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John I. Clark - One of the best experts on this subject based on the ideXlab platform.

  • The β4-β8 Groove Is an ATP-interactive Site in the α Crystallin Core Domain of the Small Heat Shock Protein, Human αB Crystallin
    Journal of Molecular Biology, 2006
    Co-Authors: Joy G. Ghosh, Scott A. Houck, Catalin E. Doneanu, John I. Clark
    Abstract:

    The site for ATP interactions in human αB crystallin, the archetype of small heat-shock proteins, was identified and characterized to resolve the controversial role of ATP in the function of small heat-shock proteins. Comparative sequence alignments identified the αB crystallin sequence, 82KHFSPEELKVKVLGD96 as a Walker-B ATP-binding Motif that is found in several ATP-binding proteins, including five molecular chaperones. Fluorescence resonance energy transfer and mass spectrometry using a novel fluorescent ATP analog, 8-azido-ATP-[γ]-1-naphthalenesulfonic acid-5(2-aminoethylamide) (azido-ATP-EDANS) and a cysteine mutant of human αB crystallin (S135C) conjugated with a fluorescent acceptor, eosin-5-maleimide (EMA) identified the β4-β8 groove as the ATP interactive site in αB crystallin. A 44% decrease in the emitted fluorescence of azido-ATP-EDANS at the absorption maximum of S135C-EMA and a corresponding 50% increase in the fluorescence emission of S135C-EMA indicated a close spatial relationship between azido-ATP-EDANS and the center of the β8 strand (131LTITSSLS138). Liquid chromatography, electrospray ionization mass spectrometry identified two peptide fragments of the αB crystallin Walker-B Motif photo-affinity-labeled with azido-ATP-EDANS confirming the β4-β8 groove as an ATP interactive site. The results presented here clearly establish the β4-β8 groove as the ATP interactive region in αB crystallin, and are in contrast to the existing paradigm that classifies small heat-shock proteins as ATP-independent chaperones.

  • The β4-β8 Groove Is an ATP-interactive Site in the α Crystallin Core Domain of the Small Heat Shock Protein, Human αB Crystallin
    Journal of Molecular Biology, 2006
    Co-Authors: Joy G. Ghosh, Scott A. Houck, Catalin E. Doneanu, John I. Clark
    Abstract:

    The site for ATP interactions in human αB crystallin, the archetype of small heat-shock proteins, was identified and characterized to resolve the controversial role of ATP in the function of small heat-shock proteins. Comparative sequence alignments identified the αB crystallin sequence, 82KHFSPEELKVKVLGD96 as a Walker-B ATP-binding Motif that is found in several ATP-binding proteins, including five molecular chaperones. Fluorescence resonance energy transfer and mass spectrometry using a novel fluorescent ATP analog, 8-azido-ATP-[γ]-1-naphthalenesulfonic acid-5(2-aminoethylamide) (azido-ATP-EDANS) and a cysteine mutant of human αB crystallin (S135C) conjugated with a fluorescent acceptor, eosin-5-maleimide (EMA) identified the β4-β8 groove as the ATP interactive site in αB crystallin. A 44% decrease in the emitted fluorescence of azido-ATP-EDANS at the absorption maximum of S135C-EMA and a corresponding 50% increase in the fluorescence emission of S135C-EMA indicated a close spatial relationship between azido-ATP-EDANS and the center of the β8 strand (131LTITSSLS138). Liquid chromatography, electrospray ionization mass spectrometry identified two peptide fragments of the αB crystallin Walker-B Motif photo-affinity-labeled with azido-ATP-EDANS confirming the β4-β8 groove as an ATP interactive site. The results presented here clearly establish the β4-β8 groove as the ATP interactive region in αB crystallin, and are in contrast to the existing paradigm that classifies small heat-shock proteins as ATP-independent chaperones.

Joy G. Ghosh - One of the best experts on this subject based on the ideXlab platform.

