Dheerendra Yadav

Fabien Lacombat

Nadia Dozova

Fabrice Rappaport

Pascal Plaza

Agathe Espagne

Real-Time Monitoring of Chromophore Isomerization and Deprotonation During the Photoactivation of the Fluorescent Protein Dronpa

Keywords: Green Fluorescent Protein, photochromism, cis/trans photoisomerization, proton transfer, femtosecond UV-visible spectroscopy, nanosecond UV-visible spectroscopy

Dronpa is a GFP-related photochromic fluorescent protein used as probe in superresolution microscopy. It is known that the photochromic reaction involves cis/trans isomerization of the chromophore and protonation/deprotonation of its phenol group, but the sequence in time of the two steps and their characteristic timescales are still the subject of much debate. We report here a comprehensive UV-visible transient absorption spectroscopy study of the photoactivation mechanism of Dronpa, covering all relevant timescales from ~100 fs to milliseconds. The Dronpa-2 variant was also studied and showed the same behavior. By carefully controlling the excitation energy to avoid multiphoton processes, we could measure both the spectrum and the anisotropy of the first photoactivation intermediate. We show that the observed few nanometer blue-shift of this intermediate is characteristic for a neutral cis chromophore, and that its anisotropy of ~0.2 is in good agreement with the reorientation of the transition dipole moment expected upon isomerization. These data constitute the first clear evidence that trans→cis isomerization of the chromophore precedes its deprotonation and occurs on the picosecond timescale, concomitantly to the excited-state decay. We found the deprotonation step to follow in ~10 μs and lead directly from the neutral cis intermediate to the final state.

INTRODUCTION

Photochromic fluorescent proteins are proteins homologous to the Green Fluorescent Protein (GFP) developed in the last decade, which can be switched back and forth between a fluorescent state (ON state) and a nonfluorescent state (OFF state) by irradiation at two wavelengths. These distinctive photophysical properties have led to the emergence of novel imaging techniques based on the optical modulation of fluorescence signals, such as super-resolution fluorescence imaging techniques (RESOLFT, [START_REF] Hofmann | Breaking the Diffraction Barrier in Fluorescence Microscopy at Low Light Intensities by Using Reversibly Photoswitchable Proteins[END_REF] PALM, 2 pcSOFI 3 ), repeated photoactivation for protein tracking, [START_REF] Ando | Regulated Fast Nucleocytoplasmic Shuttling Observed by Reversible Protein Highlighting[END_REF] photochromic FRET (pcFRET) [START_REF] Subach | Red Fluorescent Protein with Reversibly Photoswitchable Absorbance for Photochromic FRET[END_REF] and optical lock-in detection microscopy (OLID). [START_REF] Marriott | Optical Lock-In Detection Imaging Microscopy for Contrast-Enhanced Imaging in Living Cells[END_REF] Due to the wealth of applications, the molecular mechanisms responsible for photochromism in fluorescent proteins are arousing considerable interest. The three-dimensional structures of the ON and OFF states of several photochromic fluorescent proteins have been determined by Xray crystallography, showing that in most cases ON-OFF photoswitching is based on cis/trans isomerization of the chromophore coupled with proton transfer. [START_REF] Andresen | Structure and Mechanism of the Reversible Photoswitch of a Fluorescent Protein[END_REF][START_REF] Andresen | Structural basis for reversible photoswitching in Dronpa[END_REF][START_REF] Brakemann | Molecular Basis of the Light-Driven Switching of the Photochromic Fluorescent Protein Padron[END_REF][START_REF] Henderson | Structural Basis for Reversible Photobleaching of a Green Fluorescent Protein Homologue[END_REF][START_REF] Regis Faro | Low-Temperature Chromophore Isomerization Reveals the Photoswitching Mechanism of the Fluorescent Protein Padron[END_REF] This is however only a static picture, and questions remain regarding the order in which these two events happen, their characteristic timescales and detailed mechanisms.

