title: A General Strategy for the Semisynthesis of Ratiometric Fluorescent Sensor Proteins with Increased Dynamic Range.
abstract: We demonstrate how a combination of self-labeling protein tags and unnatural amino acid technology permits the semisynthesis of ratiometric fluorescent sensor proteins with unprecedented dynamic range in vitro and on live cells. To generate such a sensor, a binding protein is labeled with a fluorescent competitor of the analyte using SNAP-tag in conjugation with a second fluorophore that is positioned in vicinity of the binding site of the binding protein using unnatural amino acid technology. Binding of the analyte by the sensor displaces the tethered fluorescent competitor from the binding protein and disrupts fluorescence resonance energy transfer between the two fluorophores. Using this design principle, we generate a ratiometric fluorescent sensor protein for methotrexate that exhibits large dynamic ranges both in vitro (ratio changes up to 32) and on cell surfaces (ratio change of 13). The performance of these semisynthetic sensor proteins makes them attractive for applications in basic research and diagnostics.
authors.affiliation: Institute of Chemical Sciences and Engineering (ISIC), Institute of Bioengineering, National Centre of Competence in Research (NCCR) Chemical Biology, École Polytechnique Fédérale de Lausanne (EPFL) , 1015 Lausanne, Switzerland.
authors.forename: Lin
authors.initials: L
authors.lastname: Xue
authors.affiliation: Institute of Chemical Sciences and Engineering (ISIC), Institute of Bioengineering, National Centre of Competence in Research (NCCR) Chemical Biology, École Polytechnique Fédérale de Lausanne (EPFL) , 1015 Lausanne, Switzerland.
authors.forename: Efthymia
authors.initials: E
authors.lastname: Prifti
authors.affiliation: Institute of Chemical Sciences and Engineering (ISIC), Institute of Bioengineering, National Centre of Competence in Research (NCCR) Chemical Biology, École Polytechnique Fédérale de Lausanne (EPFL) , 1015 Lausanne, Switzerland.
authors.forename: Kai
authors.initials: K
authors.lastname: Johnsson
mesh_terms: D002232:Carbocyanines / Q000737:chemistry; D002462:Cell Membrane; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D057809:HEK293 Cells; D006801:Humans; D008727:Methotrexate / Q000032:analysis*; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis* / Q000235:genetics / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D014295:Trimethoprim / Q000378:metabolism
chemical_list: D002232:Carbocyanines; D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; C085321:cyanine dye 5; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate