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• W2069968267 abstract "Desulfitobacterium dehalogenans can use chlorinated aromatics including polychlorinated biphenyls as electron acceptors in a process called dehalorespiration. Expression of the cpr gene cluster involved in this process is regulated by CprK, which is a member of the CRP/FNR (cAMP-binding protein/fumarate nitrate reduction regulatory protein) family of helix-turn-helix transcriptional regulators. High affinity interaction of the chlorinated aromatic compound with the effector domain of CprK triggers binding of CprK to an upstream target DNA sequence, which leads to transcriptional activation of the cpr gene cluster. When incubated with oxygen or diamide, CprK undergoes inactivation; subsequent treatment with dithiothreitol restores activity. Using mass spectrometry, this study identifies two classes of redox-active thiol groups that form disulfide bonds upon oxidation. Under oxidative conditions, Cys105, which is conserved in FNR and most other CprK homologs, forms an intramolecular disulfide bond with Cys111, whereas an intermolecular disulfide bond is formed between Cys11 and Cys200. SDS-PAGE and site-directed mutagenesis experiments indicate that the Cys11/Cys200 disulfide bond links two CprK subunits in an inactive dimer. Isothermal calorimetry and intrinsic fluorescence quenching studies show that oxidation does not change the affinity of CprK for the effector. Therefore, reversible redox inactivation is manifested at the level of DNA binding. Our studies reveal a strategy for limiting expression of a redox-sensitive pathway by using a thiol-based redox switch in the transcription factor. Desulfitobacterium dehalogenans can use chlorinated aromatics including polychlorinated biphenyls as electron acceptors in a process called dehalorespiration. Expression of the cpr gene cluster involved in this process is regulated by CprK, which is a member of the CRP/FNR (cAMP-binding protein/fumarate nitrate reduction regulatory protein) family of helix-turn-helix transcriptional regulators. High affinity interaction of the chlorinated aromatic compound with the effector domain of CprK triggers binding of CprK to an upstream target DNA sequence, which leads to transcriptional activation of the cpr gene cluster. When incubated with oxygen or diamide, CprK undergoes inactivation; subsequent treatment with dithiothreitol restores activity. Using mass spectrometry, this study identifies two classes of redox-active thiol groups that form disulfide bonds upon oxidation. Under oxidative conditions, Cys105, which is conserved in FNR and most other CprK homologs, forms an intramolecular disulfide bond with Cys111, whereas an intermolecular disulfide bond is formed between Cys11 and Cys200. SDS-PAGE and site-directed mutagenesis experiments indicate that the Cys11/Cys200 disulfide bond links two CprK subunits in an inactive dimer. Isothermal calorimetry and intrinsic fluorescence quenching studies show that oxidation does not change the affinity of CprK for the effector. Therefore, reversible redox inactivation is manifested at the level of DNA binding. Our studies reveal a strategy for limiting expression of a redox-sensitive pathway by using a thiol-based redox switch in the transcription factor. Desulfitobacterium dehalogenans is a Gram-positive bacterium that can recognize and use chlorinated aromatics, like polychlorinated biphenyls and chlorohydroxyphenyl acetate (CHPA), 4The abbreviations used are: CHPA, 3-chloro-4-hydroxyphenylacetate; EMSA, electrophoretic mobility shift assay; ITC, isothermal titration calorimetry; DTT, dithiothreitol; IAM, iodoacetamide; VP, vinyl pyridine; MS, mass spectrometry. as electron acceptors (1Wiegel J. Wu Q.Z. FEMS Microbiol. Ecol. 2000; 32: 1-15Crossref PubMed Google Scholar, 2Wiegel J. Zhang X. Wu Q. Appl. Environ. Microbiol. 1999; 65: 2217-2221Crossref PubMed Google Scholar) in an energy yielding process called dehalorespiration (3Smidt H. de Vos W.M. Annu. Rev. Microbiol. 2004; 58: 43-73Crossref PubMed Scopus (397) Google Scholar, 4Utkin I. Woese C. Wiegel J. Int. J. Syst. Bacteriol. 