Semisynthetic fluorescent sensor proteins based on self-labeling protein tags

Area of Science:

• Biochemistry
• Molecular Biology
• Cellular Imaging

Background:

• Genetically encoded fluorescent sensors allow metabolite concentration probing in living cells.
• Current methods require proteins with metabolite-induced conformational changes, limiting generality.
• A need exists for more versatile and broadly applicable biosensing tools.

Purpose of the Study:

• To develop a novel, generalizable approach for creating fluorescent biosensors.
• To overcome limitations of existing genetically encoded fluorescent sensor proteins.
• To enable probing of previously inaccessible metabolites using a modular design.

Main Methods:

• Constructed biosensors using SNAP-tag, a fluorescent protein, and a metabolite-binding protein.
• Synthetically labeled SNAP-tag with a fluorophore-containing ligand for the binding protein.
• Utilized metabolite-induced displacement of the ligand to induce conformational changes.
• Measured ratiometric changes in fluorescence intensity for readout.

Main Results:

• Developed semisynthetic fluorescent biosensors with a modular design.
• Achieved ratiometric fluorescence changes upon metabolite binding.
• Observed ratiometric changes comparable to existing genetically encoded sensors.
• Demonstrated facile generation of sensors at various wavelengths.

Conclusions:

• The SNAP-tag based semisynthetic approach overcomes limitations of traditional fluorescent biosensors.
• This method allows for the creation of versatile ratiometric fluorescent sensors.
• These biosensors are valuable tools for probing metabolite concentrations in living cells, including previously inaccessible ones.