A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching

Abstract

VOLUME 29 NUMBER 10 OCTOBER 2011 nature biotechnology A r t i c l e sFluorescent proteins (FPs) 1 whose fluorescence can be reversibly or irreversibly switched by optical irradiation have opened new opportunities for the imaging of cells. They have facilitated in vivo protein-tracking schemes 2,3 , applications based on singlemolecule observations 4,5 and fluorescence microscopy with subdiffraction resolution [6][7][8][9][10] .Still, photoswitchable proteins have not displayed their full potential, because proteins that are just photoactivatable 11-13 can be switched only once, which implies that repeated measurements with the same molecule are impossible. On the other hand, photochromic or reversibly switchable fluorescent proteins (RSFPs) can be repeatedly photoswitched between a fluorescent and a nonfluorescent state by irradiation with light of two different wavelengths. However, in all previously characterized RSFPs, the wavelength used for generating the fluorescence emission is identical to one of the wavelengths used for switching the fluorescence on or off. The result is a complex interlocking of switching and fluorescence readout [14][15][16][17][18][19][20][21][22] , impeding or even precluding many applications, including fluorescence nanoscopy (super-resolution microscopy). Hence, the identification of an RSFP in which the generation of fluorescence is disentangled from switching has long been pursued. RESULTS Generation of the RSFP DreiklangNumerous GFP variants exhibit some degree of (generally undesirable) reversible photoswitching 4,23,24 . We found that the fluorescence of the yellow fluorescent protein Citrine 25,26 , a derivative of GFP, can be reversibly modulated to a small extent by alternate irradiation with light of 365 nm (on switching) and 405 nm (off switching), whereas fluorescence is excited at 515 nm. However, the achievable contrast was low, especially at pH values >6, rendering the reversible switching of Citrine unusable (Supplementary Fig. 1).To further develop this unusual switching behavior, we performed extensive random mutagenesis as well as directed PCR-mediated mutagenesis on a plasmid encoding Citrine. We transformed Escherichia coli with the plasmid, and screened with an automated home-built fluorescence microscope for bacterial colonies expressing fluorescent proteins whose fluorescence was excited with green light (515 nm) and which could be reversibly photoswitched from a fluorescent state to a long-lived nonfluorescent state by irradiation with near-UV (405 nm) light and back to a fluorescent state by UV (365 nm) light (Fig. 1a). In several consecutive screening rounds ~70,000 individual clones were analyzed. Finally, we identified a mutant differing from Citrine at four positions (Citrine-V61L, F64I, Y145H, N146D) ( Supplementary Fig. 2), which can be effectively switched and excited to fluoresce. We named this switchable fluorescent protein Dreiklang, the German word for a three-note chord in music.At thermal equilibrium, Dreiklang adopts the brightly fluorescent ...

Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate

“…Alexa 647, Dronpa, Dreinklang and rsEGFP) 3,[18][19][20] and (ii) irreversibly photoactivatable and photoconvertible (e.g. PA-GFP, PA-mCherry, mEos2, mEos3.1, mEos3.2, Dendra2, mClavGR2 and mMaple) [21][22][23][24][25][26][27] .…”

Fluorescence nanoscopy. Methods and applications

“…STED imaging can be performed on fusion constructs of fluorescent proteins that also span most of the visible spectrum: green (GFP [112]), yellow (YFP [113] and citrine [114]) and far-red emitting proteins (TagRFP657 [115] and the tetrametric E2-Crimson [116]). Several reversible photoswitchable proteins have also been demonstrated suitable for RESOLFT imaging, exploiting their reversible switching behaviour to deplete the periphery of the focal spot (Dronpa and its counterpart Padron derivative [15,22], rsEGFP2 [23] and Dreiklang [117]). …”

Imaging the cell surface and its organization down to the level of single molecules

“…This has been used to explore lipid organization on the cell surface [55]. The optimization of the fluorophores for STED has led to the reduction in the laser powers needed, reducing concerns about photodamage caused by the lasers [56].…”