Sensing of remote oxyanion binding at the DNA binding domain of the molybdate-dependent transcriptional regulator, ModE

David H. Boxer, Han Zhang, David G. Gourley, William N. Hunter, Sharon M. Kelly, Nicholas C. Price

    Research output: Contribution to journalArticle

    5 Citations (Scopus)

    Abstract

    The molybdate-dependent transcriptional regulator ModE of Escherichia coli displays a large (50%) quenching of its intrinsic tryptophan fluorescence on binding molybdate. The changes in fluorescence have been exploited to analyse the binding of molybdate to ModE. Utilising site-directed mutagenesis, a series of phenylalanine substitutions for the three tryptophans of ModE (Trp49, Trp131 and Trp186) have been constructed, to yield three mono-Trp-containing derivatives. This has allowed an assessment to be made of the contribution of each of the three tryptophans to the spectral changes observed on binding molybdate; these are most distinctive for Trp186. Linkage between the DNA-binding and molybdate-binding sites (some 55 Å apart) is shown by (a) the small, but definite, effect of molybdate on the fluorescence of Trp49 which is located at the DNA-binding winged helix–turn–helix domain, and (b) the finding that the binding of either ligand is enhanced in the presence of the other. The studies demonstrate that the mono-Trp derivatives of ModE could be useful tools with which to study the signal transduction processes specifically associated with molybdate-dependent transcriptional regulation and that this approach may have wider implications for analysis of other regulated systems.
    Original languageEnglish
    Pages (from-to)2829-2837
    Number of pages9
    JournalOrganic and Biomolecular Chemistry
    Volume2
    Issue number19
    DOIs
    Publication statusPublished - 2004

    Fingerprint

    molybdates
    regulators
    deoxyribonucleic acid
    DNA
    tryptophan
    Tryptophan
    Fluorescence
    fluorescence
    Derivatives
    mutagenesis
    Signal transduction
    Mutagenesis
    phenylalanine
    Escherichia
    Site-Directed Mutagenesis
    molybdate
    Phenylalanine
    linkages
    Escherichia coli
    Quenching

    Cite this

    Boxer, David H. ; Zhang, Han ; Gourley, David G. ; Hunter, William N. ; Kelly, Sharon M. ; Price, Nicholas C. / Sensing of remote oxyanion binding at the DNA binding domain of the molybdate-dependent transcriptional regulator, ModE. In: Organic and Biomolecular Chemistry. 2004 ; Vol. 2, No. 19. pp. 2829-2837.
    @article{3f970f7b219940638509b5edbc2b2012,
    title = "Sensing of remote oxyanion binding at the DNA binding domain of the molybdate-dependent transcriptional regulator, ModE",
    abstract = "The molybdate-dependent transcriptional regulator ModE of Escherichia coli displays a large (50{\%}) quenching of its intrinsic tryptophan fluorescence on binding molybdate. The changes in fluorescence have been exploited to analyse the binding of molybdate to ModE. Utilising site-directed mutagenesis, a series of phenylalanine substitutions for the three tryptophans of ModE (Trp49, Trp131 and Trp186) have been constructed, to yield three mono-Trp-containing derivatives. This has allowed an assessment to be made of the contribution of each of the three tryptophans to the spectral changes observed on binding molybdate; these are most distinctive for Trp186. Linkage between the DNA-binding and molybdate-binding sites (some 55 {\AA} apart) is shown by (a) the small, but definite, effect of molybdate on the fluorescence of Trp49 which is located at the DNA-binding winged helix–turn–helix domain, and (b) the finding that the binding of either ligand is enhanced in the presence of the other. The studies demonstrate that the mono-Trp derivatives of ModE could be useful tools with which to study the signal transduction processes specifically associated with molybdate-dependent transcriptional regulation and that this approach may have wider implications for analysis of other regulated systems.",
    author = "Boxer, {David H.} and Han Zhang and Gourley, {David G.} and Hunter, {William N.} and Kelly, {Sharon M.} and Price, {Nicholas C.}",
    note = "dc.publisher: Royal Society of Chemistry",
    year = "2004",
    doi = "10.1039/B404185B",
    language = "English",
    volume = "2",
    pages = "2829--2837",
    journal = "Organic and Biomolecular Chemistry",
    issn = "1477-0520",
    publisher = "Royal Society of Chemistry",
    number = "19",

    }

    Sensing of remote oxyanion binding at the DNA binding domain of the molybdate-dependent transcriptional regulator, ModE. / Boxer, David H.; Zhang, Han; Gourley, David G.; Hunter, William N.; Kelly, Sharon M.; Price, Nicholas C.

    In: Organic and Biomolecular Chemistry, Vol. 2, No. 19, 2004, p. 2829-2837.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Sensing of remote oxyanion binding at the DNA binding domain of the molybdate-dependent transcriptional regulator, ModE

    AU - Boxer, David H.

    AU - Zhang, Han

    AU - Gourley, David G.

    AU - Hunter, William N.

    AU - Kelly, Sharon M.

    AU - Price, Nicholas C.

    N1 - dc.publisher: Royal Society of Chemistry

    PY - 2004

    Y1 - 2004

    N2 - The molybdate-dependent transcriptional regulator ModE of Escherichia coli displays a large (50%) quenching of its intrinsic tryptophan fluorescence on binding molybdate. The changes in fluorescence have been exploited to analyse the binding of molybdate to ModE. Utilising site-directed mutagenesis, a series of phenylalanine substitutions for the three tryptophans of ModE (Trp49, Trp131 and Trp186) have been constructed, to yield three mono-Trp-containing derivatives. This has allowed an assessment to be made of the contribution of each of the three tryptophans to the spectral changes observed on binding molybdate; these are most distinctive for Trp186. Linkage between the DNA-binding and molybdate-binding sites (some 55 Å apart) is shown by (a) the small, but definite, effect of molybdate on the fluorescence of Trp49 which is located at the DNA-binding winged helix–turn–helix domain, and (b) the finding that the binding of either ligand is enhanced in the presence of the other. The studies demonstrate that the mono-Trp derivatives of ModE could be useful tools with which to study the signal transduction processes specifically associated with molybdate-dependent transcriptional regulation and that this approach may have wider implications for analysis of other regulated systems.

    AB - The molybdate-dependent transcriptional regulator ModE of Escherichia coli displays a large (50%) quenching of its intrinsic tryptophan fluorescence on binding molybdate. The changes in fluorescence have been exploited to analyse the binding of molybdate to ModE. Utilising site-directed mutagenesis, a series of phenylalanine substitutions for the three tryptophans of ModE (Trp49, Trp131 and Trp186) have been constructed, to yield three mono-Trp-containing derivatives. This has allowed an assessment to be made of the contribution of each of the three tryptophans to the spectral changes observed on binding molybdate; these are most distinctive for Trp186. Linkage between the DNA-binding and molybdate-binding sites (some 55 Å apart) is shown by (a) the small, but definite, effect of molybdate on the fluorescence of Trp49 which is located at the DNA-binding winged helix–turn–helix domain, and (b) the finding that the binding of either ligand is enhanced in the presence of the other. The studies demonstrate that the mono-Trp derivatives of ModE could be useful tools with which to study the signal transduction processes specifically associated with molybdate-dependent transcriptional regulation and that this approach may have wider implications for analysis of other regulated systems.

    U2 - 10.1039/B404185B

    DO - 10.1039/B404185B

    M3 - Article

    VL - 2

    SP - 2829

    EP - 2837

    JO - Organic and Biomolecular Chemistry

    JF - Organic and Biomolecular Chemistry

    SN - 1477-0520

    IS - 19

    ER -