The prototype of photochromic fluorescent proteins is Dronpa, a green-emitting protein from a Pectiniidae coral. [START_REF] Ando | Regulated Fast Nucleocytoplasmic Shuttling Observed by Reversible Protein Highlighting[END_REF] Like other GFP-related proteins, Dronpa is made of an 11-stranded β-barrel containing a covalently attached phydroxybenzylideneimidazolinone chromophore. [START_REF] Andresen | Structural basis for reversible photoswitching in Dronpa[END_REF][START_REF] Wilmann | The 1.7 A Crystal Structure of Dronpa: a Photoswitchable Green Fluorescent Protein[END_REF][START_REF] Stiel | 1.8 A Bright-State Structure of the Reversibly Switchable Fluorescent Protein Dronpa Guides the Generation of Fast Switching Variants[END_REF] In the equilibrated ON state and at neutral pH, the central ethylene bridge of the chromophore adopts a cis configuration and the phenol group is deprotonated, leading to green fluorescence peaking at 517 nm and a main absorption band at 503 nm. [START_REF] Ando | Regulated Fast Nucleocytoplasmic Shuttling Observed by Reversible Protein Highlighting[END_REF][START_REF] Andresen | Structural basis for reversible photoswitching in Dronpa[END_REF][START_REF] Wilmann | The 1.7 A Crystal Structure of Dronpa: a Photoswitchable Green Fluorescent Protein[END_REF][START_REF] Stiel | 1.8 A Bright-State Structure of the Reversibly Switchable Fluorescent Protein Dronpa Guides the Generation of Fast Switching Variants[END_REF][START_REF] Mizuno | Light-Dependent Regulation of Structural Flexibility in a Photochromic Fluorescent Protein[END_REF][START_REF] Li | A Theoretical Study on the Nature of On-and Off-States of Reversibly Photoswitching Fluorescent Protein Dronpa: Absorption, Emission, Protonation and Raman[END_REF] Upon irradiation in this band, Dronpa converts to the OFF state, which absorbs maximally at 390 nm and corresponds to the trans-phenol form of the chromophore [START_REF] Ando | Regulated Fast Nucleocytoplasmic Shuttling Observed by Reversible Protein Highlighting[END_REF][START_REF] Andresen | Structural basis for reversible photoswitching in Dronpa[END_REF][START_REF] Wilmann | The 1.7 A Crystal Structure of Dronpa: a Photoswitchable Green Fluorescent Protein[END_REF][START_REF] Stiel | 1.8 A Bright-State Structure of the Reversibly Switchable Fluorescent Protein Dronpa Guides the Generation of Fast Switching Variants[END_REF][START_REF] Mizuno | Light-Dependent Regulation of Structural Flexibility in a Photochromic Fluorescent Protein[END_REF][START_REF] Li | A Theoretical Study on the Nature of On-and Off-States of Reversibly Photoswitching Fluorescent Protein Dronpa: Absorption, Emission, Protonation and Raman[END_REF] (Scheme 1). The ON state can be regenerated by irradiation of the OFF state at ~390 nm, or recovers spontaneously in the dark with a half-life of 14 hours. [START_REF] Ando | Regulated Fast Nucleocytoplasmic Shuttling Observed by Reversible Protein Highlighting[END_REF] The ON-OFF switching therefore involves cis/trans isomerization and protonation/deprotonation of the chromophore. In addition, spatial rearrangements of some amino acids have been reported to occur in the vicinity of the chromophore during the transition, [START_REF] Andresen | Structural basis for reversible photoswitching in Dronpa[END_REF] and the non-fluorescent character of the OFF state has been ascribed to a higher degree of flexibility of the chromophore and its environment than in the ON state. 14 The photoswitching mechanisms of Dronpa have been the object of a few studies by femtosecond spectroscopy, [START_REF] Fron | Ultrafast Excited-State Dynamics of the Photoswitchable Protein Dronpa[END_REF][START_REF] Warren | Ground-State Proton Transfer in the Photoswitching Reactions of the Fluorescent Protein Dronpa[END_REF][START_REF] Lukacs | Protein Photochromism Observed by Ultrafast Vibrational Spectroscopy[END_REF] aimed at elucidating the nature and kinetics of the primary reaction steps. Due to the long excited-state lifetime of the ON state (3.6 ns [START_REF] Habuchi | Reversible Single-Molecule Photoswitching in the GFP-like Fluorescent Protein Dronpa[END_REF] ), these studies mainly concern the photoinduced dynamics of the OFF state, and hence, the OFF→ON photoswitching, or photoactivation. Fron et al. [START_REF] Fron | Ultrafast Excited-State Dynamics of the Photoswitchable Protein Dronpa[END_REF] reported the first femtosecond kinetic traces of OFF-state Dronpa in the UVvisible range. This study revealed several kinetic components in the picosecond time range, expected from the non-fluorescent character of the OFF state. A 4 ps component, slowed down twofold upon H/D exchange, was assigned to an excited-state proton transfer (ESPT), which was proposed to be the first reaction step, as in GFP. [START_REF] Chattoraj | Ultra-Fast Excited State Dynamics in Green Fluorescent Protein: Multiple States and Proton Transfer[END_REF] Two groups also recently reported femtosecond IR spectra of Dronpa and Dronpa-2, a Dronpa variant bearing the single M159T mutation and characterized by a more efficient photoswitching. [START_REF] Stiel | 1.8 A Bright-State Structure of the Reversibly Switchable Fluorescent Protein Dronpa Guides the Generation of Fast Switching Variants[END_REF][START_REF] Ando | Highlighted Generation of Fluorescence Signals Using Simultaneous Two-Color Irradiation on Dronpa Mutants[END_REF] Warren et al. [START_REF] Warren | Ground-State Proton Transfer in the Photoswitching Reactions of the Fluorescent Protein Dronpa[END_REF] observed a dominant excited-state decay of OFF-state Dronpa in 9 ps, followed by a long-lived photoproduct, the spectral signature of which did not correspond to that of a phenolate chromophore. They concluded, at odds with Fron et al., that chromophore deprotonation is a ground-state process, and proposed that trans→cis photoisomerization is the first step and occurs in 9 ps. Lukacs et al. [START_REF] Lukacs | Protein Photochromism Observed by Ultrafast Vibrational Spectroscopy[END_REF] obtained very similar data for OFF-state Dronpa-2, covering however a wider spectral range. Their data also showed no evidence for ESPT. They however questioned the occurrence of the trans→cis isomerization in the picosecond time-range, arguing that, at variance with their observations, this should lead to a blue-shift of the C=O mode on the 100-ps timescale. They proposed instead that the primary step is a conformational reorganization of the protein around the chromophore, which would prepare for longer timescale proton transfer and trans→cis isomerization.

