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Biochemistry. 2015 Aug 18;54(32):5054-62. doi: 10.1021/acs.biochem.5b00449. Epub 2015 Aug 4.

Development of a Reagentless Biosensor for Inorganic Phosphate, Applicable over a Wide Concentration Range.

Author information

1
†The Francis Crick Institute, Mill Hill Laboratory, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom.
2
‡MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom.

Abstract

A fluorescent reagentless biosensor for inorganic phosphate (Pi), based on the E. coli PstS phosphate binding protein, was redesigned to allow measurements of higher Pi concentrations and at low, substoichiometric concentrations of biosensor. This was achieved by weakening Pi binding of the previous biosensor, and different approaches are described that could enable this change in properties. The readout, providing response to the Pi concentration, is delivered by tetramethylrhodamine fluorescence. In addition to two cysteine mutations for rhodamine labeling at positions 17 and 197, the final variant had an I76G mutation in the hinge region between the two lobes that make up the protein. Upon Pi binding, the lobes rotate on this hinge and the mutation on the hinge lowers affinity ∼200-fold, with a dissociation constant now in the tens to hundreds micromolar range, depending on solution conditions. The signal change on Pi binding was up to 9-fold, depending on pH. The suitability of the biosensor for steady-state ATPase assays was demonstrated with low biosensor usage and its advantage in ability to cope with Pi contamination.

PMID:
26199994
PMCID:
PMC4741387
DOI:
10.1021/acs.biochem.5b00449
[Indexed for MEDLINE]
Free PMC Article

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