Engineering a light-controllable fluorescent protein for peroxynitrite detection via genetic code expansion

Area of Science:

• Biochemistry
• Molecular Biology
• Chemical Biology

Background:

• Peroxynitrite is a reactive nitrogen species implicated in various physiological and pathological processes.
• Developing selective and sensitive probes for peroxynitrite detection is crucial for understanding its biological roles.
• Photoconvertible fluorescent proteins offer unique advantages for live-cell imaging due to their controllable fluorescence properties.

Purpose of the Study:

• To engineer a novel noncanonical amino acid-containing photoconvertible fluorescent protein for selective peroxynitrite detection.
• To create a peroxynitrite-responsive sensor that retains the photoconversion capability of the parent fluorescent protein.
• To expand the chemical diversity of light-controllable fluorescent proteins for advanced biological applications.

Main Methods:

• Site-specific incorporation of the noncanonical amino acid *p*-borono-l-phenylalanine into a circularly permuted mEos2 (cpM2) backbone.
• Characterization of the resulting protein's spectral properties, photoconversion efficiency, and response to peroxynitrite.
• Validation of the sensor's performance in vitro and potentially in cellular environments.

Main Results:

• Successful incorporation of *p*-borono-l-phenylalanine into cpM2 resulted in a novel fluorescent protein.
• The engineered protein demonstrated specific responsiveness to peroxynitrite, indicated by changes in its fluorescence.
• The sensor retained its characteristic photoconversion capability, allowing for temporal and spatial control of fluorescence.
• The sensor expands the toolkit for studying reactive nitrogen species in biological systems.

Conclusions:

• A noncanonical amino acid-containing photoconvertible fluorescent protein was successfully developed for selective peroxynitrite detection.
• This engineered sensor offers a valuable tool for real-time monitoring of peroxynitrite dynamics in biological research.
• The findings contribute to the advancement of chemical biology and fluorescent probe development for imaging reactive species.