Wilson Muse - Academia.edu (original) (raw)
Papers by Wilson Muse
Journal of Bacteriology, Mar 1, 1998
The nitrogen assimilation control gene, nac, was detected in Escherichia coli but not in Salmonel... more The nitrogen assimilation control gene, nac, was detected in Escherichia coli but not in Salmonella typhimurium by Southern blotting, using a probe from the Klebsiella aerogenes nac (nac K) gene. The E. coli nac gene (nac E) was isolated from a cosmid clone by complementation of a nac mutation in K. aerogenes. nac E was fully functional in this complementation assay. DNA sequence analysis showed considerable divergence between nac E and nac K , with a predicted amino acid sequence identity of only 79% and most of the divergence in the C-terminal half of the protein sequence. The total predicted size of NAC E is 305 amino acids, the same as for NAC K. A null mutation, nac-28, was generated by reverse genetics. Mutants bearing nac-28 have a variety of phenotypes related to nitrogen metabolism, including slower growth on cytosine, faster growth on arginine, and suppression of the failure of an Ntr-constitutive mutant to grow with serine as sole nitrogen source. In addition to a loss of nitrogen regulation of histidase formation, nac-28 mutants also showed a loss of a weak repression of glutamate dehydrogenase formation. This repression was unexpected because it is balanced by a NACindependent activation of glutamate dehydrogenase formation during nitrogen-limited growth. Attempts to purify NAC E by using methods established for NAC K failed, and NAC E appears to be degraded with a half-life at 30°C as short as 15 min during inhibition of protein synthesis.
The nac (nitrogen assimilation control) gene isolated from Escherichia coli complemented nac muta... more The nac (nitrogen assimilation control) gene isolated from Escherichia coli complemented nac mutants in Klebsiella aerogenes. The nucleotide sequence of the gene was 70% identical to the nac gene from K. aerogenes. E. coli mutants deleted for the nac gene failed to activate a heterologous hutH gene from K. aerogenes. Primer extension experiments showed that nac transcription initiated at a unique adenine residue and was regulated by nitrogen availability in an NTR-dependent manner. NAC regulates two targets in E. coli. NAC binds the codBA operon promoter region and activates transcription both in vivo and in vitro. NAC binds at -59 bp from the start of transcription in this promoter, in contrast to the binding at -64 bp seen at NAC activated operons in Klebsiella aerogenes. Mutations in the NAC binding site of the codBA promoter that eliminated binding also eliminated NAC regulation. A second target, the gltBDF operon (which encodes glutamate synthase) was repressed by NAC. NAC binds weakly to the gltBDF promoter but the repression seen here may be indirect as NAC regulation persisted after deletion of the apparent NAC binding site. NAC$\sp{E}$ was insoluble in salt concentrations from 25 mM to 1.2 M. N-terminal fusions to a 56 amino acid leader or the maltose binding protein (MBP) resulted in soluble but transcriptionally inactive proteins still able to bind DNA. MBP-NAC fusions activated transcription following removal of the MBP tag, but degraded quickly. Addition of a 6 histidine tag to the C-terminus of NAC$\sp{E}$ increased activity in vivo, but not solubility in vitro. Carboxy terminal deletions of the NAC protein retaining as few as 99 N-terminal amino acids (of 305) retained significant in vivo activity. The properties of NAC proteins retaining 100, 120 and 129 N-terminal amino acids suggest that the N-terminal 100 amino acids of NAC contain domains for DNA binding, activation and dimerization that are locked in an activation competent conformation.Ph.D.Biological SciencesGeneticsMicrobiologyMolecular biologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/130077/2/9712046.pd
Includes bibliographical references (pages [140]-155)The techniques that would lead to a physical... more Includes bibliographical references (pages [140]-155)The techniques that would lead to a physical restriction map of B. megaterium were investigated. Methods of isolating whole chromosomal DNA were determined and optimized, and parameters for separation of large DNA molecules using Transverse Alternating Field Electrophoresis were established. Several rare cutting enzymes were tested on intact B. megaterium genomic DNA. Only two were found that could be used for physical mapping; the enzymes NotI and SfiI yielded 25 fragments and 7-8 fragments, respectively. Several new Tn917 and Tn917- 1acZ-cat insertional mutants were also isolated, including a unique tyr mutant. Using a Tn917 probe to hybridize with the DNA from the insertional mutants digested with NotI and SfiI, a partial map was formed. Six previously unmapped genetic loci were mapped within at least 0.1-5% of their actual physical distance on the chromosome. Additional mapping strategies were found to be unsuccessful, includi...
