Serial Femtosecond Crystallography and Ultrafast Absorption

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Serial Femtosecond Crystallography and Ultrafast Absorption Spectroscopy of the Photoswitchable Fluorescent Protein IrisFP Jacques-Philippe Colletier, Michel Sliwa, François-Xavier Gallat, Michihiro Sugahara, Virginia Guillon, Giorgio Schirò, Nicolas Coquelle, Joyce Woodhouse, Laure Roux, Guillaume Gotthard, Antoine Royant, Lucas Martinez Uriarte, Cyril Ruckebusch, Yasumasa Joti, Martin Byrdin, Eiichi Mizohata, Eriko Nanga, Tomoyuki Tanaka, Kensuke Tono, Makina Yabashi, Virgile Adam, Marco Cammarata, Ilme Schlichting, Dominique Bourgeois, and Martin Weik J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.5b02789 • Publication Date (Web): 11 Feb 2016 Downloaded from http://pubs.acs.org on February 12, 2016

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Serial Femtosecond Crystallography and Ultrafast Absorption Spectroscopy of the Photoswitchable Fluorescent Protein IrisFP Jacques-Philippe Colletier1,*,‡, Michel Sliwa2,*,‡, François-Xavier Gallat1, Michihiro Sugahara3, Virginia Guillon1, Giorgio Schiro1, Nicolas Coquelle1, Joyce Woodhouse1, Laure Roux1, Guillaume Gotthard1,4, Antoine Royant1,4, Lucas Martinez Uriarte2, Cyril Ruckebusch2, Yasumasa Joti5, Martin Byrdin1, Eiichi Mizohata6, Eriko Nango3, Tomoyuki Tanaka3, Kensuke Tono5, Makina Yabashi3, Virgile Adam1, Marco Cammarata7, Ilme Schlichting8, Dominique Bourgeois1, Martin Weik1,* 1

Institut de Biologie Structurale, Univ. Grenoble Alpes, CEA, CNRS, F-38044 Grenoble, France Laboratoire de Spectrochimie Infrarouge et Raman, CNRS, University of Lille, 59655 Villeneuve d'Ascq, France 3 RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan 4 ESRF, 6 rue Jules Horowitz, BP 220, 38043 Grenoble Cedex, France 5 Japan Synchrotron Radiation Research Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5198, Japan 6 Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Osaka 565-0871, Japan 7 Department of Physics, UMR UR1-CNRS 6251, University of Rennes 1, Rennes, France 8 Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120 Heidelberg, Germany 2

Corresponding Author

[email protected], [email protected] , [email protected] Author Contributions

‡These authors contributed equally.

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ABSTRACT: Reversibly photoswitchable fluorescent proteins find growing applications in cell biology, yet mechanistic details, in particular on the ultra-fast photochemical time scale, remain unknown. We employed time-resolved pump-probe absorption spectroscopy on the reversibly photoswitchable fluorescent protein IrisFP in solution to study photoswitching from the nonfluorescent (off) to the fluorescent (on) state. Evidence is provided for the existence of several intermediate states on the pico- and microsecond time scales that are attributed to chromophore isomerization and proton transfer, respectively. Kinetic modeling favors a sequential mechanism with the existence of two excited state intermediates with lifetimes of 2 and 15 ps, the second of which controls the photoswitching quantum yield. In order to support that IrisFP is suited for time-resolved experiments aiming at a structural characterization of these ps intermediates, we used serial femtosecond crystallography at an X-ray free electron laser and solved the structure of IrisFP in its on state. Sample consumption was minimized by embedding crystals in mineral grease, in which they remain photoswitchable. Our spectroscopic and structural results pave the way for time-resolved serial femtosecond crystallography aiming at characterizing the structure of ultrafast intermediates in reversibly photoswitchable fluorescent proteins.

