Ground-state proton transfer in the photoswitching reactions of the fluorescent protein Dronpa

Warren, Mark M.; Kaucikas, Marius; Fitzpatrick, Ann; Champion, P. M.; Sage, J. Timothy; Thor, Jasper J. van

doi:10.1038/ncomms2460

Cited by 71 publications

(185 citation statements)

References 26 publications

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“…The 9 ps time constant, measured in 2 H 2 O at pD 7.8, was also in agreement with fluorescence measurements, which gave a 14 ps time constant in 1 H 2 O at pH 7.4 with excitation at 390nm and detection at 440 nm 9 . The IR difference spectrum of the primary photoproduct prominently lacks phenolate modes, thus excluding the possibility of excited state proton transfer, and was stable up to 100ps of the delays reported 10 . A recent study reported TR-IR measurements of the off state of the M159T mutant of Dronpa and came to very different conclusions 16 .…”

Section: As In the Aequorea Victoriamentioning

confidence: 87%

“…The fundamental mechanisms and sequence of events were investigated previously for the reversible photoswitching reactions of the wild type Dronpa fluorescent protein using visible-pump infrared-probe spectroscopy 10 . Previous proposals for the off-on switching invoked excited state proton transfer (ESPT) in addition to trans-cis photoisomerisation 9,11,12 .…”

Section: As In the Aequorea Victoriamentioning

confidence: 99%

“…Additionally, in the very similar asFP595 reversible photoswitchable fluorescent protein from the sea anemone Anemonia sulcata (which has a Met-Tyr-Gly derived chromophore) it was proposed that the chromophore is present as a 5 zwitterion in the trans form and that excited state proton transfer proceeds from the imidazolinone nitrogen 14,15 . FTIR difference spectroscopy identified the on and off states as the cis anion and the trans neutral chromophore, respectively 10 . Considering the high structural and spectroscopic similarities with Dronpa, these assignments could possibly also apply to the asFP595 on and off states.…”

Section: As In the Aequorea Victoriamentioning

confidence: 99%

“…Blue excitation of the photoaccumulated off state results in a dominant 9ps excited state decay component, with formation of a primary photoproduct that was assigned to the cis neutral chromophore on the basis of TR-IR measurements 10 . The 9 ps time constant, measured in 2 H 2 O at pD 7.8, was also in agreement with fluorescence measurements, which gave a 14 ps time constant in 1 H 2 O at pH 7.4 with excitation at 390nm and detection at 440 nm 9 .…”

Section: As In the Aequorea Victoriamentioning

confidence: 99%

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Photoisomerization and Proton Transfer in the Forward and Reverse Photoswitching of the Fast-Switching M159T Mutant of the Dronpa Fluorescent Protein

2014

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The fast-switching M159T mutant of the reversibly photoswitchable fluorescent protein Dronpa has an enhanced yield for the on-to-off reaction. The forward and reverse photoreactions proceed via cis-trans and trans-cis photoisomerization, yet protonation and deprotonation of the hydroxyphenyl oxygen of the chromophore is responsible for the majority of the resulting spectroscopic contrast. Ultrafast visible-pump, infrared-probe spectroscopy was used to detect the picosecond, nanosecond, as well as metastable millisecond intermediates. Additionally, static FTIR difference measurements of the Dronpa-M159T mutant correspond very closely to those of the wild type Dronpa, identifying the p-hydroxybenzylidene-imidazolinone chromophore in the cis anion and trans neutral forms in the bright "on" and dark "off" states, respectively. Green excitation of the on state is followed by dominant radiative decay with characteristic time constants of 1.9 ps, 185 ps, and 1.1 ns, and additionally reveals spectral changes belonging to the species decaying with a 1.1 ns time constant, associated with both protein and chromophore modes. A 1 ms measurement of the on state identifies bleach features that correspond to those seen in the static off-minus-on Fourier transform infrared (FTIR) difference spectrum, indicating that thermal protonation of the hydroxyphenyl oxygen proceeds within this time window. Blue excitation of the off state directly resolves the formation of the primary photoproduct with 0.6 and 14 ps time constants, which is stable on the nanosecond time scale. Assignment of the primary photoproduct to the cis neutral chromophore in the electronic ground state is supported by the frequency positions expected relative to those for the nonplanar distorted geometry for the off state. A 1 ms measurement of the off state corresponds closely with the on-minus-off FTIR difference spectrum, indicating thermal deprotonation and rearrangement of the Arg66 side chain to be complete.

