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Development, Characterization, and Structural Analysis of a Genetically Encoded Red Fluorescent Peroxynitrite
Yu Pang1,2, Mian Huang3, Yichong Fan1,4
1Center for Membrane and Cell Physiology, University of Virginia, Charlottesville, Virginia 22908, United States.
ACS Chemical Biology
|May 15, 2023
Summary
Researchers developed a selective red fluorescent biosensor, pnRFP, for peroxynitrite detection. This advance expands the toolkit for sensing reactive nitrogen species in biological systems.
Area of Science:
- Biochemistry
- Molecular Biology
- Biophysics
Background:
- Boronic acid-based fluorescent molecules are used to detect reactive oxygen and nitrogen species.
- Achieving specificity in sensing these species remains a challenge.
- Genetically encoded green fluorescent peroxynitrite biosensors were previously developed using p-boronophenylalanine (pBoF).
Purpose of the Study:
- To engineer a selective red fluorescent peroxynitrite biosensor.
- To introduce pBoF into an enhanced circularly permuted red fluorescent protein (ecpApple).
- To characterize the structure and sensing mechanism of the new biosensor.
Main Methods:
- Genetic incorporation of pBoF into ecpApple.
- In vitro and live mammalian cell characterization of responsive mutants.
- X-ray crystallography, 11B-NMR, and computational studies for structural and mechanistic insights.
Main Results:
- Two responsive ecpApple mutants were created.
- One mutant senses both peroxynitrite and hydrogen peroxide.
- The pnRFP mutant demonstrates selective peroxynitrite sensing with a 'turn-off' fluorescence response.
- Structural analysis revealed the boron atom's geometry and the chromophore's twisting upon reaction.
Conclusions:
- A selective red fluorescent peroxynitrite biosensor (pnRFP) was successfully developed.
- The study provides mechanistic insights into the biosensor's response.
- This work expands the color options for genetically encoded biosensors and highlights protein scaffold versatility.