  • The β4-β8 Groove Is an ATP-interactive Site in the α Crystallin Core Domain of the Small Heat Shock Protein, Human αB Crystallin
    Journal of Molecular Biology, 2006
    Co-Authors: Joy G. Ghosh, Scott A. Houck, Catalin E. Doneanu, John I. Clark
    Abstract:

    The site for ATP interactions in human αB crystallin, the archetype of small heat-shock proteins, was identified and characterized to resolve the controversial role of ATP in the function of small heat-shock proteins. Comparative sequence alignments identified the αB crystallin sequence, 82KHFSPEELKVKVLGD96 as a Walker-B ATP-binding Motif that is found in several ATP-binding proteins, including five molecular chaperones. Fluorescence resonance energy transfer and mass spectrometry using a novel fluorescent ATP analog, 8-azido-ATP-[γ]-1-naphthalenesulfonic acid-5(2-aminoethylamide) (azido-ATP-EDANS) and a cysteine mutant of human αB crystallin (S135C) conjugated with a fluorescent acceptor, eosin-5-maleimide (EMA) identified the β4-β8 groove as the ATP interactive site in αB crystallin. A 44% decrease in the emitted fluorescence of azido-ATP-EDANS at the absorption maximum of S135C-EMA and a corresponding 50% increase in the fluorescence emission of S135C-EMA indicated a close spatial relationship between azido-ATP-EDANS and the center of the β8 strand (131LTITSSLS138). Liquid chromatography, electrospray ionization mass spectrometry identified two peptide fragments of the αB crystallin Walker-B Motif photo-affinity-labeled with azido-ATP-EDANS confirming the β4-β8 groove as an ATP interactive site. The results presented here clearly establish the β4-β8 groove as the ATP interactive region in αB crystallin, and are in contrast to the existing paradigm that classifies small heat-shock proteins as ATP-independent chaperones.

  • The β4-β8 Groove Is an ATP-interactive Site in the α Crystallin Core Domain of the Small Heat Shock Protein, Human αB Crystallin
    Journal of Molecular Biology, 2006
    Co-Authors: Joy G. Ghosh, Scott A. Houck, Catalin E. Doneanu, John I. Clark
    Abstract:

    The site for ATP interactions in human αB crystallin, the archetype of small heat-shock proteins, was identified and characterized to resolve the controversial role of ATP in the function of small heat-shock proteins. Comparative sequence alignments identified the αB crystallin sequence, 82KHFSPEELKVKVLGD96 as a Walker-B ATP-binding Motif that is found in several ATP-binding proteins, including five molecular chaperones. Fluorescence resonance energy transfer and mass spectrometry using a novel fluorescent ATP analog, 8-azido-ATP-[γ]-1-naphthalenesulfonic acid-5(2-aminoethylamide) (azido-ATP-EDANS) and a cysteine mutant of human αB crystallin (S135C) conjugated with a fluorescent acceptor, eosin-5-maleimide (EMA) identified the β4-β8 groove as the ATP interactive site in αB crystallin. A 44% decrease in the emitted fluorescence of azido-ATP-EDANS at the absorption maximum of S135C-EMA and a corresponding 50% increase in the fluorescence emission of S135C-EMA indicated a close spatial relationship between azido-ATP-EDANS and the center of the β8 strand (131LTITSSLS138). Liquid chromatography, electrospray ionization mass spectrometry identified two peptide fragments of the αB crystallin Walker-B Motif photo-affinity-labeled with azido-ATP-EDANS confirming the β4-β8 groove as an ATP interactive site. The results presented here clearly establish the β4-β8 groove as the ATP interactive region in αB crystallin, and are in contrast to the existing paradigm that classifies small heat-shock proteins as ATP-independent chaperones.

Tarek Msadek - One of the best experts on this subject based on the ideXlab platform.

