title: Robust red FRET sensors using self-associating fluorescent domains.
abstract: Elucidation of subcellular signaling networks by multiparameter imaging is hindered by a lack of sensitive FRET pairs spectrally compatible with the classic CFP/YFP pair. Here, we present a generic strategy to enhance the traditionally poor sensitivity of red FRET sensors by developing self-associating variants of mOrange and mCherry that allow sensors to switch between well-defined on- and off states. Requiring just a single mutation of the mFruit domain, this new FRET pair improved the dynamic range of protease sensors up to 10-fold and was essential to generate functional red variants of CFP-YFP-based Zn(2+) sensors. The large dynamic range afforded by the new red FRET pair allowed simultaneous use of differently colored Zn(2+) FRET sensors to image Zn(2+) over a broad concentration range in the same cellular compartment.
authors: Laboratory of Chemical Biology, Department of Biomedical Engineering, Eindhoven University of Technology , P.O. Box 513, 5600MB Eindhoven, The Netherlands.
authors: Laurens H
authors: LH
authors: Lindenburg
authors: Anne M
authors: AM
authors: Hessels
authors: Eduard H T M
authors: EH
authors: Ebberink
authors: Remco
authors: R
authors: Arts
authors: Maarten
authors: M
authors: Merkx
mesh_terms: D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D054852:Small Molecule Libraries; D015032:Zinc / Q000737:chemistry; D000097573:Red Fluorescent Protein
chemical_list: D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D054852:Small Molecule Libraries; D015032:Zinc; D000097573:Red Fluorescent Protein; C413663:fluorescent protein 583
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