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  • 94
    Agilent technologies 1260 hplc system
    1260 Hplc System, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 94/100, based on 1925 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies 1260 infinity hplc system
    1260 Infinity Hplc System, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 99/100, based on 4317 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies agilent 1260 hplc
    Metal analysis and radical generation in the presence of chelator. a) Representative TXRF spectra measured for Mf R2 (blue, at 664 μM) and Fe-reconstituted class Ia Ec R2 (orange, at 635 μM), on the 5 to 10 keV energy range. The spectra have been scaled using the peak size of the Ga internal standard and offset slightly in the Y-direction for clarity. K-level X-ray emission lines are indicated with arrows. For elements, where both Kα and Kβ lines are present, they are specified. Otherwise, arrows indicate Kα lines. Experiments were repeated three times. b) Concentrations of Mn, Fe, Co, Ni, Cu and Zn were measured in the active Mf R2, Mf NrdI and Mf <t>R1</t> protein solutions and in their respective buffers, as well as in a solution of E. coli class Ia R2 protein reconstituted with Fe in vitro . Mean concentrations and SD of measurements on 3 independently prepared samples for each sample are reported. The concentrations were converted to metal to protein molar ratio. The measurements show that none of the Mf RNR proteins contain a significant amount of metal as opposed to Ec R2a which as expected contains on the order of 2 metal ions per monomer also after a desalting step. Buffer 1 is the buffer system used for Mf R2, i.e. 25 mM HEPES-Na pH 7, 50 mM NaCl. Buffer 2 is the buffer system used for Mf R1 and Mf NrdI, i.e. 25 mM Tris-HCl pH 8, 50 mM NaCl. The protein purification involves a Ni-affinity step which is likely the reason for nickel being the dominating metal species in the sample. c) <t>HPLC</t> based in vitro assays show that RNR activity can be restored after Mf R2 is quenched by hydroxyurea. Mf R2 is regenerated by the addition of Mf NrdI followed by redox cycling with dithionite and oxygen containing buffer (green) (see main ). Reactivation and activity are observed also in the presence of a metal chelator (EDTA 0.3 mM, blue). Addition of extra metals (0.2 mM of each Mn, Fe, Co, Ni, Cu, Zn) does not improve the activity recovery (pink). Fig. 2d
    Agilent 1260 Hplc, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 99/100, based on 3721 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Agilent technologies 1260 series hplc system
    Metal analysis and radical generation in the presence of chelator. a) Representative TXRF spectra measured for Mf R2 (blue, at 664 μM) and Fe-reconstituted class Ia Ec R2 (orange, at 635 μM), on the 5 to 10 keV energy range. The spectra have been scaled using the peak size of the Ga internal standard and offset slightly in the Y-direction for clarity. K-level X-ray emission lines are indicated with arrows. For elements, where both Kα and Kβ lines are present, they are specified. Otherwise, arrows indicate Kα lines. Experiments were repeated three times. b) Concentrations of Mn, Fe, Co, Ni, Cu and Zn were measured in the active Mf R2, Mf NrdI and Mf <t>R1</t> protein solutions and in their respective buffers, as well as in a solution of E. coli class Ia R2 protein reconstituted with Fe in vitro . Mean concentrations and SD of measurements on 3 independently prepared samples for each sample are reported. The concentrations were converted to metal to protein molar ratio. The measurements show that none of the Mf RNR proteins contain a significant amount of metal as opposed to Ec R2a which as expected contains on the order of 2 metal ions per monomer also after a desalting step. Buffer 1 is the buffer system used for Mf R2, i.e. 25 mM HEPES-Na pH 7, 50 mM NaCl. Buffer 2 is the buffer system used for Mf R1 and Mf NrdI, i.e. 25 mM Tris-HCl pH 8, 50 mM NaCl. The protein purification involves a Ni-affinity step which is likely the reason for nickel being the dominating metal species in the sample. c) <t>HPLC</t> based in vitro assays show that RNR activity can be restored after Mf R2 is quenched by hydroxyurea. Mf R2 is regenerated by the addition of Mf NrdI followed by redox cycling with dithionite and oxygen containing buffer (green) (see main ). Reactivation and activity are observed also in the presence of a metal chelator (EDTA 0.3 mM, blue). Addition of extra metals (0.2 mM of each Mn, Fe, Co, Ni, Cu, Zn) does not improve the activity recovery (pink). Fig. 2d