1994; 44: 612-619Crossref PubMed Scopus (190) Google Scholar). Dehalorespiration couples the two-electron reduction of the chlorinated aromatic compound and elimination of the chloride group (dehalogenation) to oxidation of membrane-associated electron carriers and to proton translocation, ultimately resulting in ATP generation (3Smidt H. de Vos W.M. Annu. Rev. Microbiol. 2004; 58: 43-73Crossref PubMed Scopus (397) Google Scholar, 5Mohn W.W. Tiedje J.M. Arch. Microbiol. 1991; 157: 1-6Crossref Scopus (64) Google Scholar, 6van De Pasqq B.A. Jansen S. Dijkema C. Schraa G. de Vos W.M. Stams A.J. Appl. Environ. Microbiol. 2001; 67: 3958-3963Crossref PubMed Scopus (29) Google Scholar, 7Holliger C. Wohlfarth G. Diekert G. FEMS Rev. 1999; 22: 383-398Crossref Google Scholar). Polychlorinated biphenyls and other haloaromatics are xenobiotics that are produced by industry and also are biologically generated (8Gribble G.W. Chemosphere. 2003; 52: 289-297Crossref PubMed Scopus (454) Google Scholar). Exploiting the ability of dehalorespiring microbes to initiate degradation by removal of the halogen group is a potential strategy for bioremediation of these toxic compounds. Induction of dehalorespiration activity occurs when desulfitobacteria are exposed to a chlorinated aromatic compound that can serve as a dehalogenation substrate (3Smidt H. de Vos W.M. Annu. Rev. Microbiol. 2004; 58: 43-73Crossref PubMed Scopus (397) Google Scholar). Induction is mediated through the transcriptional regulatory protein, CprK. Binding the chloroaromatic compound as an effector molecule triggers a conformational change in CprK, which promotes productive interaction with a nearly palindromic DNA sequence called the “FNR-like box” located upstream of four proximal transcriptional units in the cpr regulon (9Pop S.M. Kolarik R.J. Ragsdale S.W. J. Biol. Chem. 2004; 279: 49910-49918Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The cpr regulon contains eight genes, cprT, cprK, cprZ, cprE, cprB, cprA, cprC, and cprD, that encode the dehalogenase (CprA), CprK, and a number of proteins assigned as chaperones and other components of the dehalorespiration process. The role of CprK as a transcriptional activator of the cpr dehalorespiration genes and its interaction with the FNR-like box had been predicted earlier by Smidt et al. (10Smidt H. van Leest M. van der Oost J. deVos W.M. J. Bacteriol. 2000; 182: 5683-5691Crossref PubMed Scopus (78) Google Scholar) when the cpr regulon was first described. This proposal was confirmed by in vivo and in vitro DNA binding studies, which also demonstrated that CprK only functions under reducing conditions (9Pop S.M. Kolarik R.J. Ragsdale S.W. J. Biol. Chem. 2004; 279: 49910-49918Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Exposure to oxygen or incubation with a reagent (diamide) that oxidizes dithiols to their disulfide form inhibits binding of CprK to the FNR-like box sequence, and treating the oxidized protein with dithiothreitol reverses the oxidative inactivation (9Pop S.M. Kolarik R.J. Ragsdale S.W. J. Biol. Chem. 2004; 279: 49910-49918Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The cellular rationale for redox regulation of CprK activity may relate to the oxygen sensitivity of the dehalogenase CprA, which is a metalloenzyme containing two iron-sulfur clusters and a modified form of vitamin B12 (11Krasotkina J. Walters T. Maruya K.A. Ragsdale S.W. J. Biol. Chem. 2001; 276: 40991-40997Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Because it would be wasteful to produce CprA under oxidative conditions, it would be advantageous to control cpr transcription as a function of the redox state of the cell. In this study, the mechanism of redox regulation of CprK activity was examined. Oxidation of CprK was shown by internal quenching of fluorescence experiments and by isothermal calorimetry measurements to have no affect on the affinity of CprK for effector, strongly suggesting that oxidative inactivation is exhibited at the level of DNA binding. By mass spectrometry, nonreducing SDS-PAGE, and site-directed mutagenesis studies, the reversible redox inactivation was localized at one intermolecular and one intramolecular disulfide bond. Cloning, Overexpression, and Purification of CprK—DNA isolation and manipulation were performed using standard techniques (12Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Plasmid DNA was purified with