TOC GRAPHICS

KEYWORDS : photochemistry, fluorescent proteins, isomerization, chromophore, X-ray free electron laser, ultrafast spectroscopy



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Fluorescent proteins are widely-used genetically encoded markers in live biological cells1. A subgroup of them, so-called reversibly switchable fluorescent proteins (RSFPs)2, provide essential tools in advanced fluorescence nanoscopy of live cells (PALM, RESOLFT)3. RSFPs can be repeatedly toggled back and forth between a fluorescent (on) and a non-fluorescent (off) state by irradiation with light at two different wavelengths. The most extensively studied RSFP is Dronpa4, whose photoswitching mechanism involves chromophore isomerization between cis (on state) and trans (off state) along with proton transfer (off state neutral; on state anionic) as shown by X-ray crystallography5-6 and absorption spectroscopy7. Time-resolved ultrafast spectroscopic experiments on Dronpa and its mutants showed that several photoswitching intermediates exist with lifetimes on the pico- (ps) and the microsecond (µs) time scales813 . Whether isomerization and proton transfer occur sequentially or in a concerted manner yet remains a matter of debate. On the one hand, consensus has emerged recently that ps intermediates along the off-toon switching pathway reflect ultra-fast chromophore isomerization in the excited state, while µs intermediates result in chromophore deprotonation as a thermally driven ground-state process9, 12. On the other hand, a recent study13 argues that ps-intermediate states do not originate from isomerization, which instead would happen like proton transfer on the slower time scale. Ambiguities and open questions also exist in theoretical calculations14-15, e.g. concerning the existence of a twisted intramolecular charge transfer state14 and whether isomerization during photoswitching involves one-bond rotation14 or a hula twist mechanism15. So far, threedimensional structures of picosecond intermediates in either Dronpa or any other switchable fluorescent protein remain elusive. If available, they would provide first structural insight into early events of photoswitching and could validate one or several of the photoswitching mechanisms hypothesized based on spectroscopic experiments. Here we focused our attention on IrisFP16, a peculiar RSFP that is both photoconvertible from green (emission maximum: 516 nm) to red (emission maximum: 581 nm) and, in each of these states, reversibly photoswitchable between an on- and an off-state16 (Figure S1). The green off-to-on photoswitching mechanism of IrisFP was studied on both the fs-ps and the µs-ms time scales, using time-resolved absorption spectroscopy in H2O and D2O. Ultra-fast and slow processes were detected that we tentatively attribute to excited-state chromophore isomerization and ground-state proton transfer, respectively. However, only structural information may ultimately confirm or invalidate this premise. Structural elucidation of ultra-fast intermediates is not feasible using synchrotron sources, where the time resolution is limited to 100 ps. Rather, an X-ray free electron laser (XFEL) needs to be used, with pulse lengths of 5 to 50 fs, i.e. a duration

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well below the lifetime of these intermediate states. In order to prove the feasibility of such experiments, we employed serial femtosecond crystallography (SFX17-18) at an XFEL to determine the static structure of IrisFP in its green fluorescent on-state. Together, these photophysical and structural data lay a solid ground for carrying out time-resolved SFX experiments aiming at the determination of ultra-short photoswitching intermediates in RSFPs. As in Dronpa, photoswitching in IrisFP involves cistrans isomerization of the chromophore and substantial rearrangements of residues in the chromophore cavity as demonstrated by the 3D structures of the various dark (off) and fluorescent (on) states solved at a resolution between 1.8 and 2.2 Å by cryo-crystallography at a synchrotron16. In addition, switching is accompanied by a change in the chromophore protonation state as shown by UV-vis spectroscopy, i.e. the green anionic cis species absorbs at 488 nm and is fluorescent, whereas the green protonated trans state absorbs at 390 nm and does not fluoresce16. Compared to Dronpa, IrisFP is characterized by a higher off-to-on switching quantum yield (15% compared to 7%19), making it a priori more suitable for time-resolved SFX experiments. Ultrafast photodynamics of IrisFP in solution were monitored on the 100 fs to 1 ns time scale by means of femtosecond transient absorption spectroscopy upon excitation of the protein in its green off state (100 fs pulse, 400 nm; see Supplementary Materials and Methods). Upon excitation, the transient spectrum features two negative and two positive bands (Figure 1a and Figure S2). The positive bands (maxima at 320 and 445 nm) are characteristic of the absorption by excited state species. The negative band in the 350–415 nm region with a maximum at 382 nm corresponds to the depopulation of the green trans protonated off ground state (Figure S3), whereas the broad one in the 490-690 nm region is assigned to the stimulated emission of excited state species. The latter features a maximum at 565 nm, a long tail up to 700 nm and a shoulder at 518 nm. The shoulder originates from those molecules that are in the green cis anionic on state (about 10%) in the photostationary state of the solution (Figure S3). A global decay analysis of the transient spectra reveals components with characteristic times of 96 fs, 2.02 ps and 15.3 ps (Figure 1b, Figure S4). The fastest component characterizes the slight decay of the two positive bands within a few hundred femtoseconds (Figure 1a) and can be assigned to ultrafast intramolecular vibrational relaxation within the chromophore. The 2.02 ps and 15.3 ps components characterize the global reduction of the transient signals, with stimulated emission maxima at 600 and 540 nm, respectively (Figure 1b). These two ps components can be assigned to two excited-state species (I1*, I2*) that are formed and decay either sequentially or in parallel.


