title: A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching.
abstract: Photoswitchable fluorescent proteins have enabled new approaches for imaging cells, but their utility has been limited either because they cannot be switched repeatedly or because the wavelengths for switching and fluorescence imaging are strictly coupled. We report a bright, monomeric, reversibly photoswitchable variant of GFP, Dreiklang, whose fluorescence excitation spectrum is decoupled from that for optical switching. Reversible on-and-off switching in living cells is accomplished at illumination wavelengths of ∼365 nm and ∼405 nm, respectively, whereas fluorescence is elicited at ∼515 nm. Mass spectrometry and high-resolution crystallographic analysis of the same protein crystal in the photoswitched on- and off-states demonstrate that switching is based on a reversible hydration/dehydration reaction that modifies the chromophore. The switching properties of Dreiklang enable far-field fluorescence nanoscopy in living mammalian cells using both a coordinate-targeted and a stochastic single molecule switching approach.
authors: Max Planck Institute for Biophysical Chemistry, Department of NanoBiophotonics, Göttingen, Germany.
authors: Tanja
authors: T
authors: Brakemann
authors: Andre C
authors: AC
authors: Stiel
authors: Gert
authors: G
authors: Weber
authors: Martin
authors: M
authors: Andresen
authors: Ilaria
authors: I
authors: Testa
authors: Tim
authors: T
authors: Grotjohann
authors: Marcel
authors: M
authors: Leutenegger
authors: Uwe
authors: U
authors: Plessmann
authors: Henning
authors: H
authors: Urlaub
authors: Christian
authors: C
authors: Eggeling
authors: Markus C
authors: MC
authors: Wahl
authors: Stefan W
authors: SW
authors: Hell
authors: Stefan
authors: S
authors: Jakobs
mesh_terms: D000818:Animals; D001709:Biotechnology / Q000379:methods*; D002522:Chlorocebus aethiops; D005453:Fluorescence; D036681:Fluorescence Recovery After Photobleaching; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D008853:Microscopy; D008958:Models, Molecular; D036103:Nanotechnology; D020033:Protein Isoforms / Q000378:metabolism; D014709:Vero Cells
chemical_list: D020033:Protein Isoforms; D049452:Green Fluorescent Proteins