title: Engineering redox-sensitive linkers for genetically encoded FRET-based biosensors.
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.
authors.affiliation: Institute for Genomic Biology, Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA. viadimer@uiuc.edu
authors.forename: Vladimir L
authors.initials: VL
authors.lastname: Kolossov
authors.forename: Bryan Q
authors.initials: BQ
authors.lastname: Spring
authors.forename: Anna
authors.initials: A
authors.lastname: Sokolowski
authors.forename: John E
authors.initials: JE
authors.lastname: Conour
authors.forename: Robert M
authors.initials: RM
authors.lastname: Clegg
authors.forename: Paul J A
authors.initials: PJ
authors.lastname: Kenis
authors.forename: H Rex
authors.initials: HR
authors.lastname: Gaskins
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D010084:Oxidation-Reduction; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity
chemical_list: D011994:Recombinant Proteins; D003545:Cysteine