We report the optimization of a novel redox-sensitive probe with enhanced dynamic range and an exceptionally wellpositioned oxidative midpoint redox potential. The present work characterizes factors that contribute to the improved Förster resonance energy transfer (FRET) performance of this green fluorescent protein (GFP)-based redox sensor. The a-helical linker, which separates the FRET donor and acceptor, has been extended in the new probe and leads to a decreased FRET efficiency in the linker's reduced, 'FRET-off' state. Unexpectedly, the FRET efficiency is increased in the new linker's oxidized, 'FRET-on' state compared with the parent probe, in spite of the longer linker sequence. The combination of a lowered baseline 'FRET-off' and an increased 'FRET-on' signal significantly improves the dynamic range of the probe for a more robust discrimination of its reduced and oxidized linker states. Mutagenesis of the cysteine residues within the α-helix linker reveals the importance of the fourth, C-terminal cysteine and the relative insignificance of the second cysteine in forming the disulfide bridge to clamp the linker into the high-FRET, oxidized state. To further optimize the performance of the redox probe, various cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP) FRET pairs, placed at opposite ends of the improved redox linker (RL7), were quantitatively compared and exchanged. We found that the CyPet/YPet and ECFP/YPet FRET pairs when attached to RL7 do not function well as sensitive redox probes due to a strong tendency to form heterodimers, which disrupt the α-helix. However, monomeric versions of CyPet and YPet (mCyPet and mYPet) eliminate dimerization and restore redox sensitivity of the probe. The best performing probe, ECFP-RL7-EYFP, exhibits an approximately six-fold increase in FRET efficiency in vitro when passing from the oxidized to the reduced state. We determined the midpoint redox potential of the probe to be -143±6 mV, which is ideal for measuring glutathione (GSH/GSSG) redox potentials in oxidative compartments of mammalian cells (e.g. the endoplasmic reticulum).

Original languageEnglish (US)
Pages (from-to)681-691
Number of pages11
JournalExperimental Biology and Medicine
Issue number6
StatePublished - Jun 2011


  • Förster resonance energy transfer
  • Genetically encoded biosensor
  • Glutathione
  • Green fluorescent protein variants
  • Redox potential
  • Redox-sensitive linker

ASJC Scopus subject areas

  • General Biochemistry, Genetics and Molecular Biology


Dive into the research topics of 'Development of a high-dynamic range, GFP-based FRET probe sensitive to oxidative microenvironments'. Together they form a unique fingerprint.

Cite this