Molecular BioSystems, 2008
This article cites 26 articles, 18 of which can be accessed free
Abstract: The identification of new antibacterial targets is urgently needed to address multidrug... more Abstract: The identification of new antibacterial targets is urgently needed to address multidrug resistant and latent tuberculosis infection. Sulfur metabolic pathways are essential for survival and the expression of virulence in many pathogenic bacteria, including Mycobacterium tuberculosis. In addition, microbial sulfur metabolic pathways are largely absent in humans and therefore, represent unique targets for therapeutic intervention. In this review, we summarize our current understanding of the enzymes associated with the production of sulfated and reduced sulfur-containing metabolites in Mycobacteria. Small molecule inhibitors of these catalysts represent valuable chemical tools that can be used to investigate the role of sulfur metabolism throughout the Mycobacterial lifecycle and may also represent new leads for drug development. In this light, we also summarize recent progress in the development of inhibitors of sulfur metabolism enzymes.
This article cites 16 articles, 11 of which can be accessed free
This article cites 39 articles, 23 of which can be accessed free at:
The nitrogen assimilation control gene, nac, was detected in Escherichia coli but not in Salmonel... more The nitrogen assimilation control gene, nac, was detected in Escherichia coli but not in Salmonella typhimurium by Southern blotting, using a probe from the Klebsiella aerogenes nac (nac K) gene. The E. coli nac gene (nac E) was isolated from a cosmid clone by complementation of a nac mutation in K. aerogenes. nac E was fully functional in this complementation assay. DNA sequence analysis showed considerable divergence between nac E and nac K , with a predicted amino acid sequence identity of only 79% and most of the divergence in the C-terminal half of the protein sequence. The total predicted size of NAC E is 305 amino acids, the same as for NAC K. A null mutation, nac-28, was generated by reverse genetics. Mutants bearing nac-28 have a variety of phenotypes related to nitrogen metabolism, including slower growth on cytosine, faster growth on arginine, and suppression of the failure of an Ntr-constitutive mutant to grow with serine as sole nitrogen source. In addition to a loss of nitrogen regulation of histidase formation, nac-28 mutants also showed a loss of a weak repression of glutamate dehydrogenase formation. This repression was unexpected because it is balanced by a NACindependent activation of glutamate dehydrogenase formation during nitrogen-limited growth. Attempts to purify NAC E by using methods established for NAC K failed, and NAC E appears to be degraded with a half-life at 30°C as short as 15 min during inhibition of protein synthesis.
Journal of bacteriology, 1999
The nitrogen assimilation control protein (NAC) from Klebsiella aerogenes or Escherichia coli (NA... more The nitrogen assimilation control protein (NAC) from Klebsiella aerogenes or Escherichia coli (NACK or NACE, respectively) is a transcriptional regulator that is both necessary and sufficient to activate transcription of the histidine utilization (hut) operon and to repress transcription of the glutamate dehydrogenase (gdh) operon in K. aerogenes. Truncated NAC polypeptides, generated by the introduction of stop codons within the nac open reading frame, were tested for the ability to activate hut and repress gdh in vivo. Most of the NACK and NACE fragments with 100 or more amino acids (wild-type NACK and NACE both have 305 amino acids) were functional in activating hut and repressing gdh expression in vivo. Full-length NACK and NACE were isolated as chimeric proteins with the maltose-binding protein (MBP). NACK and NACE released from such chimeras were able to activate hut transcription in a purified system in vitro, as were NACK129 and NACE100 (a NACK fragment of 129 amino acids an...
