Nitric Oxide Sensing by a Blue Fluorescent Protein

Area of Science:

• Biochemistry
• Cell Biology
• Molecular Biology

Background:

• S-nitrosylation of cysteine residues is a key post-translational modification for protein regulation.
• Fluorescent proteins (FPs) are vital tools in cell biology, but their response to nitric oxide (NO) is unexplored.
• Cysteine residues are crucial for chromophore formation in Green Fluorescent Protein (GFP)-derived FPs.

Purpose of the Study:

• To investigate the effect of nitric oxide on the fluorescence properties of blue-emitting fluorescent protein mTagBFP2.
• To determine if S-nitrosylation of cysteine residues mediates NO-induced changes in FP fluorescence.
• To explore the potential of mTagBFP2 as a genetically encoded NO sensor.

Main Methods:

• Exposure of mTagBFP2 to varying concentrations of nitric oxide.
• Measurement of fluorescence quantum yield and lifetime.
• Site-directed mutagenesis of cysteine residues in mTagBFP2.
• Expression of mTagBFP2 in mammalian cells for in vivo sensing.

Main Results:

• Micromolar concentrations of NO caused a ~30% reduction in mTagBFP2 fluorescence quantum yield and lifetime.
• Fluorescence was restored upon treatment with Na-dithionite, indicating a reversible modification.
• Mutating cysteine residues abolished NO sensitivity, confirming their role in the observed effect.
• mTagBFP2 successfully sensed exogenously generated NO in living mammalian cells.

Conclusions:

• Fluorescent proteins, including mTagBFP2, exhibit sensitivity to nitric oxide via S-nitrosylation of cysteine residues.
• mTagBFP2 can serve as a basis for developing new genetically encoded NO sensors.
• Fluorescence lifetime imaging of mTagBFP2 offers a promising approach for NO detection in biological systems.