Engineering redox-sensitive linkers for genetically encoded FRET-based biosensors

Exp Biol Med (Maywood). 2008 Feb;233(2):238-48. doi: 10.3181/0707-RM-192.


The ability to sense intracellular or intraorganellar reduction/oxidation conditions would provide a powerful tool for studying normal cell proliferation, differentiation, and apoptosis. Genetically encoded biosensors enable monitoring of the intracellular redox environment. We report the development of chimeric polypeptides useful as redox-sensitive linkers in conjunction with Förster resonance energy transfer (FRET). Alpha-helical linkers differing in length were combined with motifs that are sensitive to the redox state of the environment. The first category of linkers included a redox motif found in the thioredoxin family of oxidoreductases. This motif was flanked by two alpha-helices of equal length. The second and third categories of redox linkers were composed of alpha-helices with embedded adjacent and dispersed vicinal cysteine residues, respectively. The linkers containing redox switches were placed between a FRET pair of enhanced cyan and yellow fluorescent proteins and these constructs were tested subsequently for their efficacy. A robust method of FRET analysis, the (ratio)(A) method, was used. This method uses two fluorescence spectra performed directly on the FRET construct without physical separation of the fluorophores. The cyan/yellow construct carrying one of the designed redox linkers, RL5, exhibited a 92% increase in FRET efficiency from its reduced to oxidized states. Responsiveness of the cyan-RL5-yellow construct to changes in the intracellular redox environment was confirmed in mammalian cells by flow cytometry.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Biosensing Techniques / methods*
  • CHO Cells
  • Cricetinae
  • Cricetulus
  • Cysteine / genetics
  • Cysteine / metabolism
  • Flow Cytometry
  • Fluorescence Resonance Energy Transfer / methods*
  • Oxidation-Reduction
  • Protein Engineering / methods*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sensitivity and Specificity


  • Recombinant Proteins
  • Cysteine