a QIAprep spin miniprep kit (Qiagen). DNA fragments were purified from agarose gels using a QIAquick gel extraction kit (Qiagen). Construction of the overexpression plasmid and purification of CprK were described previously (9Pop S.M. Kolarik R.J. Ragsdale S.W. J. Biol. Chem. 2004; 279: 49910-49918Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Site-directed Mutagenesis of CprK—Cys11 and Cys200 were substituted with serine residues using the QuikChange site-directed mutagenesis protocol from Stratagene (La Jolla, CA). The pQE60::cprK plasmid was the template for PCRs using primers from Integrated DNA Technologies. DNA Sequencing—The DNA sequences of all PCR-generated DNA fragments were confirmed by automated sequencing of both strands by the Genomics Core Research Facility (University of Nebraska-Lincoln) with a Beckman/Coulter CEQ2000XL 8-capillary DNA sequencer using dye terminator chemistry. Alkylation of Cysteine Residues—Reduced (with 10 mm dithiothreitol (DTT)) and oxidized (lacking DTT) CprK samples at a concentration of 2 mg/ml in 0.5 ml of Buffer A (8 m urea, 50 mm Tris-HCl, pH 8.2, 1 mm Na-EDTA) were used for a two-step alkylation reaction (iodoacetamide (IAM) followed by 4-vinylpyridine (VP)). The first alkylation step involved incubating CprK with 50 mm IAM (final concentration) for 15 min at room temperature. The IAM stock solution was prepared by adding 100 mg of IAM to a solution containing 0.5 m sodium acetate, 2 mm Na-EDTA and incubating at 65 °C until the crystals were dissolved. The mixture was allowed to equilibrate slowly to room temperature. The IAM was protected from light at all times to avoid degradation and production of free iodine radicals, which are potent oxidants. The reaction was quenched by adding 5 ml of cold acetone with 1 n HCl (98:2) followed by centrifuging at 2000 × g for 5 min. The precipitate was washed three times by repeated suspension in a cold acetone with 1 n HCl and H2O solution (98:2:10) and centrifugation as above. After the third wash, precipitated CprK was dissolved in 0.5 ml of Buffer A and reduced by incubating at 37 °C for 30 min with 10 mm DTT. Then the sample was either digested with trypsin overnight at room temperature using a ratio of protein to trypsin of 5:1 and analyzed by mass spectroscopy or treated with a second alkylating agent. The second alkylation step was performed by adding 2 μl of 9.5 m VP (three times at 20-min intervals) to 0.5 ml of DTT-reduced CprK (above) and incubating for a total of 60 min at room temperature. The protein was precipitated, washed with cold acetone and 1 n HCl, resuspended in Buffer A containing 10 mm DTT as described above, digested with trypsin as just described, and analyzed by mass spectrometry. Mass Spectrometric Analysis—The trypsin-digested samples were injected onto a C18 PepMap100, 75 μm × 15 cm, 3-μm 100 Å column (LC Packings, Sunnyvale, CA). Tryptic peptides were then separated using a linear gradient elution from 0.3% formic acid in H2O to 0.3% formic acid in acetonitrile at a flow rate of 170 nanoliter/min. The nano-LC (liquid chromatography)/MS/MS analysis of tryptic peptides was performed using a Q-Star XL mass spectrometer (Applied Biosystems Inc.). The masses of tryptic peptides and modifications were matched with the MASCOT search engine. The two tables show the predicted relative molecular masses of the Cys-containing peptides based on their sequences and the experimental relative molecular masses, which were calculated from the experimentally observed m/z values. These tables also show the relative intensities of these peptides, which were calculated from their peak heights at a particular m/z generated from the selected ion chromatogram. Isothermal Titration Calorimetry Experiments (ITC)—ITC experiments were performed at 25 °C using a VP-ITC isothermal titration calorimeter (MicroCal Inc., Northhampton, MA). The reaction cell (∼1.4 ml) was filled with a 0.038 mm solution of purified CprK that had been dialyzed against 50 mm Tris-HCl, pH 7.5, 300 mm NaCl, without DTT, and degassed by vacuum aspiration for 5 min prior to loading. The titrated ligand CHPA (initial concentration, 0.5 mm) was dissolved in the CprK dialysis buffer. The stirring speed was 300 rpm, and the thermal power of 60 injections of 3 μl