Abstract

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). α-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 α-helices of equal length. The second and third categories of redox linkers were composed of α-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.

Original languageEnglish (US)
Pages (from-to)238-248
Number of pages11
JournalExperimental Biology and Medicine
Volume233
Issue number2
DOIs
StatePublished - Feb 1 2008

Keywords

  • Alpha-helical linker
  • Förster resonance energy transfer (FRET)
  • FRET efficiency measurements
  • Genetically encoded biosensor
  • Green fluorescent protein (GFP) variants
  • Redox-sensitive switch

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Fingerprint Dive into the research topics of 'Engineering redox-sensitive linkers for genetically encoded FRET-based biosensors'. Together they form a unique fingerprint.

  • Cite this