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Identification of cognate ligands for the Escherichia coli phnD protein product and engineering of a reagentless fluorescent biosensor for phosphonates

Duke University Medical Center, Department of Biochemistry, Durham, North Carolina 27710, USA

(RECEIVED February 24, 2006; FINAL REVISION March 31, 2006; ACCEPTED March 31, 2006)

The Escherichia coli phnD gene is hypothesized to code for the periplasmic binding component of a phosphonate uptake system. Here we report the characterization of the phosphonate-binding properties of the phnD protein product. We find that PhnD exhibits high affinity for 2-aminoethylphosphonate (5 nM), the most commonly occurring natural phosphonate produced by lower eukaryotes, but also binds several other phosphonates with micromolar affinities. A significant number of man-made phosphonates, such as insecticides and chemical warfare agents, are chemical threats and environmental pollutants. Consequently, there is an interest in developing methods for the detection and bioremediation of phosphonates. Bacterial periplasmic-binding proteins have been utilized for developing reagentless biosensors that report analytes by coupling ligand-binding events to changes in the emission properties of a covalently conjugated environmentally-sensitive fluorophore. Several PhnD conjugates described here show large changes in fluorescence upon binding to methylphosphonate (MP), with two conjugates exhibiting up to 50% decrease in emission intensity. Since MP is the final degradation product of many nerve agents, these PhnD conjugates can function as components in a biosensor system for chemical warfare agents.

Keywords: fluorescent biosensor; Escherichia coli phnD; methyl phosphonate; nerve agent degradation product; periplasmic-binding protein; 2-aminoethylphosphonate

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.062135206.

Abbreviations: PhnD, phosphonate-binding protein; PBP, periplasmic-binding protein; MP, methylphosphonate; 2-AEP, 2-aminoethylphosphonate; AMP, aminomethylphosphonate; IAF, iodoacetamidofluorescein; NBD, N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole; EMPA, ethylmethylphosphonic acid; IMPA, isopropylmethylphosphonic acid; PMPA, pinacolylmethylphosphonic acid.

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