    1260 Series Hplc System, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 91/100, based on 463 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies 1260 high performance liquid chromatography hplc
    Metal analysis and radical generation in the presence of chelator. a) Representative TXRF spectra measured for Mf R2 (blue, at 664 μM) and Fe-reconstituted class Ia Ec R2 (orange, at 635 μM), on the 5 to 10 keV energy range. The spectra have been scaled using the peak size of the Ga internal standard and offset slightly in the Y-direction for clarity. K-level X-ray emission lines are indicated with arrows. For elements, where both Kα and Kβ lines are present, they are specified. Otherwise, arrows indicate Kα lines. Experiments were repeated three times. b) Concentrations of Mn, Fe, Co, Ni, Cu and Zn were measured in the active Mf R2, Mf NrdI and Mf <t>R1</t> protein solutions and in their respective buffers, as well as in a solution of E. coli class Ia R2 protein reconstituted with Fe in vitro . Mean concentrations and SD of measurements on 3 independently prepared samples for each sample are reported. The concentrations were converted to metal to protein molar ratio. The measurements show that none of the Mf RNR proteins contain a significant amount of metal as opposed to Ec R2a which as expected contains on the order of 2 metal ions per monomer also after a desalting step. Buffer 1 is the buffer system used for Mf R2, i.e. 25 mM HEPES-Na pH 7, 50 mM NaCl. Buffer 2 is the buffer system used for Mf R1 and Mf NrdI, i.e. 25 mM Tris-HCl pH 8, 50 mM NaCl. The protein purification involves a Ni-affinity step which is likely the reason for nickel being the dominating metal species in the sample. c) <t>HPLC</t> based in vitro assays show that RNR activity can be restored after Mf R2 is quenched by hydroxyurea. Mf R2 is regenerated by the addition of Mf NrdI followed by redox cycling with dithionite and oxygen containing buffer (green) (see main ). Reactivation and activity are observed also in the presence of a metal chelator (EDTA 0.3 mM, blue). Addition of extra metals (0.2 mM of each Mn, Fe, Co, Ni, Cu, Zn) does not improve the activity recovery (pink). Fig. 2d
    1260 High Performance Liquid Chromatography Hplc, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 89/100, based on 167 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies 1260 infinity bio inert hplc system
    Metal analysis and radical generation in the presence of chelator. a) Representative TXRF spectra measured for Mf R2 (blue, at 664 μM) and Fe-reconstituted class Ia Ec R2 (orange, at 635 μM), on the 5 to 10 keV energy range. The spectra have been scaled using the peak size of the Ga internal standard and offset slightly in the Y-direction for clarity. K-level X-ray emission lines are indicated with arrows. For elements, where both Kα and Kβ lines are present, they are specified. Otherwise, arrows indicate Kα lines. Experiments were repeated three times. b) Concentrations of Mn, Fe, Co, Ni, Cu and Zn were measured in the active Mf R2, Mf NrdI and Mf <t>R1</t> protein solutions and in their respective buffers, as well as in a solution of E. coli class Ia R2 protein reconstituted with Fe in vitro . Mean concentrations and SD of measurements on 3 independently prepared samples for each sample are reported. The concentrations were converted to metal to protein molar ratio. The measurements show that none of the Mf RNR proteins contain a significant amount of metal as opposed to Ec R2a which as expected contains on the order of 2 metal ions per monomer also after a desalting step. Buffer 1 is the buffer system used for Mf R2, i.e. 25 mM HEPES-Na pH 7, 50 mM NaCl. Buffer 2 is the buffer system used for Mf R1 and Mf NrdI, i.e. 25 mM Tris-HCl pH 8, 50 mM NaCl. The protein purification involves a Ni-affinity step which is likely the reason for nickel being the dominating metal species in the sample. c) <t>HPLC</t> based in vitro assays show that RNR activity can be restored after Mf R2 is quenched by hydroxyurea. Mf R2 is regenerated by the addition of Mf NrdI followed by redox cycling with dithionite and oxygen containing buffer (green) (see main ). Reactivation and activity are observed also in the presence of a metal chelator (EDTA 0.3 mM, blue). Addition of extra metals (0.2 mM of each Mn, Fe, Co, Ni, Cu, Zn) does not improve the activity recovery (pink). Fig. 2d
