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

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

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  • 1Institute for Genomic Biology, Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA. viadimer@uiuc.edu

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). 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.

PMID:
18222979
[PubMed - indexed for MEDLINE]

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