Journal of bacteriology, 1998
The nitrogen assimilation control gene, nac, was detected in Escherichia coli but not in Salmonel... more The nitrogen assimilation control gene, nac, was detected in Escherichia coli but not in Salmonella typhimurium by Southern blotting, using a probe from the Klebsiella aerogenes nac (nacK) gene. The E. coli nac gene (nacE) was isolated from a cosmid clone by complementation of a nac mutation in K. aerogenes. nacE was fully functional in this complementation assay. DNA sequence analysis showed considerable divergence between nacE and nacK, with a predicted amino acid sequence identity of only 79% and most of the divergence in the C-terminal half of the protein sequence. The total predicted size of NAC(E) is 305 amino acids, the same as for NAC(K). A null mutation, nac-28, was generated by reverse genetics. Mutants bearing nac-28 have a variety of phenotypes related to nitrogen metabolism, including slower growth on cytosine, faster growth on arginine, and suppression of the failure of an Ntr-constitutive mutant to grow with serine as sole nitrogen source. In addition to a loss of nit...
Journal of bacteriology, 1992
Oxidation of the thiol functional group in cysteine (Cys-SH) to sulfenic (Cys-SOH), sulfinic (Cys... more Oxidation of the thiol functional group in cysteine (Cys-SH) to sulfenic (Cys-SOH), sulfinic (Cys-SO2H) and sulfonic acids (Cys-SO3H) is emerging as an important post-translational modification that can activate or deactivate the function of many proteins. Changes in thiol oxidation state have been implicated in a wide variety of cellular processes and correlate with disease states but are difficult to monitor
Molecular BioSystems, 2008
Oxidation of the thiol functional group in cysteine (Cys-SH) to sulfenic (Cys-SOH), sulfinic (Cys... more Oxidation of the thiol functional group in cysteine (Cys-SH) to sulfenic (Cys-SOH), sulfinic (Cys-SO 2 H) and sulfonic acids (Cys-SO 3 H) is emerging as an important post-translational modification that can activate or deactivate the function of many proteins. Changes in thiol oxidation state have been implicated in a wide variety of cellular processes and correlate with disease states but are difficult to monitor in a physiological setting because of a lack of experimental tools. Here, we describe a method that enables live cell labeling of sulfenic acidmodified proteins. For this approach, we have synthesized the probe DAz-1, which is chemically selective for sulfenic acids and cell permeable. In addition, DAz-1 contains an azide chemical handle that can be selectively detected with phosphine reagents via the Staudinger ligation for identification, enrichment and visualization of modified proteins. Through a combination of biochemical, mass spectrometry and immunoblot approaches we characterize the reactivity of DAz-1 and highlight its utility for detecting protein sulfenic acids directly in mammalian cells. This novel method to isolate and identify sulfenic acid-modified proteins should be of widespread utility for elucidating signaling pathways and regulatory mechanisms that involve oxidation of cysteine residues. † Electronic supplementary information (ESI) available: Synthesis, protease activity, cell viability plots and Western blots. See
Journal of Biological Chemistry, 1998
Disulfide bonds are important for the structure and stability of many proteins. In prokaryotes th... more Disulfide bonds are important for the structure and stability of many proteins. In prokaryotes their formation is catalyzed by the Dsb proteins. The DsbA protein acts as a direct donor of disulfides to newly synthesized periplasmic proteins. Genetic evidence suggests that a second protein called DsbB acts to specifically reoxidize DsbA. Here we demonstrate the direct reoxidation of DsbA by DsbB. We have developed a fluorescence assay that allows us to directly follow the reoxidation of DsbA. We show that membranes containing catalytic amounts of DsbB can rapidly reoxidize DsbA to completion. The reaction strongly depends on the presence of oxygen, implying that oxygen serves as the final electron acceptor for this disulfide bond formation reaction. Membranes from a dsbB null mutant display no DsbA reoxidation activity. The ability of DsbB to reoxidize DsbA fits Michaelis-Menten behavior with DsbA acting as a high affinity substrate for DsbB with a K m ؍ 10 M. The in vitro reconstitution described here is the first biochemical analysis of DsbB and allows us to study the major pathway of disulfide bond formation in Escherichia coli.