  • A six amino acid deletion, partially overlapping the VanSB G2 ATP-binding Motif, leads to constitutive glycopeptide resistance in VanB-type Enterococcus faecium
    Molecular Microbiology, 2003
    Co-Authors: Florence Depardieu, Patrice Courvalin, Tarek Msadek
    Abstract:

    Enterococcus faecium clinical isolate BM4524, resistant to vancomycin and susceptible to teicoplanin, harboured a chromosomal vanB cluster, including the vanSB/vanRB two-component system regulatory genes. Enterococcus faecium strain BM4525, isolated two weeks later from the same patient, was resistant to high levels of both glycopeptides. The ddl gene of BM4525 had a 2 bp insertion leading to an impaired d-alanine:d-alanine ligase. Sequencing of the vanB operon in BM4525 also revealed an 18 bp deletion in the vanSB gene designated vanSBDelta. The resulting six amino acid deletion partially overlapped the G2 ATP-binding domain of the VanSBDelta histidine kinase leading to constitutive expression of the resistance genes. Sequence analysis indicated that the deletion occurred between two tandemly arranged heptanucleotide direct repeats, separated by 11 base-pairs. The VanSB, VanSBDelta and VanRB proteins were overproduced in Escherichia coli and purified. In vitro autophosphorylation of the VanSB and VanSBDelta histidine kinases and phosphotransfer to the VanRB response regulator did not differ significantly. However, VanSBDelta was deficient in VanRB phosphatase activity leading to accumulation of phosphorylated VanRB. Increased glycopeptide resistance in E. faecium BM4525 was therefore a result of the lack of production of d-alanyl-d-alanine ending pentapeptide and to constitutive synthesis of d-alanyl-d-lactate terminating peptidoglycan precursors, following loss of d-alanine:d-alanine ligase and of VanSB phosphatase activity respectively. We suggest that the heptanucleotide direct repeat in vanSB may favour the appearance of high level constitutively expressed vancomycin resistance through a 'slippage' type of genetic rearrangement in VanB-type strains.

  • A six amino acid deletion, partially overlapping the VanSB G2 ATP-binding Motif, leads to constitutive glycopeptide resistance in VanB-type Enterococcus faecium.
    Molecular microbiology, 2003
    Co-Authors: Florence Depardieu, Patrice Courvalin, Tarek Msadek
    Abstract:

    Summary Enterococcus faecium clinical isolate BM4524, resistant to vancomycin and susceptible to teicoplanin, harboured a chromosomal vanB cluster, including the vanS B / vanR B two-component system regulatory genes. Enterococcus faecium strain BM4525, isolated two weeks later from the same patient, was resistant to high levels of both glycopeptides. The ddl gene of BM4525 had a 2 bp insertion leading to an impaired d -alanine: d -alanine ligase. Sequencing of the vanB operon in BM4525 also revealed an 18 bp deletion in the vanS B gene designated vanS BΔ . The resulting six amino acid deletion partially overlapped the G2 ATP-binding domain of the VanS BΔ histidine kinase leading to constitutive expression of the resistance genes. Sequence analysis indicated that the deletion occurred between two tandemly arranged heptanucleotide direct repeats, separated by 11 base-pairs. The VanS B , VanS BΔ and VanR B proteins were overproduced in Escherichia coli and purified. In vitro autophosphorylation of the VanS B and VanS BΔ histidine kinases and phosphotransfer to the VanR B response regulator did not differ significantly. However, VanS BΔ was deficient in VanR B phosphatase activity leading to accumulation of phosphorylated VanR B . Increased glycopeptide resistance in E. faecium BM4525 was therefore a result of the lack of production of d -alanyl- d -alanine ending pentapeptide and to constitutive synthesis of d -alanyl- d -lactate terminating peptidoglycan precursors, following loss of d -alanine: d -alanine ligase and of VanS B phosphatase activity respectively. We suggest that the heptanucleotide direct repeat in vanS B may favour the appearance of high level constitutively expressed vancomycin resistance through a ‘slippage’ type of genetic rearrangement in VanB-type strains.

