title: Development of a Reagentless Biosensor for Inorganic Phosphate, Applicable over a Wide Concentration Range.
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.
authors.affiliation: †The Francis Crick Institute, Mill Hill Laboratory, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom.
authors.forename: Claudia
authors.initials: C
authors.lastname: Solscheid
authors.affiliation: †The Francis Crick Institute, Mill Hill Laboratory, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom.
authors.forename: Simone
authors.initials: S
authors.lastname: Kunzelmann
authors.affiliation: †The Francis Crick Institute, Mill Hill Laboratory, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom.
authors.forename: Colin T
authors.initials: CT
authors.lastname: Davis
authors.affiliation: ‡MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom.
authors.forename: Jackie L
authors.initials: JL
authors.lastname: Hunter
authors.affiliation: ‡MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom.
authors.forename: Annie
authors.initials: A
authors.lastname: Nofer
authors.affiliation: †The Francis Crick Institute, Mill Hill Laboratory, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom.
authors.forename: Martin R
authors.initials: MR
authors.lastname: Webb
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D015374:Biosensing Techniques / Q000379:methods*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D033902:Periplasmic Binding Proteins / Q000737:chemistry / Q000235:genetics; D028044:Phosphate-Binding Proteins / Q000737:chemistry / Q000235:genetics; D010710:Phosphates / Q000032:analysis*; D011487:Protein Conformation; D015202:Protein Engineering; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012235:Rhodamines / Q000737:chemistry; D013050:Spectrometry, Fluorescence
chemical_list: D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D033902:Periplasmic Binding Proteins; D028044:Phosphate-Binding Proteins; D010710:Phosphates; C090133:PstS protein, E coli; D011994:Recombinant Proteins; D012235:Rhodamines; C005358:tetramethylrhodamine