was recorded every 240 s. Thermogram analysis was performed using Origin 7.0 software supplied with the instrument. Intrinsic Fluorescence Quenching Experiments—Fluorescence spectroscopy was performed at 20 °C with an OLIS RSM1000F instrument. The starting concentration of CprK (both oxidized and reduced) was 3.0 mm, and fluorescence intensity was measured at 325.6 nm following the addition of CHPA. The excitation was set at 280 nm with an excitation slit of 3.16 nm, an emission slit of 1.24 nm, and a grating of 600 lines/mm. The data were plotted fit to one-site (Equations 1 and 2) and two-site and three-site (Equation 3) models to determine the dissociation constants and to statistically assess the binding model. In these equations, F is the measured fluorescence, and x is calculated from the quadratic Equation 2, where b equals (E + L + Kd), i.e. the sum of E (total concentration of enzyme), L (total ligand concentration), and Kd (dissociation constant); and c is the product of the concentrations of L*E. In Equation 3, which describes a bi- or multi-phasic titration curve, Kdn represents the dissociation constant(s) (i.e. Kd1 and Kd2 for a biphasic curve), F0 is the initial fluorescence, and F and L are defined as for the one-site model (i.e. F1 and F2 designate the amplitudes of the first and second phases of a biphasic titration, respectively).F=(Fmax*Kd)/(x+Kd)(Eq. 1) x=0.5[(-b+(b2+4c))]0.5(Eq. 2) F=F0+Σ{Fn*Kdn/(Kdn+L)}(Eq. 3) Nonreducing SDS-PAGE Analysis—Purified proteins from the nickel-nitrilotriacetic acid metal affinity column were dialyzed against a solution of 50 mm Tris, pH 7.5, 300 mm NaCl to remove imidazole. The dialyzed protein was then incubated with 15 mm diamide, 15 mm H2O2, and/or 10 mm DTT for 5 min at room temperature. After incubation, nonreducing loading buffer (60 mm Tris-Cl, pH 6.8, 1% SDS, 10% glycerol, 0.01% bromphenol blue) was added, and a 12% SDS-PAGE experiment was run. Electrophoretic Mobility Shift Assay (EMSA)—A 90-bp fragment containing the CprB promoter region was prepared, 5′-end-labeled by [γ-32P]ATP (3000 Ci/mmol; Amersham Biosciences), and used in EMSA experiments as described earlier (9Pop S.M. Kolarik R.J. Ragsdale S.W. J. Biol. Chem. 2004; 279: 49910-49918Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The reaction mixture contained gel shift binding 1-buffer (20% glycerol, 5 mm MgCl2, 2.5 mm EDTA, 2.5 mm DTT, 250 mm NaCl, 50 mm Tris-HCl, pH 7.5, 0.25 mg/ml poly(dI-dC)-poly(dI-dC); final concentrations), CHPA (Sigma-Aldrich), and purified CprK. The reaction was initiated by adding the 32P-labeled DNA fragment, and the mixture was incubated for 20 min at room temperature. The mixtures were separated by electrophoresis, followed by drying and autoradiography as described (9Pop S.M. Kolarik R.J. Ragsdale S.W. J. Biol. Chem. 2004; 279: 49910-49918Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). CprK-Effector Interactions—The results of previous EMSA and ITC experiments showed that CprK binds the effector molecule CHPA with high affinity (3.5 ± 0.4 μm), which promotes DNA binding to a specific palindromic sequence (9Pop S.M. Kolarik R.J. Ragsdale S.W. J. Biol. Chem. 2004; 279: 49910-49918Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). CprK was shown to lose DNA binding activity when it is oxidized by diamide or by extended incubation in air and to regain DNA binding activity when it is incubated with DTT (9Pop S.M. Kolarik R.J. Ragsdale S.W. J. Biol. Chem. 2004; 279: 49910-49918Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). CprK contains a single tryptophan residue (Trp106), which is adjacent to Cys105. When CprK is incubated with CHPA, the fluorescence of this tryptophan residue is quenched, providing a way to measure effector binding affinity. The titration of DTT-reduced CprK with CHPA was fit to Equation 1, which describes a one-site model, and to Equation 3 for two- and three-site models (Fig. 1). The data density was sufficient to rule out one- and two-site models and to support a three-site binding model, based on the goodness of fit parameter (R2 = 0.992 for the three-site versus 0.981 for the two-site and 0.919 for the one-site model). The three Kd values are 0.04 μm, 1.5, and 300 μm (Table 1). Approximately 50% of the total fluorescence quenching