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. 2020 Feb 6;11(1):741.
doi: 10.1038/s41467-020-14537-0.

Photoswitching mechanism of a fluorescent protein revealed by time-resolved crystallography and transient absorption spectroscopy

Joyce Woodhouse  1 , Gabriela Nass Kovacs #  2 , Nicolas Coquelle #  1   3 , Lucas M Uriarte #  4 , Virgile Adam #  1 , Thomas R M Barends  2 , Martin Byrdin  1 , Eugenio de la Mora  1 , R Bruce Doak  2 , Mikolaj Feliks  5 , Martin Field  1   6 , Franck Fieschi  1 , Virginia Guillon  1 , Stefan Jakobs  7 , Yasumasa Joti  8 , Pauline Macheboeuf  1 , Koji Motomura  9 , Karol Nass  2 , Shigeki Owada  10 , Christopher M Roome  2 , Cyril Ruckebusch  4 , Giorgio Schirò  1 , Robert L Shoeman  2 , Michel Thepaut  1 , Tadashi Togashi  8 , Kensuke Tono  8 , Makina Yabashi  10 , Marco Cammarata  11 , Lutz Foucar  2 , Dominique Bourgeois  1 , Michel Sliwa  12 , Jacques-Philippe Colletier  1 , Ilme Schlichting  13 , Martin Weik  14
Affiliations

Photoswitching mechanism of a fluorescent protein revealed by time-resolved crystallography and transient absorption spectroscopy

Joyce Woodhouse et al. Nat Commun. .