Despite conflicting conclusions, these studies all point to photoactivation not being completed on the timescale of a few hundreds of picoseconds, which implies that some steps in the overall process must have escaped detection so far. Concerning the exact mechanism of the reaction, no clear spectral signature of trans→cis isomerization has been reported until now, and the different models that were proposed diverge about the timescale of chromophore deprotonation. Although the most recent IR data [START_REF] Warren | Ground-State Proton Transfer in the Photoswitching Reactions of the Fluorescent Protein Dronpa[END_REF][START_REF] Lukacs | Protein Photochromism Observed by Ultrafast Vibrational Spectroscopy[END_REF] suggest it is a slow, ground-state process, it has not been observed directly and its kinetics remains to be determined.

In this context, we have reinvestigated the photoactivation dynamics of Dronpa and Dronpa-2 by UV-visible transient absorption spectroscopy. To gain further insights into the mechanism of this process, we have obtained broadband femtosecond spectra in the 350 to 740 nm range, as well as the corresponding anisotropy data, in the first nanosecond of the reaction. The temporal observation window has been extended to milliseconds using nanosecond-resolved pump-probe spectroscopy, to cover all relevant timescales. The transient spectra are compared with steady-state difference spectra associated with chromophore trans→cis isomerization and deprotonation, which are also reported. Time-dependent anisotropy changes are discussed in terms of chromophore isomerization and reorientation with respect to the β-barrel. This approach allows us identifying without ambiguity the main photoactivation intermediate, and proposing a clear mechanistic model, including the sequence in time of the cis/trans isomerization and proton transfer steps and their characteristic timescales.

METHODS

Expression plasmid, mutagenesis, protein expression and purification

The expression plasmid for His-tagged Dronpa was obtained by inserting the coding sequence of Dronpa (Amalgaam) into the pProEx HTa expression vector (Invitrogen) between the NcoI and HindIII restriction sites. The M159T mutation was introduced using the QuickChange Site-Directed Mutagenesis Kit (Stratagene). The proteins were expressed in the Top10 E. coli strain (Invitrogen) and purified by Ni-NTA affinity chromatography, as described in Ref. 22. Purity was checked by SDS-PAGE. The purified proteins were finally dialyzed against Tris-H 2 SO 4 buffer (50 mM, pH 8.0) and concentrated by ultrafiltration (Vivaspin, 10-kDa MWCO), before storage at -20°C.

Preparation and basic photophysical characterization of the protein samples

Deuterated Dronpa and Dronpa-2 samples were prepared by exchanging the storage buffer against deuterated Tris-H 2 SO 4 (50 mM, pD 8.0) using the same concentrators as above. They were kept 24 hours at room temperature before use, in order to ensure full H/D exchange. OFF-state samples were prepared by irradiating equilibrated ON-state protein solutions with continuous bluegreen light obtained by filtering the output of a Xenon lamp (Linos LQX1800) with a long-pass colored glass filter (Schott GG475). Depending on the protein concentration, 1 to 15 min of illumination were typically required to achieve full photoconversion.

Protein samples containing the chromophore in its four possible forms (cis/trans, protonated/deprotonated) were prepared at the same concentration. For this purpose, the storage buffer was first exchanged against 2 mM Tris-H 2 SO 4 of pH 8.0, in order to facilitate pH adjustments. Half of the sample was then converted to the OFF state by blue-green illumination, while the other half was kept in the ON state. The pH of the ON-and OFF-state solutions was finally adjusted to 4.0 or 11.0 by 30 times dilution in 50 mM acetate or CAPS buffer of the appropriate pH. The absorption spectra of the diluted solutions were measured after four hours of pH equilibration. The same strategy of dilution and equilibration in 50-mM phosphate and CAPS buffers was used to obtain absorption spectra of OFFstate Dronpa every 0.5 pH unit for pK a determination.

The molar extinction coefficients of Dronpa and Dronpa-2 were determined by diluting the protein samples (~200 µM in Tris-H 2 SO 4 pH 8.0) by a factor ten in 0.1 M HCl, which resulted in denaturation of the protein and protonation of the chromophore. [START_REF] Niwa | Chemical nature of the light emitter of the Aequorea green fluorescent protein[END_REF] The chromophore concentration in the denatured samples was calculated using the molar extinction coefficient of the neutral cis GFP chromophore. [START_REF] Voliani | Cis-Trans Photoisomerization of Fluorescent Protein Chromophores[END_REF] Photoactivation quantum yields were determined spectrophotometrically under continuous irradiation, by comparison with the photolysis of the chemical actinometer DMAD [START_REF] Cox | Photophysical and Photochemical Properties of Sterically Hindered Aryldiazonium Salts[END_REF] (p-(dimethylamino)benzenediazonium), following a standard method 26,[START_REF] Thiagarajan | DNA repair by photolyase: a novel substrate with low background absorption around 265 nm for transient absorption studies in the UV[END_REF] detailed in §4 of the Supporting Information (SI). All absorption spectra were recorded with a Cary 300 spectrophotometer (Varian). The fluorescence spectra were measured using a fully corrected Fluoromax-3 spectrofluorimeter (Jobin Yvon).

