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Development, Characterization, and Structural Analysis of a Genetically Encoded Red Fluorescent Peroxynitrite

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Researchers developed a selective red fluorescent biosensor, pnRFP, for peroxynitrite detection. This advance expands the toolkit for sensing reactive nitrogen species in biological systems.

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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.