Journal of Bacteriology, 2003
Transcription of the cytosine deaminase (codBA) operon of Escherichia coli is regulated by nitrog... more Transcription of the cytosine deaminase (codBA) operon of Escherichia coli is regulated by nitrogen, with about three times more codBA expression in cells grown in nitrogen-limiting medium than in nitrogen-excess medium. -Galactosidase expression from codBp-lacZ operon fusions showed that the nitrogen assimilation control protein NAC was necessary for this regulation. In vitro transcription from the codBA promoter with purified RNA polymerase was stimulated by the addition of purified NAC, confirming that no other factors are required. Gel mobility shifts and DNase I footprints showed that NAC binds to a site centered at position ؊59 relative to the start site of transcription and that mutants that cannot bind NAC there cannot activate transcription. When a longer promoter region (positions ؊120 to ؉67) was used, a double footprint was seen with a second 26-bp footprint separated from the first by a hypersensitive site. When a shorter fragment was used (positions ؊83 to ؉67), only the primary footprint was seen. Nevertheless, both the shorter and longer fragments showed NAC-mediated regulation in vivo. Cytosine deaminase expression in Klebsiella pneumoniae was also regulated by nitrogen in a NAC-dependent manner. K. pneumoniae differs from E. coli in having two cytosine deaminase genes, an intervening open reading frame between the codB and codA orthologs, and a different response to hypoxanthine which increased cod expression in K. pneumoniae but decreased it in E. coli.
International Journal of Mass Spectrometry, 2011
Mycothiol (MSH), the primary low-molecular weight thiol produced in mycobacteria, acts to protect... more Mycothiol (MSH), the primary low-molecular weight thiol produced in mycobacteria, acts to protect the cell from oxidative stress and to maintain redox homeostasis, notably in the pathogenic Mycobacterium tuberculosis in the course of human infection. The mycothiol disulfide reductase (Mtr) enzyme reduces the oxidized form of mycothiol, mycothione (MSSM), back to MSH, however its role in bacterial viability is not clear. In this study, we sought to determine the MSH levels of wild-type (WT) and Mtr mutant mycobacteria during oxidative stress. We describe a rapid method for the relative quantification of MSH using high-sensitivity mass spectrometry (MS) with selected ion monitoring (SIM). This method uses only minimal sample cleanup, and does not require advanced chromatographic equipment or fluorescent compounds. MSH levels decreased in the Mtr mutant only upon treatment with peroxide, and the results were consistent between our method and previously-described thiol quantification methods. Our results indicate that our MS-based method is a useful, high-throughput alternative tool for the quantification of MSH from mycobacteria.
Cell, 1999
Disulfide bond formation is catalyzed in vivo by DsbA and DsbB. Here we reconstitute this oxidati... more Disulfide bond formation is catalyzed in vivo by DsbA and DsbB. Here we reconstitute this oxidative folding system using purified components. We have found the sources of oxidative power for protein folding and show how disulfide bond formation is linked to cellular metabolism. We find that disulfide bond formation and the electron transport chain are directly coupled. DsbB uses quinones as electron acceptors, allowing various choices for electron transport to support disulfide bond formation. Electrons flow via cytochrome bo oxidase to oxygen under aerobic conditions or via cytochrome bd oxidase under partially anaerobic conditions. Under truly anaerobic conditions, menaquinone shuttles electrons to alternate final electron acceptors such as fumarate. This flexibility reflects the vital nature of the disulfide catalytic system.