Femtosecond spectroscopy data acquisition and analysis

Broadband femtosecond transient absorption spectra were recorded in the 350 to 740 nm spectral range following 388 nm excitation using the pump-probe with whitelight continuum technique, as described in Ref. 28. To access both the isotropic transient absorption signal and the anisotropy, we fixed the polarization of the pump at 45° of that of the probe and recorded alternatively the parallel and perpendicular probe components using motorized analyzers placed after the sample. The excitation energy was set to ~30 nJ/pulse (5 µW, 166 Hz), focused on a surface of ~7×10 4 µm 2 (FWHM), which was low enough a fluence to avoid multi-photon processes (see SI, §2). OFF-state Dronpa or Dronpa-2 samples (~250 µM in 50 mM Tris-H 2 SO 4 of pH 8.0) were placed in a 1-mm optical path cuvette, which was translated in two dimensions in its own plane, in order to minimize spatial overlap between consecutive laser shots. The gradual conversion of the samples to their ON state due to repetitive laser excitation was compensated by ~10 s periods of blue-green irradiation every ~1500 pump shots, which regenerated the OFF state. The blue-green light was obtained as described above, and switched on and off synchronously with femtosecond data acquisition using a computercontrolled mechanical shutter. The parallel and perpendicular data were averaged over ~9000 pump shots (corresponding to 6 scans of the whole kinetics) and corrected for the chirp of the probe beam, which was independently measured by recording cross-phase modulation [START_REF] Ekvall | Cross Phase Modulation Artifact in Liquid Phase Transient Absorption Spectroscopy[END_REF] in the pure solvent.

The isotropic differential absorbance spectra were constructed from the measured parallel and perpendicular spectra using Eq. 2 below. They were globally fitted by the sum of four exponentials and a plateau, convoluted with a Gaussian function, using a home-written Mathematica (Wolfram) routine. The Gaussian represented the instrument response function (IRF), the FWHM of which was found to be 140 fs. The procedure used dimensional reduction and noise filtering by singular value decomposition (SVD). The number of retained singular values at the stage of analysis of the truncated data matrix varied from four to six, which was enough to ensure that no information loss could alter subsequent global fitting. The fit was in fact made in two steps. In the first one the full data set was analyzed and the cross-phase modulation (XPM) artifact [START_REF] Ekvall | Cross Phase Modulation Artifact in Liquid Phase Transient Absorption Spectroscopy[END_REF] occurring during pump-probe overlap was empirically accounted for by a sum of the IRF Gaussian and its first and second derivatives. In the final step times below 200 fs were removed in order to get rid of minor spectral contaminations due to XPM. It was checked that the time constants of both steps were fully compatible. The average amplitude of the residues after fitting was on the order of 5×10 -5 OD. The decay-associated difference spectrum (DADS), that is, the amplitude spectrum of each component was calculated over the entire experimental spectral range.

Transient anisotropy measurements

It is well known that the process of photoselection creates a temporary orientational heterogeneity in a sample. [START_REF] Lakowicz | Principles of Fluorescence Spectroscopy[END_REF] In transient absorption spectroscopy, this orientational heterogeneity can be measured by the anisotropy (r), a quantity defined with the components of the signal parallel (ΔA par ) and perpendicular (ΔA perp ) to the polarization of the excitation, as follows:

perp par perp par A A A A r        2 (1)
In this expression, the denominator simply corresponds to three times the isotropic signal (ΔA iso ), that is, the signal that would be observed for a non-polarized sample:

3 2 perp par iso A A A      (2)
Brownian rotational diffusion leads to a global decay of the anisotropy, the kinetics of which depends on the size and shape of the rotating system. For globular proteins, a single average rotational correlation time (θ) is usually resolved:

   t r t r   exp ) ( 0 (3)
where r 0 is the intrinsic anisotropy, measured before rotational diffusion takes place. Dronpa is expected to have the same rotational correlation time as GFP, that is, 16 ns. [START_REF] Volkmer | One-and Two-Photon Excited Fluorescence Lifetimes and Anisotropy Decays of Green Fluorescent Proteins[END_REF] This value determines the temporal window in which polarization effects can be detected. Rotational diffusion was in practice neglected in the few hundreds of picoseconds time window probed with the femtosecond setup. We will hence identify r with r 0 in the following.

When a single electronic transition contributes to the transient signal, the intrinsic anisotropy is determined by the angle α between its transition dipole moment and that of the transition excited by the laser:

5 1 cos 3 2    r (4)
r varies between 0.4 (α = 0) and -0.2 (α = 90°). When several electronic transitions contribute to the signal, the total anisotropy (r) is related to the individual anisotropies (r i ) of these transitions by the simple rule:

iso iso i i A A r r     , (5)
with ΔA i,iso the contribution of the i th transition to the isotropic signal. Compensations between the different contributions to ΔA iso can lead to a small denominator and hence to anisotropy values outside the range -0.2 ≤ r ≤ 0.4.

In the following, we will focus on the anisotropy associated with the long-lived component of our global kinetic analysis (see §2.3). This anisotropy was constructed from the parallel and perpendicular DADS of the plateau (DADS par , resp. DADS perp ), obtained by simultaneous global fitting of ΔA par and ΔA perp with the same fit function, as follows:

perp par perp par DADS DADS DADS DADS r 2    (6)

Nanosecond spectroscopy

The photoinduced absorption changes of OFF-state Dronpa and Dronpa-2 in the 5 ns to 10 ms time range were measured using a home-built nanosecond spectrometer described in Ref. 32. In this setup, the pump and probe pulses are conveyed to the sample through optical fibers, which scramble polarizations. The samples (~25 µM in 50 mM Tris-H 2 SO 4 of pH 8.0) were placed in standard 1×1 cm cuvettes and excited at 425 nm on a surface of ~1 cm 2 with 0.7 mJ (7 mW, 10 Hz), 5 ns pulses from a Nd:YAG pumped optical parametric oscillator (OPO). The absorption changes were probed with a second OPO tuned at selected wavelengths in the visible (460 to 530 nm) in order to detect contributions from anionic chromophore forms, and in the UV (380 nm) to probe neutral forms. The signal was averaged over six to twelve pump flashes for each probe wavelength and pump-probe delay. The samples were maintained in their initial OFF states during data accumulation by continuous irradiation with blue-green light from a filtered Xenon lamp, as in femtosecond spectroscopy. Data analysis was performed using Origin 7 (OriginLab).