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Figure 1: (a) Ultrafast transient absorption spectra of IrisFP in the green trans protonated off state at pH 7. Spectra were recorded at different time delays following a 400-nm laser femtosecond excitation; the region near 400 nm is a blind zone due to strong laser scattering. (b) Decay associated spectra obtained by global fitting with three exponential functions (96 fs +/- 20 fs, 2.02 ps +/0.05 ps, 15.3 ps +/-0.3 ps) and a constant. (c) The upper panel shows microsecond transient absorption spectra of IrisFP in the green trans protonated off state recorded at different time delays following a 410-nm laser excitation. For a comparison, the lower panel shows two steady state difference spectra calculated between the green cis anionic on state at pH 7 and the green trans protonated off state at pH 7, on the one hand, and the green cis protonated state at pH 5 and the green trans protonated off state at pH 7, on the other hand. (d) Kinetics traces and multi-exponential fit at 490 nm for IrisFP in D2O and H2O on the microsecond time scale. Time-resolved UV-visible transient absorption was then followed from 10 ns up to the ms time scale in laser flash-photolysis experiments. The spectrum obtained 200 ns post-excitation (Figure 1c, upper panel) matches that previously reported for a green cis protonated state at pH 516 (Figure 1c, lower panel). The spectrum remains unchanged up to a few µs, and then slowly (10 ms) evolves (Figure 1c, upper panel) into that characteristic of the green cis anionic on state (Figure 1c, lower panel). In D2O solution the same bands are observed, but the recovery of the green cis anionic on state on the µs-

ms time scale shows a pronounced H/D exchange effect (Figure 1d and Figure S5), demonstrating the involvement of a proton transfer. Indeed, time constants of the three exponential functions used to fit the temporal evolution at 490 nm were 51 µs (38%), 2.7 ms (17%), and 16 ms (45%) in heavy water, to compare with 21 µs (33%), 227 µs (21%) and 2.1 ms (46%) in light water (Figure 1d and Figure S5). To the contrary, femtosecond transient absorption spectra and kinetics in D2O (49.5 fs, 2.1 ps and 17.9 ps) are similar to those in H2O (96 fs, 2.02 ps and 15.3 ps; Figure S6 and S7). Therefore, we propose that spectral changes on the µs-ms time scale (Figure 1c) reflect proton transfer during the transition from a green cis protonated state to the final green cis anionic on state (Figure 2) as proposed for Dronpa9, 12. Consequently, ps spectral changes (Figure 1a) are assigned to trans-cis isomerization characterizing the transition from the trans protonated off state to a green cis protonated state (Figure 2). In order to determine whether the two ps excited states I1*, I2* decay sequentially or in parallel, two kinetic models were tested using the hybrid hard- and softmultivariate curve resolution method20. Whereas in the case of Dronpa a parallel decay from several excited states has been put forward12, for IrisFP only a sequential mechanism fitted the fs data well (Figure 2 and Figure S8). The relaxation to the ground state (either to the green trans protonated off state or to a green cis protonated state; Figure 2 and S8) thus occurs solely from the second excited state species (I2*). This species is crucial as it controls the quantum yield of trans-to-cis isomerization, which was determined to be 17% using a com-