Journal of Bacteriology, Mar 1, 1998
The nitrogen assimilation control gene, nac, was detected in Escherichia coli but not in Salmonel... more The nitrogen assimilation control gene, nac, was detected in Escherichia coli but not in Salmonella typhimurium by Southern blotting, using a probe from the Klebsiella aerogenes nac (nac K) gene. The E. coli nac gene (nac E) was isolated from a cosmid clone by complementation of a nac mutation in K. aerogenes. nac E was fully functional in this complementation assay. DNA sequence analysis showed considerable divergence between nac E and nac K , with a predicted amino acid sequence identity of only 79% and most of the divergence in the C-terminal half of the protein sequence. The total predicted size of NAC E is 305 amino acids, the same as for NAC K. A null mutation, nac-28, was generated by reverse genetics. Mutants bearing nac-28 have a variety of phenotypes related to nitrogen metabolism, including slower growth on cytosine, faster growth on arginine, and suppression of the failure of an Ntr-constitutive mutant to grow with serine as sole nitrogen source. In addition to a loss of nitrogen regulation of histidase formation, nac-28 mutants also showed a loss of a weak repression of glutamate dehydrogenase formation. This repression was unexpected because it is balanced by a NACindependent activation of glutamate dehydrogenase formation during nitrogen-limited growth. Attempts to purify NAC E by using methods established for NAC K failed, and NAC E appears to be degraded with a half-life at 30°C as short as 15 min during inhibition of protein synthesis.
The nac (nitrogen assimilation control) gene isolated from Escherichia coli complemented nac muta... more The nac (nitrogen assimilation control) gene isolated from Escherichia coli complemented nac mutants in Klebsiella aerogenes. The nucleotide sequence of the gene was 70% identical to the nac gene from K. aerogenes. E. coli mutants deleted for the nac gene failed to activate a heterologous hutH gene from K. aerogenes. Primer extension experiments showed that nac transcription initiated at a unique adenine residue and was regulated by nitrogen availability in an NTR-dependent manner. NAC regulates two targets in E. coli. NAC binds the codBA operon promoter region and activates transcription both in vivo and in vitro. NAC binds at -59 bp from the start of transcription in this promoter, in contrast to the binding at -64 bp seen at NAC activated operons in Klebsiella aerogenes. Mutations in the NAC binding site of the codBA promoter that eliminated binding also eliminated NAC regulation. A second target, the gltBDF operon (which encodes glutamate synthase) was repressed by NAC. NAC binds weakly to the gltBDF promoter but the repression seen here may be indirect as NAC regulation persisted after deletion of the apparent NAC binding site. NAC$\sp{E}$ was insoluble in salt concentrations from 25 mM to 1.2 M. N-terminal fusions to a 56 amino acid leader or the maltose binding protein (MBP) resulted in soluble but transcriptionally inactive proteins still able to bind DNA. MBP-NAC fusions activated transcription following removal of the MBP tag, but degraded quickly. Addition of a 6 histidine tag to the C-terminus of NAC$\sp{E}$ increased activity in vivo, but not solubility in vitro. Carboxy terminal deletions of the NAC protein retaining as few as 99 N-terminal amino acids (of 305) retained significant in vivo activity. The properties of NAC proteins retaining 100, 120 and 129 N-terminal amino acids suggest that the N-terminal 100 amino acids of NAC contain domains for DNA binding, activation and dimerization that are locked in an activation competent conformation.Ph.D.Biological SciencesGeneticsMicrobiologyMolecular biologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/130077/2/9712046.pd
Includes bibliographical references (pages [140]-155)The techniques that would lead to a physical... more Includes bibliographical references (pages [140]-155)The techniques that would lead to a physical restriction map of B. megaterium were investigated. Methods of isolating whole chromosomal DNA were determined and optimized, and parameters for separation of large DNA molecules using Transverse Alternating Field Electrophoresis were established. Several rare cutting enzymes were tested on intact B. megaterium genomic DNA. Only two were found that could be used for physical mapping; the enzymes NotI and SfiI yielded 25 fragments and 7-8 fragments, respectively. Several new Tn917 and Tn917- 1acZ-cat insertional mutants were also isolated, including a unique tyr mutant. Using a Tn917 probe to hybridize with the DNA from the insertional mutants digested with NotI and SfiI, a partial map was formed. Six previously unmapped genetic loci were mapped within at least 0.1-5% of their actual physical distance on the chromosome. Additional mapping strategies were found to be unsuccessful, includi...