RESULTS AND DISCUSSION

Difference spectra associated with chromophore isomerization and deprotonation; photoactivation quantum yields of Dronpa and Dronpa-2

At neutral pH, OFF-state Dronpa contains mainly the trans-phenol form of the chromophore, and ON-state Dronpa the cis-phenolate form [START_REF] Andresen | Structural basis for reversible photoswitching in Dronpa[END_REF][START_REF] Wilmann | The 1.7 A Crystal Structure of Dronpa: a Photoswitchable Green Fluorescent Protein[END_REF][START_REF] Stiel | 1.8 A Bright-State Structure of the Reversibly Switchable Fluorescent Protein Dronpa Guides the Generation of Fast Switching Variants[END_REF][START_REF] Mizuno | Light-Dependent Regulation of Structural Flexibility in a Photochromic Fluorescent Protein[END_REF][START_REF] Li | A Theoretical Study on the Nature of On-and Off-States of Reversibly Photoswitching Fluorescent Protein Dronpa: Absorption, Emission, Protonation and Raman[END_REF] (see Scheme 1). Owing to the double change in chromophore structure between the OFF and ON states, we reasoned that, in a sequential mechanistic scheme, photoactivation could potentially involve two additional forms as intermediates, namely the cis-phenol and trans-phenolate forms. In order to facilitate the interpretation of the time-resolved spectroscopy data, we set about determining the steady-state absorption spectra of the four possible chromophore forms, at the same concentration.

The spectra of the different forms were obtained by adjusting the pH of irradiated and non-irradiated Dronpa solutions and correcting remaining contributions from minority acid-base forms. They are shown in Figure 1A. A large red-shift is with no surprise associated with the deprotonation of the phenol group. More interestingly, we found that trans→cis isomerization of the phenol form also induces noticeable absorption changes (a fewnanometer blue-shift and small increase in intensity of the lowest electronic transition), while the trans-and cisphenolate forms are spectrally indistinguishable. Such a signature of trans→cis isomerization of the phenol form seems to be a general property of fluorescent proteins and their chromophores, as it is also observed for Dronpa-2 (see SI, Figure S5) as well as for several p-hydroxybenzylidene imidazolinone derivatives in solution. [START_REF] Voliani | Cis-Trans Photoisomerization of Fluorescent Protein Chromophores[END_REF][START_REF] Addison | Ultrafast Studies of the Photophysics of Cis and Trans States of the Green Fluorescent Protein Chromophore[END_REF][START_REF] Dong | Isomerization in Fluorescent Protein Chromophores Involves Addition/Elimination[END_REF] Figure 1. A) Steady-state absorption spectra of the different forms of the Dronpa chromophore: cis-and trans-phenolate (indistinguishable), trans-phenol and cis-phenol. B) Difference spectra associated with trans→cis isomerization of a phenol chromophore (cis-phenol minus trans-phenol; 10× magnified) and with deprotonation of a trans chromophore (trans-phenolate minus trans-phenol). The data were smoothed over 11 points using Savitzky-Golay algorithm with a second order polynomial.

The large red-shift of the trans-phenolate form with respect to the OFF state (trans-phenol) suggests (Förster cycle [START_REF] Förster | Die pH-Abhängigkeit der Fluoreszenz von Naphthalinderivaten[END_REF] ) that the latter is a photoacid, i.e. is prone to deprotonate in the excited-state, as is the case of the neutral state of GFP, [START_REF] Chattoraj | Ultra-Fast Excited State Dynamics in Green Fluorescent Protein: Multiple States and Proton Transfer[END_REF][START_REF] Lossau | Time-Resolved Spectroscopy of Wild-Type and Mutant Green Fluorescent Proteins Reveals Excited State Deprotonation Consistent with Fluorophore-Protein Interactions[END_REF] and more generally of hydroxyaromatic molecules. [START_REF] Agmon | Elementary Steps in Excited-State Proton Transfer[END_REF] On the other hand, we found the pK a of OFF-state Dronpa to be 10.1 ± 0.1, that is 5 pH units higher than that reported for the ON state (pK a = 5 [START_REF] Ando | Regulated Fast Nucleocytoplasmic Shuttling Observed by Reversible Protein Highlighting[END_REF] ). This large pK a shift, which is likely due to the change in local environment of the phenol group induced by cis/trans isomerization, [START_REF] Andresen | Structural basis for reversible photoswitching in Dronpa[END_REF][START_REF] Brakemann | Molecular Basis of the Light-Driven Switching of the Photochromic Fluorescent Protein Padron[END_REF] indicates that at neutral pH, trans→cis isomerization may provide the driving force for groundstate deprotonation. Based on the steady-state absorption spectra of the four chromophore forms of Dronpa, we calculated the difference spectra associated with the deprotonation of a trans chromophore (trans-phenolate minus trans-phenol) and with the trans→cis isomerization of a phenol chromophore (cis-phenol minus transphenol). These spectra, which are displayed in Figure 1B, will serve as references to interpret the time-resolved spectroscopy data presented in the next sections and establish what occurs during Dronpa photoactivation.