Catalin E. Doneanu - One of the best experts on this subject based on the ideXlab platform.

  • The β4-β8 Groove Is an ATP-interactive Site in the α Crystallin Core Domain of the Small Heat Shock Protein, Human αB Crystallin
    Journal of Molecular Biology, 2006
    Co-Authors: Joy G. Ghosh, Scott A. Houck, Catalin E. Doneanu, John I. Clark
    Abstract:

    The site for ATP interactions in human αB crystallin, the archetype of small heat-shock proteins, was identified and characterized to resolve the controversial role of ATP in the function of small heat-shock proteins. Comparative sequence alignments identified the αB crystallin sequence, 82KHFSPEELKVKVLGD96 as a Walker-B ATP-binding Motif that is found in several ATP-binding proteins, including five molecular chaperones. Fluorescence resonance energy transfer and mass spectrometry using a novel fluorescent ATP analog, 8-azido-ATP-[γ]-1-naphthalenesulfonic acid-5(2-aminoethylamide) (azido-ATP-EDANS) and a cysteine mutant of human αB crystallin (S135C) conjugated with a fluorescent acceptor, eosin-5-maleimide (EMA) identified the β4-β8 groove as the ATP interactive site in αB crystallin. A 44% decrease in the emitted fluorescence of azido-ATP-EDANS at the absorption maximum of S135C-EMA and a corresponding 50% increase in the fluorescence emission of S135C-EMA indicated a close spatial relationship between azido-ATP-EDANS and the center of the β8 strand (131LTITSSLS138). Liquid chromatography, electrospray ionization mass spectrometry identified two peptide fragments of the αB crystallin Walker-B Motif photo-affinity-labeled with azido-ATP-EDANS confirming the β4-β8 groove as an ATP interactive site. The results presented here clearly establish the β4-β8 groove as the ATP interactive region in αB crystallin, and are in contrast to the existing paradigm that classifies small heat-shock proteins as ATP-independent chaperones.

  • The β4-β8 Groove Is an ATP-interactive Site in the α Crystallin Core Domain of the Small Heat Shock Protein, Human αB Crystallin
    Journal of Molecular Biology, 2006
    Co-Authors: Joy G. Ghosh, Scott A. Houck, Catalin E. Doneanu, John I. Clark
    Abstract:

    The site for ATP interactions in human αB crystallin, the archetype of small heat-shock proteins, was identified and characterized to resolve the controversial role of ATP in the function of small heat-shock proteins. Comparative sequence alignments identified the αB crystallin sequence, 82KHFSPEELKVKVLGD96 as a Walker-B ATP-binding Motif that is found in several ATP-binding proteins, including five molecular chaperones. Fluorescence resonance energy transfer and mass spectrometry using a novel fluorescent ATP analog, 8-azido-ATP-[γ]-1-naphthalenesulfonic acid-5(2-aminoethylamide) (azido-ATP-EDANS) and a cysteine mutant of human αB crystallin (S135C) conjugated with a fluorescent acceptor, eosin-5-maleimide (EMA) identified the β4-β8 groove as the ATP interactive site in αB crystallin. A 44% decrease in the emitted fluorescence of azido-ATP-EDANS at the absorption maximum of S135C-EMA and a corresponding 50% increase in the fluorescence emission of S135C-EMA indicated a close spatial relationship between azido-ATP-EDANS and the center of the β8 strand (131LTITSSLS138). Liquid chromatography, electrospray ionization mass spectrometry identified two peptide fragments of the αB crystallin Walker-B Motif photo-affinity-labeled with azido-ATP-EDANS confirming the β4-β8 groove as an ATP interactive site. The results presented here clearly establish the β4-β8 groove as the ATP interactive region in αB crystallin, and are in contrast to the existing paradigm that classifies small heat-shock proteins as ATP-independent chaperones.