Molecular BioSystems, 2008
This article cites 26 articles, 18 of which can be accessed free
Abstract: The identification of new antibacterial targets is urgently needed to address multidrug... more Abstract: The identification of new antibacterial targets is urgently needed to address multidrug resistant and latent tuberculosis infection. Sulfur metabolic pathways are essential for survival and the expression of virulence in many pathogenic bacteria, including Mycobacterium tuberculosis. In addition, microbial sulfur metabolic pathways are largely absent in humans and therefore, represent unique targets for therapeutic intervention. In this review, we summarize our current understanding of the enzymes associated with the production of sulfated and reduced sulfur-containing metabolites in Mycobacteria. Small molecule inhibitors of these catalysts represent valuable chemical tools that can be used to investigate the role of sulfur metabolism throughout the Mycobacterial lifecycle and may also represent new leads for drug development. In this light, we also summarize recent progress in the development of inhibitors of sulfur metabolism enzymes.
This article cites 16 articles, 11 of which can be accessed free
This article cites 39 articles, 23 of which can be accessed free at:
The nitrogen assimilation control gene, nac, was detected in Escherichia coli but not in Salmonel... more The nitrogen assimilation control gene, nac, was detected in Escherichia coli but not in Salmonella typhimurium by Southern blotting, using a probe from the Klebsiella aerogenes nac (nac K) gene. The E. coli nac gene (nac E) was isolated from a cosmid clone by complementation of a nac mutation in K. aerogenes. nac E was fully functional in this complementation assay. DNA sequence analysis showed considerable divergence between nac E and nac K , with a predicted amino acid sequence identity of only 79% and most of the divergence in the C-terminal half of the protein sequence. The total predicted size of NAC E is 305 amino acids, the same as for NAC K. A null mutation, nac-28, was generated by reverse genetics. Mutants bearing nac-28 have a variety of phenotypes related to nitrogen metabolism, including slower growth on cytosine, faster growth on arginine, and suppression of the failure of an Ntr-constitutive mutant to grow with serine as sole nitrogen source. In addition to a loss of nitrogen regulation of histidase formation, nac-28 mutants also showed a loss of a weak repression of glutamate dehydrogenase formation. This repression was unexpected because it is balanced by a NACindependent activation of glutamate dehydrogenase formation during nitrogen-limited growth. Attempts to purify NAC E by using methods established for NAC K failed, and NAC E appears to be degraded with a half-life at 30°C as short as 15 min during inhibition of protein synthesis.
Journal of bacteriology, 1999
The nitrogen assimilation control protein (NAC) from Klebsiella aerogenes or Escherichia coli (NA... more The nitrogen assimilation control protein (NAC) from Klebsiella aerogenes or Escherichia coli (NACK or NACE, respectively) is a transcriptional regulator that is both necessary and sufficient to activate transcription of the histidine utilization (hut) operon and to repress transcription of the glutamate dehydrogenase (gdh) operon in K. aerogenes. Truncated NAC polypeptides, generated by the introduction of stop codons within the nac open reading frame, were tested for the ability to activate hut and repress gdh in vivo. Most of the NACK and NACE fragments with 100 or more amino acids (wild-type NACK and NACE both have 305 amino acids) were functional in activating hut and repressing gdh expression in vivo. Full-length NACK and NACE were isolated as chimeric proteins with the maltose-binding protein (MBP). NACK and NACE released from such chimeras were able to activate hut transcription in a purified system in vitro, as were NACK129 and NACE100 (a NACK fragment of 129 amino acids an...
