A biosensor for inorganic phosphate using a rhodamine-labeled phosphate binding protein

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Inorganic phosphate (Pi) is crucial in cellular processes.
• Accurate and sensitive detection of Pi is essential for biological research.
• Existing biosensors may have limitations in sensitivity or real-time application.

Purpose of the Study:

• To develop a novel, highly sensitive biosensor for inorganic phosphate (Pi).
• To utilize a double-labeling strategy with rhodamine fluorophores on a phosphate-binding protein.
• To demonstrate the application of this biosensor in monitoring enzymatic activity.

Main Methods:

• Engineered a phosphate-binding protein from Escherichia coli with two cysteine mutations.
• Labeled the mutated sites with rhodamine fluorophores, exploiting fluorescence quenching through dimer formation.
• Investigated the conformational changes upon Pi binding and their effect on fluorescence intensity.
• Measured binding kinetics and dissociation constants at 10°C.

Main Results:

• Achieved an approximately 18-fold increase in fluorescence intensity upon inorganic phosphate binding with the best mutant (A17C, A197C).
• Determined a maximum rate of fluorescence increase of 267 s⁻¹ and a dissociation rate of 6.6 s⁻¹ for Pi.
• Demonstrated real-time measurement of ATP hydrolysis during helicase activity on DNA.

Conclusions:

• The developed double-labeled protein biosensor offers a sensitive and dynamic method for detecting inorganic phosphate.
• This novel approach provides advantages in fluorescence-based sensing and has potential for various biological applications.
• The sensor enables real-time monitoring of enzymatic processes involving Pi, such as ATP hydrolysis.