PMID:15455157
| Citation |
Boxer, DH, Zhang, H, Gourley, DG, Hunter, WN, Kelly, SM and Price, NC (2004) Sensing of remote oxyanion binding at the DNA binding domain of the molybdate-dependent transcriptional regulator, ModE. Org. Biomol. Chem. 2:2829-37 |
|---|---|
| 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 angstroms 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. |
| Links |
PubMed Online version:10.1039/b404185b |
| Keywords |
Amino Acid Sequence; Anions/chemistry; Anions/metabolism; Binding Sites; DNA/metabolism; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Fluorescence; Models, Biological; Models, Molecular; Molecular Sequence Data; Molybdenum/chemistry; Molybdenum/metabolism; Oxygen/metabolism; Phenylalanine/chemistry; Phenylalanine/genetics; Regulatory Elements, Transcriptional/genetics; Transcription Factors/genetics; Transcription Factors/metabolism; Tryptophan/chemistry; Tryptophan/genetics |
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