Journal of bacteriology, 1998
The nitrogen assimilation control gene, nac, was detected in Escherichia coli but not in Salmonel... more The nitrogen assimilation control gene, nac, was detected in Escherichia coli but not in Salmonella typhimurium by Southern blotting, using a probe from the Klebsiella aerogenes nac (nacK) gene. The E. coli nac gene (nacE) was isolated from a cosmid clone by complementation of a nac mutation in K. aerogenes. nacE was fully functional in this complementation assay. DNA sequence analysis showed considerable divergence between nacE and nacK, with a predicted amino acid sequence identity of only 79% and most of the divergence in the C-terminal half of the protein sequence. The total predicted size of NAC(E) is 305 amino acids, the same as for NAC(K). A null mutation, nac-28, was generated by reverse genetics. Mutants bearing nac-28 have a variety of phenotypes related to nitrogen metabolism, including slower growth on cytosine, faster growth on arginine, and suppression of the failure of an Ntr-constitutive mutant to grow with serine as sole nitrogen source. In addition to a loss of nit...
Journal of bacteriology, 1992
Oxidation of the thiol functional group in cysteine (Cys-SH) to sulfenic (Cys-SOH), sulfinic (Cys... more Oxidation of the thiol functional group in cysteine (Cys-SH) to sulfenic (Cys-SOH), sulfinic (Cys-SO2H) and sulfonic acids (Cys-SO3H) is emerging as an important post-translational modification that can activate or deactivate the function of many proteins. Changes in thiol oxidation state have been implicated in a wide variety of cellular processes and correlate with disease states but are difficult to monitor
Molecular BioSystems, 2008
Oxidation of the thiol functional group in cysteine (Cys-SH) to sulfenic (Cys-SOH), sulfinic (Cys... more Oxidation of the thiol functional group in cysteine (Cys-SH) to sulfenic (Cys-SOH), sulfinic (Cys-SO 2 H) and sulfonic acids (Cys-SO 3 H) is emerging as an important post-translational modification that can activate or deactivate the function of many proteins. Changes in thiol oxidation state have been implicated in a wide variety of cellular processes and correlate with disease states but are difficult to monitor in a physiological setting because of a lack of experimental tools. Here, we describe a method that enables live cell labeling of sulfenic acidmodified proteins. For this approach, we have synthesized the probe DAz-1, which is chemically selective for sulfenic acids and cell permeable. In addition, DAz-1 contains an azide chemical handle that can be selectively detected with phosphine reagents via the Staudinger ligation for identification, enrichment and visualization of modified proteins. Through a combination of biochemical, mass spectrometry and immunoblot approaches we characterize the reactivity of DAz-1 and highlight its utility for detecting protein sulfenic acids directly in mammalian cells. This novel method to isolate and identify sulfenic acid-modified proteins should be of widespread utility for elucidating signaling pathways and regulatory mechanisms that involve oxidation of cysteine residues. † Electronic supplementary information (ESI) available: Synthesis, protease activity, cell viability plots and Western blots. See
Journal of Biological Chemistry, 1998
Disulfide bonds are important for the structure and stability of many proteins. In prokaryotes th... more Disulfide bonds are important for the structure and stability of many proteins. In prokaryotes their formation is catalyzed by the Dsb proteins. The DsbA protein acts as a direct donor of disulfides to newly synthesized periplasmic proteins. Genetic evidence suggests that a second protein called DsbB acts to specifically reoxidize DsbA. Here we demonstrate the direct reoxidation of DsbA by DsbB. We have developed a fluorescence assay that allows us to directly follow the reoxidation of DsbA. We show that membranes containing catalytic amounts of DsbB can rapidly reoxidize DsbA to completion. The reaction strongly depends on the presence of oxygen, implying that oxygen serves as the final electron acceptor for this disulfide bond formation reaction. Membranes from a dsbB null mutant display no DsbA reoxidation activity. The ability of DsbB to reoxidize DsbA fits Michaelis-Menten behavior with DsbA acting as a high affinity substrate for DsbB with a K m ؍ 10 M. The in vitro reconstitution described here is the first biochemical analysis of DsbB and allows us to study the major pathway of disulfide bond formation in Escherichia coli.