    1260 Infinity Bio Inert Hplc System, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 99/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies 1260 infinity binary hplc
    Metal analysis and radical generation in the presence of chelator. a) Representative TXRF spectra measured for Mf R2 (blue, at 664 μM) and Fe-reconstituted class Ia Ec R2 (orange, at 635 μM), on the 5 to 10 keV energy range. The spectra have been scaled using the peak size of the Ga internal standard and offset slightly in the Y-direction for clarity. K-level X-ray emission lines are indicated with arrows. For elements, where both Kα and Kβ lines are present, they are specified. Otherwise, arrows indicate Kα lines. Experiments were repeated three times. b) Concentrations of Mn, Fe, Co, Ni, Cu and Zn were measured in the active Mf R2, Mf NrdI and Mf <t>R1</t> protein solutions and in their respective buffers, as well as in a solution of E. coli class Ia R2 protein reconstituted with Fe in vitro . Mean concentrations and SD of measurements on 3 independently prepared samples for each sample are reported. The concentrations were converted to metal to protein molar ratio. The measurements show that none of the Mf RNR proteins contain a significant amount of metal as opposed to Ec R2a which as expected contains on the order of 2 metal ions per monomer also after a desalting step. Buffer 1 is the buffer system used for Mf R2, i.e. 25 mM HEPES-Na pH 7, 50 mM NaCl. Buffer 2 is the buffer system used for Mf R1 and Mf NrdI, i.e. 25 mM Tris-HCl pH 8, 50 mM NaCl. The protein purification involves a Ni-affinity step which is likely the reason for nickel being the dominating metal species in the sample. c) <t>HPLC</t> based in vitro assays show that RNR activity can be restored after Mf R2 is quenched by hydroxyurea. Mf R2 is regenerated by the addition of Mf NrdI followed by redox cycling with dithionite and oxygen containing buffer (green) (see main ). Reactivation and activity are observed also in the presence of a metal chelator (EDTA 0.3 mM, blue). Addition of extra metals (0.2 mM of each Mn, Fe, Co, Ni, Cu, Zn) does not improve the activity recovery (pink). Fig. 2d
    1260 Infinity Binary Hplc, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 99/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies model 1260 hplc
    Metal analysis and radical generation in the presence of chelator. a) Representative TXRF spectra measured for Mf R2 (blue, at 664 μM) and Fe-reconstituted class Ia Ec R2 (orange, at 635 μM), on the 5 to 10 keV energy range. The spectra have been scaled using the peak size of the Ga internal standard and offset slightly in the Y-direction for clarity. K-level X-ray emission lines are indicated with arrows. For elements, where both Kα and Kβ lines are present, they are specified. Otherwise, arrows indicate Kα lines. Experiments were repeated three times. b) Concentrations of Mn, Fe, Co, Ni, Cu and Zn were measured in the active Mf R2, Mf NrdI and Mf <t>R1</t> protein solutions and in their respective buffers, as well as in a solution of E. coli class Ia R2 protein reconstituted with Fe in vitro . Mean concentrations and SD of measurements on 3 independently prepared samples for each sample are reported. The concentrations were converted to metal to protein molar ratio. The measurements show that none of the Mf RNR proteins contain a significant amount of metal as opposed to Ec R2a which as expected contains on the order of 2 metal ions per monomer also after a desalting step. Buffer 1 is the buffer system used for Mf R2, i.e. 25 mM HEPES-Na pH 7, 50 mM NaCl. Buffer 2 is the buffer system used for Mf R1 and Mf NrdI, i.e. 25 mM Tris-HCl pH 8, 50 mM NaCl. The protein purification involves a Ni-affinity step which is likely the reason for nickel being the dominating metal species in the sample. c) <t>HPLC</t> based in vitro assays show that RNR activity can be restored after Mf R2 is quenched by hydroxyurea. Mf R2 is regenerated by the addition of Mf NrdI followed by redox cycling with dithionite and oxygen containing buffer (green) (see main ). Reactivation and activity are observed also in the presence of a metal chelator (EDTA 0.3 mM, blue). Addition of extra metals (0.2 mM of each Mn, Fe, Co, Ni, Cu, Zn) does not improve the activity recovery (pink). Fig. 2d
