title: Improving FRET dynamic range with bright green and red fluorescent proteins.
abstract: A variety of genetically encoded reporters use changes in fluorescence (or Förster) resonance energy transfer (FRET) to report on biochemical processes in living cells. The standard genetically encoded FRET pair consists of CFPs and YFPs, but many CFP-YFP reporters suffer from low FRET dynamic range, phototoxicity from the CFP excitation light and complex photokinetic events such as reversible photobleaching and photoconversion. We engineered two fluorescent proteins, Clover and mRuby2, which are the brightest green and red fluorescent proteins to date and have the highest Förster radius of any ratiometric FRET pair yet described. Replacement of CFP and YFP with these two proteins in reporters of kinase activity, small GTPase activity and transmembrane voltage significantly improves photostability, FRET dynamic range and emission ratio changes. These improvements enhance detection of transient biochemical events such as neuronal action-potential firing and RhoA activation in growth cones.
authors.affiliation: Department of Bioengineering, Stanford University, Stanford, California, USA.
authors.forename: Amy J
authors.initials: AJ
authors.lastname: Lam
authors.forename: François
authors.initials: F
authors.lastname: St-Pierre
authors.forename: Yiyang
authors.initials: Y
authors.lastname: Gong
authors.forename: Jesse D
authors.initials: JD
authors.lastname: Marshall
authors.forename: Paula J
authors.initials: PJ
authors.lastname: Cranfill
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Baird
authors.forename: Michael R
authors.initials: MR
authors.lastname: McKeown
authors.forename: Jörg
authors.initials: J
authors.lastname: Wiedenmann
authors.forename: Michael W
authors.initials: MW
authors.lastname: Davidson
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Schnitzer
authors.forename: Roger Y
authors.initials: RY
authors.lastname: Tsien
authors.forename: Michael Z
authors.initials: MZ
authors.lastname: Lin
mesh_terms: D001483:Base Sequence; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D020742:rhoA GTP-Binding Protein / Q000378:metabolism; D000097573:Red Fluorescent Protein
chemical_list: D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017868:Cyclic AMP-Dependent Protein Kinases; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D020742:rhoA GTP-Binding Protein; D000097573:Red Fluorescent Protein