Journal of Bacteriology, 2003
Transcription of the cytosine deaminase (codBA) operon of Escherichia coli is regulated by nitrog... more Transcription of the cytosine deaminase (codBA) operon of Escherichia coli is regulated by nitrogen, with about three times more codBA expression in cells grown in nitrogen-limiting medium than in nitrogen-excess medium. -Galactosidase expression from codBp-lacZ operon fusions showed that the nitrogen assimilation control protein NAC was necessary for this regulation. In vitro transcription from the codBA promoter with purified RNA polymerase was stimulated by the addition of purified NAC, confirming that no other factors are required. Gel mobility shifts and DNase I footprints showed that NAC binds to a site centered at position ؊59 relative to the start site of transcription and that mutants that cannot bind NAC there cannot activate transcription. When a longer promoter region (positions ؊120 to ؉67) was used, a double footprint was seen with a second 26-bp footprint separated from the first by a hypersensitive site. When a shorter fragment was used (positions ؊83 to ؉67), only the primary footprint was seen. Nevertheless, both the shorter and longer fragments showed NAC-mediated regulation in vivo. Cytosine deaminase expression in Klebsiella pneumoniae was also regulated by nitrogen in a NAC-dependent manner. K. pneumoniae differs from E. coli in having two cytosine deaminase genes, an intervening open reading frame between the codB and codA orthologs, and a different response to hypoxanthine which increased cod expression in K. pneumoniae but decreased it in E. coli.
International Journal of Mass Spectrometry, 2011
Mycothiol (MSH), the primary low-molecular weight thiol produced in mycobacteria, acts to protect... more Mycothiol (MSH), the primary low-molecular weight thiol produced in mycobacteria, acts to protect the cell from oxidative stress and to maintain redox homeostasis, notably in the pathogenic Mycobacterium tuberculosis in the course of human infection. The mycothiol disulfide reductase (Mtr) enzyme reduces the oxidized form of mycothiol, mycothione (MSSM), back to MSH, however its role in bacterial viability is not clear. In this study, we sought to determine the MSH levels of wild-type (WT) and Mtr mutant mycobacteria during oxidative stress. We describe a rapid method for the relative quantification of MSH using high-sensitivity mass spectrometry (MS) with selected ion monitoring (SIM). This method uses only minimal sample cleanup, and does not require advanced chromatographic equipment or fluorescent compounds. MSH levels decreased in the Mtr mutant only upon treatment with peroxide, and the results were consistent between our method and previously-described thiol quantification methods. Our results indicate that our MS-based method is a useful, high-throughput alternative tool for the quantification of MSH from mycobacteria.
Cell, 1999
Disulfide bond formation is catalyzed in vivo by DsbA and DsbB. Here we reconstitute this oxidati... more Disulfide bond formation is catalyzed in vivo by DsbA and DsbB. Here we reconstitute this oxidative folding system using purified components. We have found the sources of oxidative power for protein folding and show how disulfide bond formation is linked to cellular metabolism. We find that disulfide bond formation and the electron transport chain are directly coupled. DsbB uses quinones as electron acceptors, allowing various choices for electron transport to support disulfide bond formation. Electrons flow via cytochrome bo oxidase to oxygen under aerobic conditions or via cytochrome bd oxidase under partially anaerobic conditions. Under truly anaerobic conditions, menaquinone shuttles electrons to alternate final electron acceptors such as fumarate. This flexibility reflects the vital nature of the disulfide catalytic system.