    Model 1260 Hplc, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 90/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies 1260 capillary hplc system
    Metal analysis and radical generation in the presence of chelator. a) Representative TXRF spectra measured for Mf R2 (blue, at 664 μM) and Fe-reconstituted class Ia Ec R2 (orange, at 635 μM), on the 5 to 10 keV energy range. The spectra have been scaled using the peak size of the Ga internal standard and offset slightly in the Y-direction for clarity. K-level X-ray emission lines are indicated with arrows. For elements, where both Kα and Kβ lines are present, they are specified. Otherwise, arrows indicate Kα lines. Experiments were repeated three times. b) Concentrations of Mn, Fe, Co, Ni, Cu and Zn were measured in the active Mf R2, Mf NrdI and Mf <t>R1</t> protein solutions and in their respective buffers, as well as in a solution of E. coli class Ia R2 protein reconstituted with Fe in vitro . Mean concentrations and SD of measurements on 3 independently prepared samples for each sample are reported. The concentrations were converted to metal to protein molar ratio. The measurements show that none of the Mf RNR proteins contain a significant amount of metal as opposed to Ec R2a which as expected contains on the order of 2 metal ions per monomer also after a desalting step. Buffer 1 is the buffer system used for Mf R2, i.e. 25 mM HEPES-Na pH 7, 50 mM NaCl. Buffer 2 is the buffer system used for Mf R1 and Mf NrdI, i.e. 25 mM Tris-HCl pH 8, 50 mM NaCl. The protein purification involves a Ni-affinity step which is likely the reason for nickel being the dominating metal species in the sample. c) <t>HPLC</t> based in vitro assays show that RNR activity can be restored after Mf R2 is quenched by hydroxyurea. Mf R2 is regenerated by the addition of Mf NrdI followed by redox cycling with dithionite and oxygen containing buffer (green) (see main ). Reactivation and activity are observed also in the presence of a metal chelator (EDTA 0.3 mM, blue). Addition of extra metals (0.2 mM of each Mn, Fe, Co, Ni, Cu, Zn) does not improve the activity recovery (pink). Fig. 2d
    1260 Capillary Hplc System, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 88/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Metal analysis and radical generation in the presence of chelator. a) Representative TXRF spectra measured for Mf R2 (blue, at 664 μM) and Fe-reconstituted class Ia Ec R2 (orange, at 635 μM), on the 5 to 10 keV energy range. The spectra have been scaled using the peak size of the Ga internal standard and offset slightly in the Y-direction for clarity. K-level X-ray emission lines are indicated with arrows. For elements, where both Kα and Kβ lines are present, they are specified. Otherwise, arrows indicate Kα lines. Experiments were repeated three times. b) Concentrations of Mn, Fe, Co, Ni, Cu and Zn were measured in the active Mf R2, Mf NrdI and Mf R1 protein solutions and in their respective buffers, as well as in a solution of E. coli class Ia R2 protein reconstituted with Fe in vitro . Mean concentrations and SD of measurements on 3 independently prepared samples for each sample are reported. The concentrations were converted to metal to protein molar ratio. The measurements show that none of the Mf RNR proteins contain a significant amount of metal as opposed to Ec R2a which as expected contains on the order of 2 metal ions per monomer also after a desalting step. Buffer 1 is the buffer system used for Mf R2, i.e. 25 mM HEPES-Na pH 7, 50 mM NaCl. Buffer 2 is the buffer system used for Mf R1 and Mf NrdI, i.e. 25 mM Tris-HCl pH 8, 50 mM NaCl. The protein purification involves a Ni-affinity step which is likely the reason for nickel being the dominating metal species in the sample. c) HPLC based in vitro assays show that RNR activity can be restored after Mf R2 is quenched by hydroxyurea. Mf R2 is regenerated by the addition of Mf NrdI followed by redox cycling with dithionite and oxygen containing buffer (green) (see main ). Reactivation and activity are observed also in the presence of a metal chelator (EDTA 0.3 mM, blue). Addition of extra metals (0.2 mM of each Mn, Fe, Co, Ni, Cu, Zn) does not improve the activity recovery (pink). Fig. 2d

