pulse-labeled beta-lactamase, alkaline phosphatase, and OmpA are secreted but largely lack disulfide bonds. These disulfideless Proteins may represent in vivo folding intermediates, since they are protease sensitive and chase slowly into stable oxidized forms. The DsbA gene codes for a 21,000 Mr periplasmic Protein containing the sequence cys-pro-his-cys, which resembles the active sites of certain disulfide oxidoreductases. The purified DsbA Protein is capable of reducing the disulfide bonds of insulin, an activity that it shares with these disulfide oxidoreductases. Our results suggest that disulfide bond formation is facilitated by DsbA in vivo.
Tracy Raivio - One of the best experts on this subject based on the ideXlab platform.
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envelope stress responses and gram negative bacterial pathogenesis
Molecular Microbiology, 2005Co-Authors: Tracy RaivioAbstract:: The sigma(E), Cpx and Bae envelope stress responses of Escherichia coli are involved in the maintenance, adaptation and protection of the bacterial envelope in response to a variety of stressors. Recent studies indicate that the Cpx and sigma(E) stress responses exist in many Gram-negative bacterial pathogens. The envelope is of particular importance to these organisms because most virulence determinants reside in, or must transit through, this cellular compartment. The Cpx system has been implicated in expression of pili, type IV secretion systems and key virulence regulators, while the sigma(E) pathway has been shown to be critical for protection from oxidative stress and intracellular survival. Homologues of the sigma(E)- and Cpx-regulated protease DegP are essential for full virulence in numerous pathogens, and, like sigma(E), DegP appears to confer resistance to oxidative stress and intracellular survival capacity. Some pathogens contain multiple homologues of the Cpx-regulated, disulphide bond catalyst DsbA Protein, which has been demonstrated to play roles in the expression of secreted virulence determinants, type III secretion systems and pili. This review highlights recent studies that indicate roles for the sigma(E), Cpx and Bae envelope stress responses in Gram-negative bacterial pathogenesis.
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microreview envelope stress responses and gram negative bacterial pathogenesis
Molecular Microbiology, 2005Co-Authors: Tracy RaivioAbstract:Summary The σE, Cpx and Bae envelope stress responses of Escherichia coli are involved in the maintenance, adaptation and protection of the bacterial envelope in response to a variety of stressors. Recent studies indicate that the Cpx and σE stress responses exist in many Gram-negative bacterial pathogens. The envelope is of particular importance to these organisms because most virulence determinants reside in, or must transit through, this cellular compartment. The Cpx system has been implicated in expression of pili, type IV secretion systems and key virulence regulators, while the σE pathway has been shown to be critical for protection from oxidative stress and intracellular survival. Homologues of the σE– and Cpx-regulated protease DegP are essential for full virulence in numerous pathogens, and, like σE, DegP appears to confer resistance to oxidative stress and intracellular survival capacity. Some pathogens contain multiple homologues of the Cpx-regulated, disulphide bond catalyst DsbA Protein, which has been demonstrated to play roles in the expression of secreted virulence determinants, type III secretion systems and pili. This review highlights recent studies that indicate roles for the σE, Cpx and Bae envelope stress responses in Gram-negative bacterial pathogenesis.
Jennifer L Martin - One of the best experts on this subject based on the ideXlab platform.
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Biochemical characterization of CtDsbA.
2016Co-Authors: Signe Christensen, Jennifer L Martin, Morten K. Grøftehauge, Karl Byriel, Wilhelmina M. Huston, Emily Furlong, Begoña Heras, Róisín M. McmahonAbstract:A Reduction of insulin (131 μM) was measured as increase in absorbance at 650nm in 0.1mM sodium phosphate buffer, pH 7, 2mM EDTA. The reaction was performed in the absence (■) or presence of 10 μM EcDsbC (●), EcDsbA (♦) or CtDsbA (○). Representative data are shown for the absence and presence of 10 μM EcDsbA. Mean and SD are shown for two biological replicates (three biological replicates for CtDsbA). B 80 nM EcDsbA (▼) and 320 nM CtDsbA (●), MtbDsbA (■) and WpDsbA1 (▲) oxidize a fluorescently labeled Protein in the presence of 2 mM GSSG. GSSG shows only limited oxidizing activity in the absence of a DsbA Protein (■). The buffer only control (○) shows no oxidizing activity. For MtbDsbA, WpDsbA1, EcDsbA and CtDsbA mean and SD of two biological replicates are shown (for each biological replicates four technical replicates was performed.) For the buffer and GSSG only controls, mean and sd for four technical replicates are shown. C Isomerase activity was assessed as the ability to refold scrambled RNAseA. ScRNase (40 μM) was incubated in 0.1 M sodium phosphate buffer pH 7.0, 1 mM EDTA, 10 μM DTT in the absence and presence of 10 μM EcDsbA (■), EcDsbC (○) and CtDsbA (●). RNase activity was monitored as the cleavage of cCMP which leads to an increase in absorbance at 296 nm. Mean and SD for three biological replicates is shown for CtDsbA. EcDsbC and EcDsbA is able to restore ~80% and ~20% of RNaseA activity, which is equivalent to what has been reported previously [8] D Temperature induced unfolding of oxidized (○) and reduced (●) CtDsbA was determined by far UV CD spectroscopy. The thermal unfolding of CtDsbA results in an increase in molar ellipticity at 220 nm showing that the reduced form of CtDsbA is more stable than the oxidized form. Mean and SD are shown for two biological replicates.
