Converting a binding protein into a biosensing conformational switch using protein fragment exchange.

Area of Science:

• Biotechnology
• Molecular Biology
• Biophysics

Background:

• Biosensors require functional coupling of recognition domains to output modules for signal transduction.
• Existing biosensor designs often rely on protein conformational changes, which can be limited by natural allosteric mechanisms and insufficient conformational changes.
• This limits the magnitude and reproducibility of signals, such as those measured by Förster resonance energy transfer (FRET).

Purpose of the Study:

• To introduce a new method, protein fragment exchange (FREX), to overcome limitations in biosensor design.
• To develop a robust and sensitive biosensor system capable of ratiometric Förster resonance energy transfer (FRET) responses.
• To demonstrate the versatility and functionality of FREX in various biological contexts.

Main Methods:

• Protein fragment exchange (FREX) utilizes a binding protein and a duplicated fragment that exchanges positions upon ligand binding.
• Fluorescent donor and acceptor groups are strategically placed on the binding protein and fragment to enable ratiometric FRET measurements.
• The FREX system was demonstrated using fibronectin III monobody scaffolds and Alexa FRET pairs.

Main Results:

• FREX sensors exhibited ratiometric FRET changes of up to 8.6-fold.
• The sensors performed consistently in both buffer and serum environments.
• A genetically encoded variant of the FREX sensor demonstrated functionality in cell lysates and mammalian cell cultures.

Conclusions:

• Protein fragment exchange (FREX) provides a powerful strategy for creating highly responsive and reproducible biosensors.
• The FREX system overcomes limitations of traditional protein conformational change-based biosensors.
• FREX technology is adaptable for diverse applications, including in vitro and in vivo cellular imaging and diagnostics.