    Journal: Nature

    Article Title: Metal-free ribonucleotide reduction powered by a DOPA radical in Mycoplasma pathogens

    doi: 10.1038/s41586-018-0653-6

    Figure Lengend Snippet: Metal analysis and radical generation in the presence of chelator. a) Representative TXRF spectra measured for Mf R2 (blue, at 664 μM) and Fe-reconstituted class Ia Ec R2 (orange, at 635 μM), on the 5 to 10 keV energy range. The spectra have been scaled using the peak size of the Ga internal standard and offset slightly in the Y-direction for clarity. K-level X-ray emission lines are indicated with arrows. For elements, where both Kα and Kβ lines are present, they are specified. Otherwise, arrows indicate Kα lines. Experiments were repeated three times. b) Concentrations of Mn, Fe, Co, Ni, Cu and Zn were measured in the active Mf R2, Mf NrdI and Mf R1 protein solutions and in their respective buffers, as well as in a solution of E. coli class Ia R2 protein reconstituted with Fe in vitro . Mean concentrations and SD of measurements on 3 independently prepared samples for each sample are reported. The concentrations were converted to metal to protein molar ratio. The measurements show that none of the Mf RNR proteins contain a significant amount of metal as opposed to Ec R2a which as expected contains on the order of 2 metal ions per monomer also after a desalting step. Buffer 1 is the buffer system used for Mf R2, i.e. 25 mM HEPES-Na pH 7, 50 mM NaCl. Buffer 2 is the buffer system used for Mf R1 and Mf NrdI, i.e. 25 mM Tris-HCl pH 8, 50 mM NaCl. The protein purification involves a Ni-affinity step which is likely the reason for nickel being the dominating metal species in the sample. c) HPLC based in vitro assays show that RNR activity can be restored after Mf R2 is quenched by hydroxyurea. Mf R2 is regenerated by the addition of Mf NrdI followed by redox cycling with dithionite and oxygen containing buffer (green) (see main ). Reactivation and activity are observed also in the presence of a metal chelator (EDTA 0.3 mM, blue). Addition of extra metals (0.2 mM of each Mn, Fe, Co, Ni, Cu, Zn) does not improve the activity recovery (pink). Fig. 2d

    Article Snippet: The in-vitro M. florum R1-R2 catalyzed reduction of CDP to deoxy-CDP was measured in a HPLC system (Agilent 1260 Infinity) using a Waters Symmetry C18 column.

