Reversible molecular photoswitches: A key technology for nanoscience and fluorescence imaging

Sauer, Markus

doi:10.1073/pnas.0504264102

Cited by 99 publications

(65 citation statements)

References 23 publications

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“…To evaluate the properties of Dreiklang for imaging purposes, we prepared a layer of purified Dreiklang molecules and wrote complex patterns 6,30,31 into this layer showing that Dreiklang can be exploited for reversibly recording and reading information (Fig. 3a and Supplementary Movie 1).…”

Section: Use Of Dreiklang For Fluorescence Recovery After Switchingmentioning

confidence: 99%

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

et al. 2011

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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 ...

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Section: Use Of Dreiklang For Fluorescence Recovery After Switchingmentioning

confidence: 99%

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

et al. 2011

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“…Other than the photoactivatable FPs, RSFPs may be repeatedly and reversibly switched by irradiation between a fluorescent and a nonfluorescent state. Hence they exhibit unique advantages for protein tracking applications, subdiffraction microscopy, and several novel applications that had not been addressable previously (8)(9)(10)(11)(12)(13). Dronpa (14) and asFP595 (asulCP, asCP) (15) are the most prominent RSFPs.…”

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Structural basis for reversible photoswitching in Dronpa

Andresen¹,

Stiel²,

Trowitzsch

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

264

348

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Dronpa is a novel GFP-like fluorescent protein with exceptional light-controlled switching properties. It may be reversibly switched between a fluorescent on-state and a nonfluorescent off-state by irradiation with light. To elucidate the molecular basis of the switching mechanism, we generated reversibly switchable Dronpa protein crystals. Using these crystals we determined the elusive dark-state structure of Dronpa at 1.95-Å resolution. We found that the photoswitching results in a cis-trans isomerization of the chromophore accompanied by complex structural rearrangements of four nearby amino acid residues. Because of this cascade of intramolecular events, the chromophore is exposed to distinct electrostatic surface potentials, which are likely to influence the protonation equilibria at the chromophore. We suggest a comprehensive model for the light-induced switching mechanism, connecting a cascade of structural rearrangements with different protonation states of the chromophore. The recently discovered reversibly switchable FPs (RSFPs) are a further powerful class of FPs for cell biology and beyond. Other than the photoactivatable FPs, RSFPs may be repeatedly and reversibly switched by irradiation between a fluorescent and a nonfluorescent state. Hence they exhibit unique advantages for protein tracking applications, subdiffraction microscopy, and several novel applications that had not been addressable previously (8-13). Dronpa (14) and asFP595 (asulCP, asCP) (15) are the most prominent RSFPs. Like all FPs, they exhibit a GFP-like fold, namely a ␤-barrel enclosing an ␣-helix containing the autocatalytically formed chromophore. Because of its low quantum yield, the tetrameric asFP595 is only of limited use for cell biology applications, whereas Dronpa has been successfully used for several protein tracking studies (14,16,17). Dronpa is monomeric, displays favorable switching properties, and shows bright fluorescence with a remarkable fluorescence quantum yield of 0.85. Furthermore, several new Dronpa variants with accelerated switching kinetics have been described (18).Despite its tremendous potential for many applications, little is known about the molecular basis of the switching in Dronpa. Thus far competing models discussing either the light-driven regulation of the chromophoric protonation state by the surrounding protein matrix (19, 20) or postulating a cis-trans isomerization of the chromophore indirectly determining its own protonation state (18) have been suggested.To unravel this problem we generated Dronpa protein crystals that were reversibly switchable with visible light at ambient conditions. After switching the whole Dronpa crystals we determined its thus far elusive off-state structure by x-ray crystallography. We found that the primary event of the switching is a light-activated cis-trans isomerization of the chromophore together with a cascade of residue rearrangements. Because the electrostatic surface potentials in the chromophoric cis and trans cavities differ substantially, we postulate ...

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“…Only RSFPs can be used for repeated measurements of protein movements in live cells (3) and for the emerging technique of optical lock-in detection (OLID) (4). Moreover, RSFPs possess all the fundamental requirements to be useful for rewritable ultrahigh density optical data storage (5,6). In addition, RSFPs demonstrate great potential for use in recently developed superresolution microscopy techniques, such as reversible saturable optical fluorescence transition (RESOLFT) microscopy with ultralow light intensities (7,8) and photoactivated localization microscopy (PALM) (9) or stochastic optical reconstruction microscopy (STORM) (10), or fluorescence photoactivation localization microscopy (FPALM) (11) [collectively referred as (F)PALM/STORM].…”

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confidence: 99%

A unique series of reversibly switchable fluorescent proteins with beneficial properties for various applications

Chang

Zhang

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

120

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Reversibly switchable fluorescent proteins (RSFPs) have attracted widespread interest for emerging techniques including repeated tracking of protein behavior and superresolution microscopy. Among the limited number of RSFPs available, only Dronpa is widely employed for most cell biology applications due to its monomeric and other favorable photochemical properties. Here we developed a series of monomeric green RSFPs with beneficial optical characteristics such as high photon output per switch, high photostability, a broad range of switching rate, and pH-dependence, which make them potentially useful for various applications. One member of this series, mGeos-M, exhibits the highest photon budget and localization precision potential among all green RSFPs. We propose mGeos-M as a candidate to replace Dronpa for applications such as dynamic tracking, dual-color superresolution imaging, and optical lock-in detection.

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Reversible molecular photoswitches: A key technology for nanoscience and fluorescence imaging

Cited by 99 publications

References 23 publications

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

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

Structural basis for reversible photoswitching in Dronpa

A unique series of reversibly switchable fluorescent proteins with beneficial properties for various applications

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