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Genetically encoding thyronine for fluorescent detection of peroxynitrite.

Shanshan Li1, Bing Yang1, Tomonori Kobayashi1

  • 1Department of Pharmaceutical Chemistry and the Cardiovascular Research Institute, University of California San Francisco, 555 Mission Bay Boulevard South, San Francisco, CA 94158, United States.

Bioorganic & Medicinal Chemistry
|August 24, 2020
PubMed
Summary

Researchers developed a new fluorescent probe to detect peroxynitrite, a reactive oxidant. This probe uses genetically encoded thyronine (Thy) in proteins, which becomes fluorescent when reacting with peroxynitrite.

Keywords:
FluorescenceGenetic code expansionPeroxynitriteThyronineUnnatural amino acid

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • Peroxynitrite is a potent reactive oxygen species involved in crucial cellular processes like signaling and death.
  • Accurate detection of peroxynitrite is vital for understanding its biological roles.
  • Existing detection methods may have limitations in specificity or application.

Purpose of the Study:

  • To develop a novel fluorescent protein probe for the selective detection of peroxynitrite.
  • To genetically encode the unnatural amino acid thyronine (Thy) in cellular systems.
  • To establish a responsive fluorescent reporter system based on Thy incorporation.

Main Methods:

  • Evolving an orthogonal tRNAPyl/ThyRS pair for the genetic encoding of thyronine (Thy).
  • Incorporating Thy into the chromophore of green fluorescent protein (GFP) variants (sfGFP, cpsGFP).
  • Treating Thy-containing proteins with peroxynitrite and monitoring fluorescence changes.

Main Results:

  • Successful genetic encoding of Thy in both E. coli and mammalian cells.
  • Thy incorporation into GFP chromophores resulted in a non-fluorescent or weakly fluorescent reporter.
  • Peroxynitrite treatment induced O-dearylation of Thy to tyrosine, restoring GFP fluorescence.
  • The fluorescence restoration was time- and concentration-dependent, indicating selective detection.

Conclusions:

  • A novel genetically encoded fluorescent probe for selective peroxynitrite detection has been established.
  • The Thy-based reporter system offers a sensitive and specific method for monitoring peroxynitrite.
  • Genetically encoded thyronine holds potential for broader applications in redox biology research.