    Techniques: IA, In Vitro, Protein Purification, High Performance Liquid Chromatography, Activity Assay

    A new RNR subclass able to rescue an Escherichia coli strain lacking aerobic RNR. a) Sequence alignment of the new R2 protein groups to a number of standard, di-metal containing, R2 proteins. Purple background indicates the 6 normally essential metal-binding residues, only 3 of which are conserved. Two variants are observed in which 3 carboxylate metal ligands are either substituted for valine, proline and lysine (VPK variant) or for glutamine, serine and lysine (QSK variant). The normally radical harboring tyrosine residue is shown with a green background. b) Taxonomic distribution of NrdF2. QSK/VPK encoding organisms and their collected RNR class repertoire. As common for class I RNRs, several genomes encoding the QSK or VPK variant also harbor other RNRs. The QSK clusters are found only in Actinobacteria, whereas the VPK clusters are also found in Firmicutes, Tenericutes, Chlamydiae and Fusobacteria. c) Expression of the MfnrdFIE operon induced by addition of 0.1% v/v L-arabinose (green) rescued the JEM164 double knockout (Δ nrdAB , Δ nrdEF ) strain while when gene expression was suppressed with 0.1% v/v D-glucose (brown) the strain failed to recover, as did the strain lacking the vector (red). Growth curves (average of 3 experiments with SD) are shown. d) Mf NrdI activates Mf R2 in an O 2 dependent reaction. HPLC based in vitro RNR activity assays show no activity for R2 protein expressed separately in E. coli (red), while aerobic co-expression with MfnrdI and MfnrdE (green) or MfnrdI (orange) produced an active R2 protein. Anaerobic co-expression (yellow) or incubation of the active R2 with hydroxyurea (light blue) abolishes the activity. Partial activity could be regenerated by the addition of Mf NrdI and redox cycling with dithionite and oxygen, blue and maroon for one and two reduction-oxidation cycles respectively. Data points are shown for triplicate experiments.

    Journal: Nature

    Article Title: Metal-free ribonucleotide reduction powered by a DOPA radical in Mycoplasma pathogens

    doi: 10.1038/s41586-018-0653-6

    Figure Lengend Snippet: A new RNR subclass able to rescue an Escherichia coli strain lacking aerobic RNR. a) Sequence alignment of the new R2 protein groups to a number of standard, di-metal containing, R2 proteins. Purple background indicates the 6 normally essential metal-binding residues, only 3 of which are conserved. Two variants are observed in which 3 carboxylate metal ligands are either substituted for valine, proline and lysine (VPK variant) or for glutamine, serine and lysine (QSK variant). The normally radical harboring tyrosine residue is shown with a green background. b) Taxonomic distribution of NrdF2. QSK/VPK encoding organisms and their collected RNR class repertoire. As common for class I RNRs, several genomes encoding the QSK or VPK variant also harbor other RNRs. The QSK clusters are found only in Actinobacteria, whereas the VPK clusters are also found in Firmicutes, Tenericutes, Chlamydiae and Fusobacteria. c) Expression of the MfnrdFIE operon induced by addition of 0.1% v/v L-arabinose (green) rescued the JEM164 double knockout (Δ nrdAB , Δ nrdEF ) strain while when gene expression was suppressed with 0.1% v/v D-glucose (brown) the strain failed to recover, as did the strain lacking the vector (red). Growth curves (average of 3 experiments with SD) are shown. d) Mf NrdI activates Mf R2 in an O 2 dependent reaction. HPLC based in vitro RNR activity assays show no activity for R2 protein expressed separately in E. coli (red), while aerobic co-expression with MfnrdI and MfnrdE (green) or MfnrdI (orange) produced an active R2 protein. Anaerobic co-expression (yellow) or incubation of the active R2 with hydroxyurea (light blue) abolishes the activity. Partial activity could be regenerated by the addition of Mf NrdI and redox cycling with dithionite and oxygen, blue and maroon for one and two reduction-oxidation cycles respectively. Data points are shown for triplicate experiments.

    Article Snippet: The in-vitro M. florum R1-R2 catalyzed reduction of CDP to deoxy-CDP was measured in a HPLC system (Agilent 1260 Infinity) using a Waters Symmetry C18 column.

    Techniques: Sequencing, Binding Assay, Variant Assay, Expressing, Double Knockout, Plasmid Preparation, High Performance Liquid Chromatography, In Vitro, Activity Assay, Produced, Incubation