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

Claudia Solscheid,Jackie L Hunter,Simone Kunzelmann,Colin T Davis,Annie Nofer,Martin R Webb

doi:10.1021/acs.biochem.5b00449

Claudia Solscheid, Jackie L Hunter + Show 4 more

Open Access

https://doi.org/10.1021/acs.biochem.5b00449

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Journal: Biochemistry	Publication Date: Aug 4, 2015
Citations: 35	License type: publisher-specific-oa

Affiliation: The Francis Crick Institute

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.

Highlights

Inorganic phosphate (Pi) is a byproduct of numerous reactions in the cell, including metabolic reactions like fatty acid metabolism, energy transducing ATPases and cell signaling, such as by GTPases and protein phosphatases
The associated conformational change could be targeted by modifying amino acids that are in the cleft and modify its closure, but are not involved in Pi binding
There were significant differences in effects of mutations in the E. coli protein, used here, from those described for Synechococcus PBP, predicted to be a close structural homologue.[29]

Summary

Introduction

Inorganic phosphate (Pi) is a byproduct of numerous reactions in the cell, including metabolic reactions like fatty acid metabolism, energy transducing ATPases and cell signaling, such as by GTPases and protein phosphatases. A variant of rho-PBP is described here with much lower affinity for Pi, but retaining a fluorescence change on Pi binding.

Results

Conclusion