As we will see later ( §3.4), the analysis of femtosecond anisotropy data requires knowledge of the quantum yields of photoactivation. Since the photoactivation quantum yield of Dronpa-2 was not known and there was only one reference for Dronpa (Φ = 0.37 4 ), we undertook measuring these yields. Photoactivation was carried out under continuous irradiation and followed spectrophotometrically to determine its rate constant. The photon flux was deduced from the photolysis rate constant of a chemical actinometer in the same irradiation conditions (see SI §4 for more details). The results are presented in Table 1. The method actually gives access to the product of Φ by the extinction coefficient of the OFF state, ε OFF . With our ε OFF values, we obtained Φ = 7.3 ± 0.5 % for Dronpa and Φ = 13.7 ± 0.9 % for Dronpa-2 (standard deviation over four independent measurements). These yields are significantly lower than the published photoactivation yield of Dronpa. We could however confirm our light flux determination using a second chemical actinometer and a power-meter. Such relatively low quantum yield values indicate that photoactivation is in competition with other efficient pathways leading back to the OFF state. Dronpa-2 52805 13360 13.7 ± 0.9

Table 1. Molar Extinction Coefficients and

Excited-state dynamics probed by broadband femtosecond UV-visible spectroscopy

We studied the primary steps of Dronpa photoactivation by broadband femtosecond UV-visible spectroscopy. Figure 2 shows the isotropic transient absorption spectra of OFF-state Dronpa measured after 388nm excitation with a pump energy of 20 nJ/pulse (3.3 µW, 166 Hz, ~7×10 4 µm 2 spot size), for pump-probe delays ranging from 0.125 to 300 ps. It should be noted that these data significantly extend the study by Fron et al., [START_REF] Fron | Ultrafast Excited-State Dynamics of the Photoswitchable Protein Dronpa[END_REF] which was limited to absolute values of the signal at a few discrete wavelengths between 370 and 450 nm. The initial spectrum, which corresponds to a pumpprobe delay of 0.125 ps (Figure 2A), consists of four bands: a negative band dominated by ground-state bleaching at 390 nm, a first positive band dominated by excited-state absorption at 440 nm, a negative band dominated by stimulated emission from 480 to 650 nm, showing two humps at 515 and 570 nm, and a second, red excited-state absorption band beyond 650 nm. The stimulated emission band appears red-shifted compared to the steadystate emission spectrum of OFF-state Dronpa (dashed grey line in Figure 2C), due to the overlapping of its blue edge with excited-state absorption. This initial spectrum closely resembles the early transient absorption spectrum of neutral GFP reported by Kennis et al., [START_REF] Kennis | Uncovering the Hidden Ground State of Green Fluorescent Protein[END_REF] as expected for the excited phenol form of the chromophore.

The temporal evolution of transient absorption involves three phases, highlighted in Figures 2A-C. The first two phases (0.125 to 3 ps) are characterized by subtle modifications of the shape of the stimulated emission band. A decay of the blue hump is first observed between 0.125 and 0.375 ps (Figure 2A). The red excited-state absorption also partially decays on this timescale, while ground-state bleaching shows little evolution. Then, from 0.85 to 3 ps, the red hump of stimulated emission decays in turn (Figure 2B), accompanied by partial decays of ground-state bleaching and of the 440-nm excited-state absorption, while the red excited-state absorption remains stable. Finally, the third phase, from 3 to 300 ps (Figure 2C), consists in an essentially homothetic decay of all bands to a small long-lived signal which will be discussed in the following section.

It is interesting to compare the above-described evolution of the transient absorption of OFF-state Dronpa with that of neutral GFP. In the latter, a pronounced stimulated-emission band with the spectral features of an anionic chromophore develops in the picosecond timescale, due to fast excited-state deprotonation of the phenol group, while the 440-nm excited-state absorption grows. [START_REF] Kennis | Uncovering the Hidden Ground State of Green Fluorescent Protein[END_REF] The transient signal then slowly decays to zero on the nanosecond timescale, which corresponds to the excited-state lifetime of the phenolate chromophore. No such changes are observed for OFF-state Dronpa, suggesting that ESPT is not a dominant reaction pathway in this case.

The data were first fitted globally with a sum of four independent exponentials and a plateau. The lifetimes and amplitude spectra (also called DADS, for decay-associated difference spectra) of the components are given in Figure S6A. Since two of the exponentials had DADS of similar shapes, the data were then fitted with two exponentials, one biexponential function (two exponentials with wavelength-independent relative weights) and a plateau, which led to equally small residuals. The lifetimes from this second fit were 0.19 ± 0.03 ps, 2.1 ± 0.1 ps, 10 ± 1 ps (66 %) and 64 ± 7 ps (34 %). The 10 and 64-ps lifetimes correspond to the biexponential component, which describes the homothetic decay of Figure 2 C. The DADS of the different components are shown in Figure S6B. The Dronpa-2 variant showed a similar behavior, with however slightly slower kinetics. These data are presented and commented in the SI ( §1, Figures S1 andS2). We observed no significant kinetic isotope effect upon H/D exchange, in line with the absence of ESPT argued above.