Abstract

Reversibly switchable fluorescent proteins (RSFPs) serve as markers in advanced fluorescence imaging. Photoswitching from a non-fluorescent off-state to a fluorescent on-state involves trans-to-cis chromophore isomerization and proton transfer. Whereas excited-state events on the ps timescale have been structurally characterized, conformational changes on slower timescales remain elusive. Here we describe the off-to-on photoswitching mechanism in the RSFP rsEGFP2 by using a combination of time-resolved serial crystallography at an X-ray free-electron laser and ns-resolved pump-probe UV-visible spectroscopy. Ten ns after photoexcitation, the crystal structure features a chromophore that isomerized from trans to cis but the surrounding pocket features conformational differences compared to the final on-state. Spectroscopy identifies the chromophore in this ground-state photo-intermediate as being protonated. Deprotonation then occurs on the μs timescale and correlates with a conformational change of the conserved neighbouring histidine. Together with a previous excited-state study, our data allow establishing a detailed mechanism of off-to-on photoswitching in rsEGFP2.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Structure and photoswitching scheme of rsEGFP2.
a Three-dimensional structure showing an 11-stranded β-barrel embedding a chromophore held by a central α-helix. b The chromophore of rsEGFP2 in its fluorescent on-state (bottom) absorbs at 479 nm (laser 488 nm) and emits at 503 nm. Isomerization competes with fluorescence and leads the chromophore to its non-fluorescent off-state, which absorbs at 403 nm (laser 400 nm) and ultimately isomerizes back to the initial on-state. Flashes and arrows represent the color of each actinic wavelength. The width of arrows is representative of the efficiency of each isomerization.
Fig. 2
Fig. 2. Transient UV–Visible spectroscopy in H2O solution.
Time-resolved difference absorption spectra of rsEGFP2 in H2O solution (50 mM HEPES pH 8, 50 mM NaCl) recorded after a 410 nm nanosecond excitation of the trans-protonated off-state in the time windows from 100 ns to 9 ms (a–c). The spectrum without laser excitation was subtracted to calculate the difference spectra. The colored arrows (red in (a), cyan in (b) and light green in (c)) correspond to the three time constants (5.57, 36.1 and 825 µs, respectively) identified by a global fit analysis of kinetic traces for all wavelengths. d Decay associated spectra obtained by fitting the kinetic traces in panels (a–c) for all wavelengths with a weighted sum of three exponential functions.
Fig. 3
Fig. 3. Off-state structure.
Chromophore (HBI) and its neighboring residues in the rsEGFP2 laser_off structure determined by SFX without pump-laser activation are shown. a The initial laser-off model consists of a mixture of the off-state conformer (light gray carbon trace) and the residual on-state conformer (yellow carbon trace) with occupancies of 90% and 10%, respectively. 2Fobslaser_off–Fcalclaser_off (blue) and Fobslaser_off–Fcalclaser_off (green/red) maps at 1.6 Å resolution are displayed at 1σ and ±3σ, respectively. A positive Fobslaser_off –Fcalclaser_off peak between the trans (gray) and the cis (yellow) chromophores suggests the presence of an additional conformer. b Result of ensemble refinements against the laser_off dataset starting for the off-state model (chromophore 100% trans1). A third chromophore conformation and a third rotamer of His149 are revealed. (c) The final laser-off model features triple conformations of His149 and of the chromophore, i.e. His149-off and trans1 (light gray), His149-on and cis (yellow) and the additional His149-supp and trans2 (dark gray) conformations, at 65%, 10% and 25% occupancy, respectively. 2Fobslaser_off–Fcalclaser_off (blue) and Fobslaser_off–Fcalclaser_off (green/red) maps calculated from the laser-off dataset at 1.6 Å resolution are displayed at 1σ and ±3σ, respectively.
Fig. 4
Fig. 4. 10-ns intermediate-state structure.
a Q-weighted difference electron density map (Fobslaser_on_∆10ns–Fobslaser_off), determined from SFX data with and without pump-laser activation, is contoured at +3σ (green) and −3σ (red) and overlaid onto the model determined from the laser_off dataset. b Model of the laser_on_∆10 ns intermediate structure (cyan) determined by difference refinement at 1.85 Å resolution. 2Fextrapolatedlaser_on_∆10ns–Fcalc (blue, 1σ) and Fextrapolatedlaser_on_∆10ns–Fcalc maps (green/red, ±3σ, respectively) are shown.
Fig. 5
Fig. 5. Overlay of 10-ns intermediate-state and off-state structures.
The chromophore regions in the 10-ns intermediate structure (cyan) and the laser-off model (gray) are shown. For the sake of clarity, only the major conformer (65% occupancy) of the laser-off model is shown. Note that His149 is hydrogen bonded to Tyr146 in the laser-off model and to the chromophore in the 10-ns intermediate structure.
Fig. 6
Fig. 6. Overlay of 10-ns intermediate-state and on-state structures.
Overlay (a, d) of the chromophore region in the 10-ns intermediate structure (cyan, b, e) and the on-state structure (yellow, c, f) determined from SFX at room temperature (PDB entry code 5O89). The focus is on the phenol moiety of the chromophore, His149 and water W356 (a–c) and on the entire chromophore, Ser206 and Glu223 (d–f). Key distances are indicated.
Fig. 7
Fig. 7. Model for the rsEGFP2 off-to-on photoswitching process.
The bold purple bar represents the laser_on_∆10 ns intermediate structure determined by TR-SFX. Time constants correspond to those determined by femtosecond (87 ps) and nanosecond-resolved pump–probe UV–visible absorption spectroscopy (5.57, 36.1 and 825 µs) in H2O solution (50 mM HEPES pH 8, 50 mM NaCl). All three cis protonated states (purple bars) are ground-state intermediates, interconversion of which involves rearrangements of the protein matrix (orange arrows). Corresponding structural models for the chromophore and His149 as determined by X-ray crystallography are shown in panels a, b (this work) and d. Trans1 and His149-off (light gray) and trans2 and His149-supp (dark gray) of the trans-protonated off-state (a, Fig. 3c) transited to cis and His149-off-like at 10 ns (b, Fig. 4b, Fig. 6b, e). His149-off-like has moved to His149-on in the cis anionic on state (d, Fig. 6c, f). The structure whose intermediate forms in 5.57 µs remains elusive (c).
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