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Section: As In the Aequorea Victoriamentioning

confidence: 87%

Section: As In the Aequorea Victoriamentioning

confidence: 99%

Section: As In the Aequorea Victoriamentioning

confidence: 99%

Section: As In the Aequorea Victoriamentioning

confidence: 99%

See 2 more Smart Citations

Photoisomerization and Proton Transfer in the Forward and Reverse Photoswitching of the Fast-Switching M159T Mutant of the Dronpa Fluorescent Protein

2014

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“…Starting with a protonated chromophore in the trans isomeric state, the hypothesis of an excited state proton transfer (ESPT) being the driving force for back photoswitching competes with that of isomerization taking place in the protonated state of the chromophore, followed by deprotonation in the cis conformation as a ground state process. Several investigations by ultrafast infrared and UV-Vis spectroscopy on Dronpa suggest that the latter hypothesis is the correct one [40,41]. In IrisFP, transient femtosecond absorption spectroscopy suggested the existence, upon excitation of the trans protonated state, of two excited-state intermediates with lifetimes in the picosecond regime, which may represent twisted chromophore species, possibly leading to isomerization (Figure 6a).…”

Section: On-off Photoswitchingmentioning

confidence: 96%

Deciphering Structural Photophysics of Fluorescent Proteins by Kinetic Crystallography

Bourgeois

2017

IJMS

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Because they enable labeling of biological samples in a genetically-encoded manner, Fluorescent Proteins (FPs) have revolutionized life sciences. Photo-transformable fluorescent proteins (PTFPs), in particular, recently attracted wide interest, as their fluorescence state can be actively modulated by light, a property central to the emergence of super-resolution microscopy. PTFPs, however, exhibit highly complex photophysical behaviours that are still poorly understood, hampering the rational engineering of variants with improved performances. We show that kinetic crystallography combined with in crystallo optical spectroscopy, modeling approaches and single-molecule measurements constitutes a powerful tool to decipher processes such as photoactivation, photoconversion, photoswitching, photoblinking and photobleaching. Besides potential applications for the design of enhanced PTFPs, these investigations provide fundamental insight into photoactivated protein dynamics.

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Photoreversible On–Off Recording of Persistent Room‐Temperature Phosphorescence

Katsurada

Hirata

Totani

et al. 2015

Advanced Optical Materials

116

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allow the movement of the targets to be detected, even when their motion is slow. Therefore, promising materials and techniques that enable clear detection of the local motion of an individual target independent of the persistent emission around the target and autofl uorescence still need to be designed. Photoactivated fl uorescent materials have been developed for highdensity recording and super-resolution imaging. [6][7][8][9][10][11][12] Some systems containing a fl uorescent compound and photochromic compound have also been reported. [8][9][10] The fl uorescence of the emissive materials is switched off and on by effi cient energy transfer from the fl uorescent compound to the photochromic one, which is triggered by photoisomerization of the photochromic compound. If persistent luminescent materials with the ability to be photoactivated can be prepared, the resulting photoactivated persistent emission process will potentially be useful for tracking the local movement of the target independent of other persistent emitting signals around the target as well as background autofl uorescence. However, persistent luminescence is commonly observed from rare metal-doped metal oxides [ 1,2,5 ] and it is still diffi cult to fabricate photoactivated metal oxide particles. Another approach that differs from conventional metal oxide-based persistent emitters is persistent room-temperature phosphorescence (RTP), which has a lifetime approaching one second and has recently been observed from purely organic amorphous [ 13 ] and crystalline materials. [ 14 ] The amorphous materials displaying RTP are composed of a metal-free aromatic guest in an amorphous hydroxyl steroidal host, which is rigid with an oxygen barrier because of its intermolecular hydrogen bonding. The rigidity and oxygen barrier of the steroidal host minimize the nonradiative deactivation from the triplet excited state of the purely organic guest caused by thermal relaxation and oxygen diffusion, resulting in effi cient persistent RTP from the guest species. In such amorphous host-guest materials, the rigidity and oxygen barrier of the amorphous hydroxyl steroidal host around the persistent RTP guest should remain when a small amount of photochromic compound is doped into the host as another guest. Therefore, we anticipate that effi cient energy transfer processes from the guest exhibiting persistent RTP to that exhibiting photochromism should occur in the amorphous hydroxyl steroidal host through photoisomerization of the photochromic compound, which should subsequently trigger on-off switching of persistent RTP.

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Ground-state proton transfer in the photoswitching reactions of the fluorescent protein Dronpa

Cited by 71 publications

References 26 publications

Photoisomerization and Proton Transfer in the Forward and Reverse Photoswitching of the Fast-Switching M159T Mutant of the Dronpa Fluorescent Protein

Photoisomerization and Proton Transfer in the Forward and Reverse Photoswitching of the Fast-Switching M159T Mutant of the Dronpa Fluorescent Protein

Deciphering Structural Photophysics of Fluorescent Proteins by Kinetic Crystallography

Photoreversible On–Off Recording of Persistent Room‐Temperature Phosphorescence

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