With the exception of the 190-fs lifetime which had never been observed before, [START_REF]Convoluting a 140-fs gaussian by a 190[END_REF] our findings of multiple components in the picosecond time range are in accord with previous ultrafast studies by Fron et al. [START_REF] Fron | Ultrafast Excited-State Dynamics of the Photoswitchable Protein Dronpa[END_REF] and Lukacs et al. [START_REF] Lukacs | Protein Photochromism Observed by Ultrafast Vibrational Spectroscopy[END_REF] In agreement with the latter, we believe that the multiexponential character of the excited-state dynamics is in part due to the reported structural heterogeneity of the chromophore and a portion of the β-barrel in the OFF ground-state. [START_REF] Andresen | Structural basis for reversible photoswitching in Dronpa[END_REF][START_REF] Mizuno | Light-Dependent Regulation of Structural Flexibility in a Photochromic Fluorescent Protein[END_REF] The excited-state decay of GFP chromophores is indeed known to be sensitive to structural distortions. [START_REF] Conyard | Chemically Modulating the Photophysics of the GFP Chromophore[END_REF] Multiple conformations of the protein matrix may also lead to a distribution of decay rates, in particular if the decay coordinate involves a largeamplitude molecular motion, as is the case for some cis/trans isomerization reactions. [START_REF] Waldeck | Photoisomerization Dynamics of Stilbenes[END_REF] The rate of such a motion is indeed expected to depend on the available free space.

Another possible source of complex kinetics is the coupling between chromophore dynamics and protein dielectric relaxation. The protein matrix is indeed expected to respond to the instantaneous charge redistribution occurring in the chromophore upon excitation, through motions of polar and charged amino-acid side-chains and of trapped water molecules and rearrangements in chromophore-protein hydrogen bonds. [START_REF] Chang | Ultrafast Solvation Dynamics at Binding and Active Sites of Photolyases[END_REF][START_REF] Abbyad | Measurement of Solvation Responses at Multiple Sites in a Globular Protein[END_REF] This dynamicsolvation-like phenomenon often gives rise to timedependent band shifts, but may also lead to more complex spectral shape changes, on timescales ranging from ~100 fs to nanoseconds. [START_REF] Chang | Ultrafast Solvation Dynamics at Binding and Active Sites of Photolyases[END_REF][START_REF] Abbyad | Measurement of Solvation Responses at Multiple Sites in a Globular Protein[END_REF] A contribution from vibrational cooling is in addition not excluded. Although tuned at the maximum of the lowest-energy absorption band, the 388-nm excitation wavelength may indeed be moderately blue-shifted with respect to the 0-0 transition. The reshaping of the stimulated-emission band and the stability of ground-state bleaching during the 190-fs phase suggest that it corresponds to such relaxations of the initial excited-state. The 2.1 ps phase could also contain a contribution from dynamic solvation and/or vibrational cooling. The partial decay of ground-state bleaching however indicates that this phase in addition involves excitedstate decay, like the biexponential phase. It could reflect the particularly fast decay of a small subpopulation of proteins, on a timescale close to a solvation or cooling component, the biexponential component corresponding to the pure decay of other, relaxed, subpopulations. The nature of the photochemical process responsible for the excited-state decay will be clarified in the next sections, based on the assignment of the first ground-state species.

Spectral signature of the first ground-state species

In order to identify the first ground-state species (noted X) formed upon excited-state decay of OFF-state Dronpa, we closely inspected the small transient signal remaining at 300 ps. We verified that this signal stays essentially unchanged until 1.5 ns, the longest pump-probe delay accessible to our femtosecond setup. The spectrum of the plateau with which it was fitted in our global kinetic analysis (see §3.2) is shown in Figure 3A, together with that of Dronpa-2.

These long-lived spectra contain no trace of anionic absorption around 500 nm, which definitely confirms that ESPT does not occur during the preceding excited-state dynamics, in agreement with the conclusions of Warren et al. [START_REF] Warren | Ground-State Proton Transfer in the Photoswitching Reactions of the Fluorescent Protein Dronpa[END_REF] and Lukacs et al. [START_REF] Lukacs | Protein Photochromism Observed by Ultrafast Vibrational Spectroscopy[END_REF] Instead, the long-lived spectrum of Dronpa exhibits a transient absorption band below 413 nm, with a maximum around 385 nm, and a ground-state bleaching band from 413 to 465 nm. The Dronpa-2 spectrum essentially consists in a transient absorption band peaking at 390 nm and extending up to 430 nm, with very little bleaching observed beyond. As emphasized in Figure 3, these transients resemble the steady-state differ-ence spectra associated with the trans→cis isomerization of phenol chromophores (Figure 3B). More specifically, the wave-like shape of the Dronpa spectrum and the position of its bleaching band at 425 nm meet these expectations. The transient absorption band of Dronpa-2 is redshifted with respect to that of Dronpa and the amplitude of its bleaching band is much smaller, which is also well reproduced by the calculated difference spectra. These observations suggest that X is a cis-phenol form, both for Dronpa and Dronpa-2. Differences between the 300-ps spectra and the steady-state difference spectra however exist. A red-shifted position of the transient absorption bands can in particular be noticed. These differences could be due to an incomplete relaxation of the proteins around the isomerized chromophores on this relatively short timescale. Although qualitatively similar, X is therefore spectrally distinct from the relaxed cis-phenol form observed in steady-state experiments. It has to be mentioned here that the long-lived transient signal of Dronpa is very sensitive to the laser energy used for excitation. Additional absorption contributions indeed show up at 455 nm and above 525 nm for pump energies larger than 50 nJ/pulse (that is 70 µJ cm -2 given our spot size of 7×10 4 µm 2 ; see Figure S3). We attribute these contributions to chromophore radicals and solvated electrons, formed by a two-photon mechanism (see SI §2 for more details). Such a facile biphotonic ionization of OFF-state Dronpa under femtosecond excitation can hinder the detection of the first photoactivation intermediate, when the laser energy is too high. At the 20 nJ/pulse pump energy used in the present work, the contamination does however not exceed 1 % of the population of photoactivation intermediate (X) and lies below the detection limit of the experiment (see SI §2).