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parative method with a solution of Ru(bpy)3]2+ 21. I1* and I2* might either be two distinct excited states with different chromophore geometries, or represent hot and cold intermediates of the same excited state with the same chromophore geometry. Twisting around the ringbridging bonds has been reported to occur in the excited GFP chromophore in solution22. Even though the structural nature of I1* and I2* remains unknown, we speculate that such a twisting might also characterize the IrisFP chromophore in I2* (and possibly I1*), as a possible pathway for radiationless decay explaining the nonfluorescent character of this state. Only time-resolved SFX experiments23-24 revealing the three-dimensional structures of I1* and I2* will be able to confirm the exactness of the proposed photo-mechanism. Yet, whether or not such experiments are feasible on crystalline IrisFP had remained to be established.

Figure 2: General photo-mechanism of off–to-on photoswitching for green IrisFP in solution. In order to indicate the feasibility of time-resolved SFX experiments, we ambitioned carrying out static SFX on crystalline IrisFP in its on state. However, due to the small amount of available sample, liquid injection using a gas dynamic virtual nozzle could not be considered, since it typically requires tens of mg of crystalline sample. The only viable option was resorting to a recently developed injection technology, whereby microcrystals are embedded in mineral grease for presentation to the X-ray beam25. A pre-requisite was thus to verify that crystalline IrisFP embedded in the grease matrix remains indeed photoswitchable (Figure S11a). The thermal recovery to the on from the off state occurs with a half-life of 220 and 65 min in IrisFP crystals with and without grease embedding, respectively (see SI and Figure S16). The more than 3-fold slowdown of IrisFP thermal recovery in grease is in line with a recent study on Dronpa in solution, whose photoswitching kinetics has been reported to slow down when the viscosity of the surrounding solution was increased26. Using in crystallo micro-spectrophotometry, we observed slow spectral changes (time-scale of tens of minutes) after greaseembedding (Figure S11a) that are absent in a control experiment carried out on an IrisFP crystal without grease (Figure S11b). These changes are reverted upon restoring the initial humidity of crystalline IrisFP (Figure

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S11a). In order to assess a potential effect of grease embedding on the crystal structure, two datasets were collected at a synchrotron beamline by the standard oscillation method, from two IrisFP macrocrystals harvested from the same drop and flash-cooled at 100 K, either after cryo-protection and grease embedding (control 1), or after cryo-protection only (control 2). The Fourier difference electron density (Fo1-Fo2) map calculated between controls 1 and 2 (Riso 15.2 %) shows no features around the chromophore or elsewhere in the protein above a contour level of ± 3.5 σ (not shown), indicating there is no major effect of grease embedding on the structure of IrisFP at 100 K. For SFX experiments, crystals (Figure S9; 10 × 10 × 50-80 µm3) of IrisFP in its green on resting state were thus mixed with the grease and the mix containing randomly oriented protein microcrystals was streamed across the XFEL beam (pulse length < 10 fs; 7 keV photon energy; 30 Hz repetition rate) by means of syringeinjection at BL327 of SACLA28 (Figure S10). From 0.5 mg of crystalline IrisFP, 66300 diffraction patterns were collected within 37 min, of which 10899 were indexed using CrystFEL29 (overall redundancy 159, CC*: 0.98, Rsplit: 22 %; see table S1 for details). The structure was solved by molecular replacement at 2.75 Å resolution (CC* in 2.90 -2.75 Å resolution range: 0. 89; Rwork / Rfree 0.174 / 0.232; table S1), using the structure of IrisFP determined at 100 K16 as a starting model. The structure of IrisFP (tetrameric; 226 amino acids per monomer16, of which 220-222 are visible in the electron density maps) determined by SFX at room temperature is very similar to the one determined by traditional cryo-crystallography at the synchrotron (RMSD of 0.565 Å over the four monomers). In particular, the chromophore isomeric state can be determined as being cis (Figure 3a), as expected for the green fluorescent on state, and not trans (Figure 3b). Each of the three residues forming the chromophore (His62, Tyr63 and Gly64) are well defined in an electron density map calculated with a model from which the chromophore had been omitted (Figure 3a). Yet, the SFX structure displays minor changes compared to the synchrotron structure determined from a crystal embedded in grease and flashcooled at 100 K (control 1). A decrease in unit cell volume is observed at 100 K (-6%) that is reflected in a reduction of the chromophore-cavity volume by 25 % (for details see SI and Table S1). At the atomic level, the most striking difference between the SFX and the synchrotron structures is the presence of an alternate conformation in the Arg66 side chain in the SFX structure, which stabilizes the chromophore hydrogen bonding (Figure 3a). This difference and the change in the cavity volume might be due to the difference in data collection temperature. We suggest that the SFX structure determined from room-temperature data is physiologically more meaningful.