occurs in the first two phases (about 25% in each of these phases), and the remaining 50% of the intensity is lost in the third phase. At high concentrations of effector, the fluorescence is nearly completely quenched.TABLE 1Dissociation constants for the CprK-CHPA complexReduced CprKOxidized CprKITCFluorescenceITCFluorescenceKdAmpaAmplitude of the phase relative to the total change in fluorescence or heat, expressed as a percentageKdAmpKdAmpKdAmpμm%μm%μm%μm%0.02 ± 0.0152 ± 20.04 ± 0.0222 ± 50.038 ± 0.00727 ± 21.3 ± 0.248 ± 21.5 ± 0.731 ± 44.8 ± 1.61005.9 ± 1.723 ± 2300 ± 6047 ± 2260 ± 1750 ± 2a Amplitude of the phase relative to the total change in fluorescence or heat, expressed as a percentage Open table in a new tab Oxidation of CprK could disable either effector or DNA binding. To address the mode of reversible redox inactivation, intrinsic tryptophan fluorescence quenching experiments were performed to compare the affinity of oxidized versus reduced CprK for CHPA. With CprK prepared aerobically and in the absence of a reducing reagent, the total intrinsic fluorescence is ∼6.5-fold higher than that of the reduced protein. However, as with the reduced protein, the data best fit a three-site model with dissociation constants of 0.04, 5.9, and 260 μm, which are nearly identical to those determined for the reduced protein (Fig. 1 and Table 1). To further investigate the thermodynamics of effector binding to CprK, ITC experiments were performed with the oxidized and reduced protein. Given the low Kd measured by fluorescence quenching, we collected many data points across the titration curve, especially at low effector concentrations (Fig. 2). Two phases are clearly evident in the ITC profile for the reduced protein, and the two-site model provides a much better fit to the data than a single-site model, based on the chi-square values (1874 for the two-site model versus 3299 for the one-site model). The two Kd values (0.02 ± 0.01 and 1.3 ± 0.2 μm) are similar to the first two Kd values observed by fluorescence quenching experiments, and the two phases have nearly equal amplitudes (Table 1). Our focus was on the tight binding sites, so we did not titrate to high enough effector concentrations to attempt to verify the low affinity (300 μm) binding site detected by fluorescence quenching. We performed ITC studies on the oxidized protein numerous times; however, because this form of CprK is relatively unstable at the high concentration and over the long incubation time required for this experiment, the data were consistently of poorer quality than those for the reduced protein. In this case, we are unable to discriminate between a two-site and a one-site model; therefore, following Occam's razor, show the fit to the one-site model in Fig. 2b. Regardless, the ITC data indicate that the oxidized protein contains at least one high affinity site (Kd = 4.8 μm), which is in general agreement with the fluorescence quenching data. In summary, on the basis of fluorescence quenching and ITC studies, we conclude that the oxidized and reduced forms of CprK have two high affinity and one low affinity sites and that CHPA binding is not affected by the redox state of the protein. In both ITC and fluorescence experiments, the phase amplitudes of the two high affinity sites are equivalent, and the amplitude of the low affinity site in the fluorescence experiment equals the sum of the phase amplitudes of the two high affinity sites (Table 1). CHPA binding to the high affinity site(s) is largely entropy driven, with ΔS values at 310 K of -9.3 kJ/mol and -6.9 kJ/mol associated with Kd1 and Kd2, respectively (Fig. 2). Because oxidative inactivation of CprK does not measurably alter its interactions with the effector, disulfide bond formation must affect DNA binding affinity. Consistent with earlier results on redox control of CprK activity (9Pop S.M. Kolarik R.J. Ragsdale S.W. J. Biol. Chem. 2004; 279: 49910-49918Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), an EMSA performed with the oxidized protein sample after the ITC experiment showed no detectable DNA binding; however, when 10 mm DTT was incubated with the oxidized CprK-CHPA mixture for 5 min, DNA binding activity was restored (not shown). The reversibility of the redox inactivation is consistent with the hypothesis that oxidation involves the formation of one or more disulfide bonds. Another possibility is that one of the cysteine residues is oxidized to a sulfinate or sulfenate, which also would also be reversed by treatment with DTT (13Paget M.S. Buttner M.J. Annu. Rev. Genet. 