Single-fluorophore biosensors based on conformation-sensitive GFP variants.

Area of Science:

• Biochemistry
• Molecular Biology
• Neuroscience

Background:

• Green Fluorescent Protein (GFP) forms a stable barrel protecting its chromophore.
• Existing biosensors often rely on Förster Resonance Energy Transfer (FRET) or multiple wavelengths.
• Protein kinase A (PKA) and calcium dynamics are crucial cellular processes requiring precise monitoring.

Purpose of the Study:

• To develop a single-wavelength biosensor by modifying GFP.
• To investigate mutations in GFP's beta-strands that confer sensitivity to protein interactions.
• To create novel biosensors for monitoring PKA and calcium signaling with enhanced fluorescence properties.

Main Methods:

• Site-directed mutagenesis of GFP beta-strand 7.
• Engineering of PKA and calcium sensors using modified GFP.
• Characterization of fluorescence intensity and lifetime changes.
• In vivo imaging of neuronal activity in brain slices using two-photon microscopy.

Main Results:

• Mutations in beta-strand 7 made GFP fluorescence sensitive to protein domain interactions.
• A single-wavelength PKA sensor was developed, relying on intrinsic GFP fluorescence changes.
• Calcium sensors incorporating the conformation-sensitive GFP (csGFP) showed calcium-dependent fluorescence.
• The csGFP sensors demonstrated high signal-to-baseline ratios and minimal photobleaching in neuronal imaging.

Conclusions:

• Mutations conferring conformational sensitivity to GFP enable new single-wavelength biosensor designs.
• These conformation-sensitive GFP variants are valuable tools for high-resolution imaging of cellular dynamics.
• The developed biosensors provide a sensitive and robust method for studying PKA and calcium signaling in neurons.