A Genetically Encoded Calcium Ion Biosensor with an Exceptionally Large Ratiometric Response

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Ratiometric fluorescent protein (FP)-based calcium (Ca²⁺) biosensors often have limited Ca²⁺-dependent changes in emission ratio.
• Existing biosensors typically rely on Förster resonance energy transfer (FRET) between two FPs.

Purpose of the Study:

• To overcome limitations of existing ratiometric Ca²⁺ biosensors.
• To develop novel biosensors with substantially greater ratiometric changes.

Main Methods:

• Explored an alternative biosensor design strategy by hybridizing two independently optimized intensiometric single FP-based Ca²⁺ biosensors into a single protein construct.
• Utilized a shared calmodulin (CaM) and CaM-binding peptide (CBP) domain.
• Hybridized a direct-response red FP-based biosensor with an inverse-response green FP-based biosensor.

Main Results:

• Created SuiCa, a single-polypeptide Ca²⁺ biosensor.
• SuiCa exhibits exceptionally large red-to-green ratiometric fluorescence changes: ~60-fold in purified protein, ~80-fold in cell cultures, and ~37-fold in primary neurons.
• SuiCa offers advantages over co-expressed FP biosensors, including smaller gene size, fixed fluorophore stoichiometry, and a Ca²⁺-dependent response from a single shared domain.

Conclusions:

• SuiCa represents a new addition to the Ca²⁺ imaging toolbox.
• The biosensor provides bright fluorescence and large ratiometric changes.
• Offers improved Ca²⁺ detection capabilities for biological research.