NitrOFF: An Engineered Fluorescent Biosensor to Illuminate Nitrate Transport in Living Cells

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Nitrate plays a dual role in human physiology, acting as both a pollutant and a nutrient linked to nitric oxide.
• Fundamental understanding of nitrate biology, particularly uptake and storage, is limited.
• Real-time imaging of nitrate in mammalian cells is challenging due to a lack of suitable biosensors.

Purpose of the Study:

• To engineer a genetically encoded fluorescent biosensor for real-time monitoring of nitrate in mammalian cells.
• To characterize the biosensor's response to nitrate binding and its underlying structural mechanism.

Main Methods:

• Designed and engineered a biosensor (NitrOFF) by fusing EGFP and the NreA nitrate-recognition domain.
• Utilized directed evolution over seven rounds, introducing 15 mutations to optimize the biosensor.
• Determined X-ray crystal structures of both apo and nitrate-bound NitrOFF.
• Assessed NitrOFF's utility in monitoring nitrate uptake in HEK293 cells.

Main Results:

• Developed NitrOFF, a biosensor exhibiting a turn-off intensiometric response with a K_d of approximately 9 µM.
• X-ray crystallography revealed a significant conformational change (68.4°) upon nitrate binding, driven by linker helix formation.
• The structural rearrangement alters EGFP fluorescence by detaching a key strand, effectively quenching fluorescence.
• Demonstrated NitrOFF's capability to monitor exogenous nitrate uptake in human embryonic kidney cells.

Conclusions:

• NitrOFF is a highly effective fluorescent biosensor for quantifying intracellular nitrate levels.
• The structural insights provide a basis for understanding nitrate-induced allosteric regulation in biosensors.
• NitrOFF enables real-time monitoring of nitrate dynamics in mammalian cells, advancing nitrate biology research.