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structure of tcpg the DsbA Protein folding catalyst from vibrio cholerae
Journal of Molecular Biology, 1997Co-Authors: Joel A Peek, Eileen Rattigan, Ronald K Taylor, Jennifer L MartinAbstract:The efficient and correct folding of bacterial disulfide bonded Proteins in vivo is dependent upon a class of periplasmic oxidoreductase Proteins called DsbA, after the Escherichia coli enzyme. In the pathogenic bacterium Vibrio cholerae, the DsbA homolog (TcpG) is responsible for the folding, maturation and secretion of virulence factors. Mutants in which the tcpg gene has been inactivated are avirulent; they no longer produce functional colonisation pill and they no longer secrete cholera toxin. TcpG is thus a suitable target for inhibitors that could counteract the virulence of this organism, thereby preventing the symptoms of cholera. The crystal structure of oxidized TcpG (refined at a resolution of 2.1 Angstrom) serves as a starting point for the rational design of such inhibitors. As expected, TcpG has the same fold as E. coli DsbA, with which it shares similar to 40% sequence identity. Ln addition, the characteristic surface features of DsbA are present in TcpG, supporting the notion that these features play a functional role. While the overall architecture of TcpG and DsbA is similar and the surface features are retained in TcpG, there are significant differences. For example, the kinked active site helix results from a three-residue loop in DsbA, but is caused by a proline in TcpG (making TcpG more similar to thioredoxin in this respect). Furthermore, the proposed peptide binding groove of TcpG is substantially shortened compared with that of DsbA due to a six-residue deletion. Also, the hydrophobic pocket of TcpG is more shallow and the acidic patch is much less extensive than that of E. coli DsbA. The identification of the structural and surface features that are retained or are divergent in TcpG provides a useful assessment of their functional importance in these Protein folding catalysts and is an important prerequisite for the design of TcpG inhibitors. (C) 1997 Academic Press Limited.
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crystal structure of the DsbA Protein required for disulphide bond formation in vivo
Nature, 1993Co-Authors: Jennifer L Martin, James C A Bardwell, John KuriyanAbstract:Proteins that contain disulphide bonds are often slow to fold in vitro because the oxidation and correct pairing of the cysteine residues is rate limiting. The folding of such Proteins is greatly accelerated in Escherichia coli by DsbA, but the mechanism of this rate enhancement is not well understood. Here we report the crystal structure of oxidized DsbA and show that it resembles closely the ubiquitous redox Protein thioredoxin, despite very low sequence similarity. An important difference, however, is the presence of another domain which forms a cap over the thioredoxin-like active site of DsbA. The redox-active disulphide bond, which is responsible for the oxidation of substrates, is thus at a domain interface and is surrounded by grooves and exposed hydrophobic side chains. These features suggest that DsbA might act by binding to partially folded polypeptide chains before oxidation of cysteine residues.
Timothy R Hirst - One of the best experts on this subject based on the ideXlab platform.
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a homologue of the escherichia coli DsbA Protein involved in disulphide bond formation is required for enterotoxin biogenesis in vibrio cholerae
Molecular Microbiology, 1992Co-Authors: Helen M Webb, Timothy R HirstAbstract:A strain of Vibrio cholerae, which had been engineered to express high levels of the non-toxic B subunit (EtxB) of Escherichia coli heat-labile enterotoxin, was subjected to transposon (TnphoA) mutagenesis. Two chromosomal TnphoA insertion mutations of the strain were isolated that showed a severe defect in the amount of EtxB produced. The loci disrupted by TnphoA in the two mutant derivatives were cloned and sequenced, and this revealed that the transposon had inserted at different sites in the same gene. The open reading frame of the gene predicts a 200-amino-acid exported Protein, with a Cys-X-X-Cys motif characteristic of thioredoxin, Protein disulphide isomerase, and DsbA (a periplasmic Protein required for disulphide bond formation in E. coli). The V. cholerae Protein exhibited 40% identity with the DsbA Protein of E. coli, including 90% identity in the region of the active-site motif. Introduction of a plasmid encoding E. coli DsbA into the V. cholerae TnphoA derivatives was found to restore enterotoxin formation, whilst expression of Etx or EtxB in a DsbA mutant of E. coli confirmed that DsbA is required for enterotoxin formation in E. coli. These results suggest that, since each EtxB subunit contains a single intramolecular disulphide bond, a transient intermolecular interaction with DsbA occurs during toxin subunit folding which catalyses formation of the disulphide in vivo.
