CUTie2: The Attack of the Cyclic Nucleotide Sensor Clones

Area of Science:

• Biophysics
• Molecular Biology
• Cellular Imaging

Background:

• Genetically encoded Förster Resonance Energy Transfer (FRET) sensors allow sub-cellular detection of small molecules, advancing signaling pathway research.
• Designing new FRET sensors is challenging due to large polypeptide sizes, conformational variability, and reliance on empirical methods.

Purpose of the Study:

• To engineer a new-generation FRET sensor for the second messenger cyclic guanosine monophosphate (cGMP).
• To utilize a computational approach for rational FRET sensor design, applicable to other allosteric protein modules.

Main Methods:

• Sequence/structure analysis and coarse-grained molecular dynamics simulations were employed.
• In vitro experiments and biochemical characterization validated the sensor's performance and binding properties.

Main Results:

• A novel FRET sensor, CUTie2, was developed for cGMP based on Protein kinase G I (PKGI).
• Molecular dynamics simulations accurately predicted FRET efficiency, confirmed by experimental data.
• The cGMP binding module retained high affinity and selectivity comparable to the full-length PKGI protein.

Conclusions:

• The computational strategy offers a cost-effective method for custom FRET sensor design.
• This approach is generalizable for engineering sensors for various allosteric protein modules and small molecules.