Scott A. Houck - One of the best experts on this subject based on the ideXlab platform.

  • The β4-β8 Groove Is an ATP-interactive Site in the α Crystallin Core Domain of the Small Heat Shock Protein, Human αB Crystallin
    Journal of Molecular Biology, 2006
    Co-Authors: Joy G. Ghosh, Scott A. Houck, Catalin E. Doneanu, John I. Clark
    Abstract:

    The site for ATP interactions in human αB crystallin, the archetype of small heat-shock proteins, was identified and characterized to resolve the controversial role of ATP in the function of small heat-shock proteins. Comparative sequence alignments identified the αB crystallin sequence, 82KHFSPEELKVKVLGD96 as a Walker-B ATP-binding Motif that is found in several ATP-binding proteins, including five molecular chaperones. Fluorescence resonance energy transfer and mass spectrometry using a novel fluorescent ATP analog, 8-azido-ATP-[γ]-1-naphthalenesulfonic acid-5(2-aminoethylamide) (azido-ATP-EDANS) and a cysteine mutant of human αB crystallin (S135C) conjugated with a fluorescent acceptor, eosin-5-maleimide (EMA) identified the β4-β8 groove as the ATP interactive site in αB crystallin. A 44% decrease in the emitted fluorescence of azido-ATP-EDANS at the absorption maximum of S135C-EMA and a corresponding 50% increase in the fluorescence emission of S135C-EMA indicated a close spatial relationship between azido-ATP-EDANS and the center of the β8 strand (131LTITSSLS138). Liquid chromatography, electrospray ionization mass spectrometry identified two peptide fragments of the αB crystallin Walker-B Motif photo-affinity-labeled with azido-ATP-EDANS confirming the β4-β8 groove as an ATP interactive site. The results presented here clearly establish the β4-β8 groove as the ATP interactive region in αB crystallin, and are in contrast to the existing paradigm that classifies small heat-shock proteins as ATP-independent chaperones.

  • The β4-β8 Groove Is an ATP-interactive Site in the α Crystallin Core Domain of the Small Heat Shock Protein, Human αB Crystallin
    Journal of Molecular Biology, 2006
    Co-Authors: Joy G. Ghosh, Scott A. Houck, Catalin E. Doneanu, John I. Clark
    Abstract:

    The site for ATP interactions in human αB crystallin, the archetype of small heat-shock proteins, was identified and characterized to resolve the controversial role of ATP in the function of small heat-shock proteins. Comparative sequence alignments identified the αB crystallin sequence, 82KHFSPEELKVKVLGD96 as a Walker-B ATP-binding Motif that is found in several ATP-binding proteins, including five molecular chaperones. Fluorescence resonance energy transfer and mass spectrometry using a novel fluorescent ATP analog, 8-azido-ATP-[γ]-1-naphthalenesulfonic acid-5(2-aminoethylamide) (azido-ATP-EDANS) and a cysteine mutant of human αB crystallin (S135C) conjugated with a fluorescent acceptor, eosin-5-maleimide (EMA) identified the β4-β8 groove as the ATP interactive site in αB crystallin. A 44% decrease in the emitted fluorescence of azido-ATP-EDANS at the absorption maximum of S135C-EMA and a corresponding 50% increase in the fluorescence emission of S135C-EMA indicated a close spatial relationship between azido-ATP-EDANS and the center of the β8 strand (131LTITSSLS138). Liquid chromatography, electrospray ionization mass spectrometry identified two peptide fragments of the αB crystallin Walker-B Motif photo-affinity-labeled with azido-ATP-EDANS confirming the β4-β8 groove as an ATP interactive site. The results presented here clearly establish the β4-β8 groove as the ATP interactive region in αB crystallin, and are in contrast to the existing paradigm that classifies small heat-shock proteins as ATP-independent chaperones.