2003; 37: 91-121Crossref PubMed Scopus (253) Google Scholar). To distinguish between these various possibilities, chemical alkylation and mass spectrometric experiments were performed. Alkylation of Cysteine Residues and Mass Spectrometric Analysis of CprK—CprK from D. dehalogenans contains five cysteine residues: Cys11, Cys105, Cys111, Cys161, and Cys200. To assess whether any of these residues are involved in disulfide bond formation, CprK samples were prepared by exposure to air, oxidation with diamide, or reduction with 10 mm DTT. The CprK samples were denatured by urea to expose all of the Cys residues, subjected to a two-step alkylation procedure, and then analyzed by mass spectrometry. The first alkylation step involved treatment of the urea-denatured protein with IAM, which labeled Cys residues that were present in the thiolate form to generate S-carboxamidomethylcysteine. The second alkylation step involved reduction with DTT, followed by treatment with VP. Cys residues that were initially present in a disulfide bond would not be labeled by IAM during the first alkylation step; however, after reduction with DTT, those residues would react with VP and undergo conversion to S-(4-vinylpyridylethyl)cysteine. CprK samples were digested with trypsin and analyzed by mass spectrometry after either the first or second alkylation step. After the one-step alkylation reaction, 77% (airoxidized) and 84% (reduced) coverage of the CprK sequence was achieved by mass spectrometric analysis, and 83% (oxidized) and 88% (reduced) sequence coverage was obtained after the two-step procedure. The results of the one-step and two-step alkylation procedures are shown in Tables 2 and 3. In these tables, the number of IAM modifications is listed beside the peptide identification. The predicted molecular mass of each peptide, based on its sequence, was compared with the experimentally determined mass. The tables also include the intensity value, which does not measure the absolute quantity of a peptide but quantifies the relative abundance of a particular peptide under similar liquid chromatography/MS conditions.TABLE 2Mass spectrometry results of air-oxidized and reduced CprK alkylated with IAMPeptideSequenceAir-oxidizedReducedIAMIntensityIAMIntensityNo.aNumber of cysteine residues modifiedMr (calc)bRelative molecular mass based on the matched peptide sequenceMr (expt)cExperimental m/z factored by zNo.aNumber of cysteine residues modifiedMr (calc)bRelative molecular mass based on the matched peptide sequenceMr (expt)cExperimental m/z factored by zcpscps1–25AMSAEGLDKDFCGIIPDSFFPEKNDdND indicates that this peptide was not detected under these conditionsNDNDND12757.302758.1765105–114TCWFSEECLR01272.531270.46400021386.571386.494928157–167LFYECSSQGK11331.611330.57188611330.621330.57234198–203VLACLK0645.39645.37200NDNDNDND198–204VLACLKK0773.48773.46337NDNDNDND1–25 + 198–203AMSAEGLDKDFCGIIPDSFFPEK + VLACLK03343.673343.56175NDNDNDND1–25 + 198–204AMSAEGLDKDFCGIIPDSFFPEK + VLACLKK03471.763471.64227NDNDNDNDa Number of cysteine residues modifiedb Relative molecular mass based on the matched peptide sequencec Experimental m/z factored by zd ND indicates that this peptide was not detected under these conditions Open table in a new tab TABLE 3Mass spectrometry results of air-oxidized and reduced CprK modified by two-step alkylationPeptideSequenceAir-oxidizedReducedNo.aNumber of cysteine residues modifiedMr (calc)bRelative molecular mass based on the matched peptide sequenceMr (expt)cExperimental m/z factored by zIntensityNo.aNumber of cysteine residues modifiedMr (calc)bRelative molecular mass based on the matched peptide sequenceMr (expt)cExperimental m/z factored by zIntensityIAM4VPdThe number of VP modifications detected are listed in these columnsIAM4VPdThe number of VP modifications detected are listed in these