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

    6 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

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