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in its green on state presented here can be envisioned as the first time point of a molecular movie. Time-resolved SFX on the ps timescale23 has now the potential to structurally resolve details of the isomerization mechanism, such as the putative existence of twisted chromophore intermediates. Such structural snapshots will provide essential insights into the structural dynamics of a fluorescent protein during the early events of photoactivation. This, in turn, could prove essential for the rational design of more efficient fluorescent proteins for applications in biological nanoscopy. ACKNOWLEDGMENT The authors are grateful to Leonard Chavas and So Iwata for technical support at SACLA. JPC thanks Duilio Cascio, Michael Sawaya, and David Eisenberg for stimulating discussions and continuing support. MSl is grateful to JeanPierre Verwaerde and Julien Dubois for technical support in the rotating cell design and time resolved experiments. The XFEL experiments were carried out at BL3 of SACLA with the approval of the Japan Synchrotron Radiation Research Institute (JASRI; Proposal No. 2013B8046), and the synchrotron experiments at ID29 of ESRF, under long-term projects MX1464, MX1583 and MX1676 (IBS BAG). We warmly thank the SACLA and ESRF staff for assistance.

ASSOCIATED CCCONTENT

Figure 3: The IrisFP chromophore in the SFX structure is in the cis conformation. The refined cis model is overlaid in (a), featuring Arg66 in two alternate conformations, whereas the trans model16 is overlaid in (b). Unbiased 2Fobs-Fcalc (contoured in grey at 1 σ) and Fo-Fc (contoured in red and green at -3 and +3 σ, respectively) electron density maps obtained from SFX data following molecular replacement starting with the cis (a) or the trans model (b) of IrisFP. Strong Fo-Fc peaks appear on the trans chromophore and on His194 and Glu144 (b). Thus, only the cis model fits the 2Fobs-Fcalc electron density map well. Our time-resolved absorption spectroscopy experiments of IrisFP in solution provided evidence for chromophore isomerization with a yield of 17%, involving two excited states on the ps timescale. Kinetic modeling is in favor of a sequential mechanism in which the excited state I2* with a life time of 15.3 ps controls the off-toon switching quantum yield. The structural nature of the spectroscopically-resolved intermediate state remains elusive. In view of future crystallographic experiments aiming at a structural characterization of this crucial intermediate, we used serial femtosecond crystallography (SFX) at an X-ray free electron laser to solve the structure of IrisFP in its on state. The structure of IrisFP

Supporting Information. SFX and synchrotron data collection, processing and statistics, structure solution and refinement, absorption spectroscopy of IrisFP crystals, time-resolved absorption spectroscopy. The Supporting Information is available free of charge on the ACS Publications website. This information can be found on the internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author

[email protected] , [email protected] , [email protected] Author Contributions

‡These authors contributed equally. Funding Sources

The study was supported by a grant from the CNRS (PEPS SASLELX) to MW, and ANR grants to MW, MC, MSl (BioXFEL) and to DB (NoBleach). The Cryobench is a platform of the Grenoble Instruct centre (ISBG; UMS 3518 CNRS–CEA–UJF–EMBL) with support from ESRF, FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10LABX-49-01). The Chevreul Institute (FR 2638), the Ministère de l’Enseignement Supérieur et de la Recherche, the Région Nord – Pas de Calais and FEDER are acknowledged for financial support to MS.

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