columnscpscps1–25AMSAEGLDKDFCGIIPDSFFPEK102757.302758.3170102757.302757.3315010–25DFCGIIPDSFFPEK011854.891854.89226101854.891854.89120105–114TCWFSEECLR111434.611434.63180201386.571386.573163021482.641482.65999111434.611434.63279157–167LFYECSSQGK101330.621330.624000101330.621330.64300011378.661378.671906198–203VLACLK10702.41702.4294510702.41702.43400001750.45750.45200a Number of cysteine residues modifiedb Relative molecular mass based on the matched peptide sequencec Experimental m/z factored by zd The number of VP modifications detected are listed in these columns Open table in a new tab When CprK samples were reduced with 10 mm DTT and alkylated by IAM, mass spectrometric analysis revealed that Cys11, Cys105, Cys111, and Cys161 were modified with carboxamido labels. Thus, in the presence of DTT, these four Cys residues were initially present in the thiolate form (Table 2). Although when samples were analyzed after this one-step IAM alkylation experiment, a peptide covering Cys200 was not detected, all five cysteine residues were modified by IAM in the first step of the two-step alkylation of reduced CprK (Table 3). In summary, these data indicate that DTT-reduced active protein contains all five cysteine residues in the thiol or thiolate form. When the air-oxidized protein was analyzed by the one-step alkylation reaction, a peptide containing S-carboxyamidomethyl-labeled Cys161 (peptide 157-167) was recovered in high amounts (Table 2), indicating that this residue was in the thiol form under both reducing (above) and oxidizing conditions. None of the other four Cys residues (Cys11, Cys105, Cys111, and Cys200) of air-oxidized CprK were labeled by IAM. The MS analysis did not indicate the presence of sulfenate, sulfinate, or sulfonate modification of any Cys-containing peptides. An unlabeled peptide 105-114 containing Cys105 and Cys111 was identified whose mass was lower by 2.06 Da than predicted, suggesting the loss of two hydrogen atoms (Fig. 3A), which would be expected for a peptide containing a disulfide bond. A smaller amount of this peptide contained IAM modification, whereas in the DTT-reduced samples, the doubly IAM-modified peptide 105-114 was present in much higher proportion than the unmodified one (supplemental Fig. S1). These results indicate that, in the air-oxidized protein, Cys161 is a reduced thiol(ate), and Cys105 and Cys111 are linked by a disulfide bridge. The situation with Cys11 and Cys200 is addressed below. In agreement with the results of the one-step alkylation protocol, when air-oxidized CprK was subjected to the two-step procedure, peptide 105-114 contained two S-(4-pyridylethyl) modifications (Table 3). A small proportion of this peptide was found with both modifications (S-carboxyamidomethyl and S-(4-pyridylethyl)); however, the peptide containing the double modification by VP was 6-fold more abundant. Therefore, both the single and double alkylation results indicate that Cys105 and Cys111 form a disulfide bond in air-oxidized CprK. This disulfide bridge is most likely intramolecular because these residues are close in the primary sequence. Furthermore, we could not locate a peak with the mass expected for two covalently linked 105-114 peptides during analysis of the single alkylation experiment. The two-step procedure also results in IAM modification of the peptide containing Cys161, with a smaller amount of VP modification. Based on the results with the one-step procedure, we assume that the minor amount of Cys161 labeled by VP simply reflects incomplete IAM labeling, although we cannot rule out a minor proportion of Cys161 being involved in a disulfide link with another residue. MS analysis of the peptides containing Cys11 and Cys200 was more complicated. When the oxidized CprK sample was treated with only IAM, we did not observe a peptide containing Cys11 (Table 2). However, as shown in the last two rows of Table 2, we observed two peaks with masses corresponding to the sum of t" @default.
• W2069968267 created "2016-06-24" @default.
• W2069968267 creator A5061447121 @default.
• W2069968267 creator A5080113233 @default.
• W2069968267 creator A5080730574 @default.
• W2069968267 creator A5078350256 @default.
• W2069968267 date "2006-09-01" @default.
• W2069968267 modified "2023-09-26" @default.
• W2069968267 title "Transcriptional Activation of Dehalorespiration" @default.
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