Anisotropy of the first ground-state species

X-ray crystallography studies showed that cis/trans isomerization of Dronpa is accompanied by a large reorientation of the chromophore with respect to the β-barrel 8 (Scheme 2). Such a large molecular reorientation is expected to translate into a large change in direction of the transition dipole moment (α angle), and thus, into a significant change of the absorption anisotropy (see §2. It should be pointed out at this stage that the long-lived transient signal of Dronpa results from two strongly overlapping contributions with different intrinsic anisotropies: the pure absorption of X and ground-state bleaching. The isotropic signal ΔA iso can in particular be written as follows:

         OFF X iso A A A    (7)
where A X is the isotropic absorption of X and A OFF the isotropic absorption of the missing OFF ground-state population. In such a case, Eq. 5 applies, and measured anisotropy r can be expressed as:

            iso OFF OFF X X A A r A r r      (8)
with r X and r OFF the individual anisotropies of the two bands. In this expression, r and ΔA iso are known experimentally, and r OFF takes the standard value of 0.4 expected when the same electronic transition is both excited and probed (Eq. 4 with α = 0). A X and A OFF are however not known independently, and decomposing ΔA iso into its components as in Eq. 7 is not straightforward due to the strong spectral overlap and absence of precise knowledge of the spectrum of X. A OFF was therefore determined independently from the pump-probe data, in order to extract the anisotropy of X. A OFF involves the molar extinction coefficient of the OFF state (ε OFF , in mol -1 L cm -1 ), the quantum yield of formation of the intermediate (Φ X ), the optical path of the cuvette (l, cm) and the concentration of excited Dronpa molecules (C exc , in mol L -1 ):

exc X OFF OFF C l A    (9)
If X is a cis isomer, as suggested by its spectral signature, thermal conversion back to the trans, OFF state is expected to be rather inefficient. The cis isomer is indeed known to be more stable than the trans, both in the protein [START_REF] Wilmann | The 1.7 A Crystal Structure of Dronpa: a Photoswitchable Green Fluorescent Protein[END_REF][START_REF] Stiel | 1.8 A Bright-State Structure of the Reversibly Switchable Fluorescent Protein Dronpa Guides the Generation of Fast Switching Variants[END_REF] and in solution. [START_REF] Voliani | Cis-Trans Photoisomerization of Fluorescent Protein Chromophores[END_REF] For this reason, we made the assumption that Φ X is equal to the overall photoactivation quantum yield, Φ (Table 1). C exc was estimated independently from the pump-probe data, based on measurements of the laser beam profiles at the sample position (see SI, §3). The amplitude of ground-state bleaching at time zero could indeed not be used for that purpose due to overlapping with excited-state absorption.

The decomposition of the isotropic signal is shown in Figure 4A. The absorption of X (red line), obtained by subtracting the bleaching contribution from the total signal, is blue-shifted by a few nanometers with respect to the initial OFF state (black dashed line), in agreement with our prediction for a cis-phenol form (Figure 1A). Knowing A X , A OFF and r OFF , we calculated the anisotropy spectrum of X using Eq. 8. This spectrum is shown in Figure 4B (red line). The anisotropy of X is constant throughout the whole absorption band, in good agreement with a single electronic transition being probed. It is significantly lower than r OFF = 0.4 (black dashed line), which indicates a large reorientation of the transition dipole moment with respect to the OFF state. The same bleaching subtraction procedure was applied to Dronpa-2, leading to a similarly low anisotropy for the intermediate (Figure S7). The average values of r X for Dronpa and Dronpa-2 over the 370-410 nm range of maximum absorption of X are given in Table 2, with error bars reflecting the experimental errors on C exc and Φ. The reorientation of the transition dipole moment in X with respect to the initial OFF state (α angle in Scheme 2) can be deduced from these anisotropies using Eq. 4. We find α = 30 ± 3° for Dronpa and α = 39 ± 4° for Dronpa-2. The reorientation angle expected for a fully isomerized neutral chromophore can on the other hand be predicted on the basis of the X-ray structures of the ON and OFF states of Dronpa [START_REF] Andresen | Structural basis for reversible photoswitching in Dronpa[END_REF][START_REF] Wilmann | The 1.7 A Crystal Structure of Dronpa: a Photoswitchable Green Fluorescent Protein[END_REF][START_REF] Stiel | 1.8 A Bright-State Structure of the Reversibly Switchable Fluorescent Protein Dronpa Guides the Generation of Fast Switching Variants[END_REF] and of the directions of the transition dipole moments of the cis and trans isomers of the chromophore. These directions are unfortunately not directly available for the neutral chromophore. Olsen et al. calculated the transition dipole moments of the cis and trans anionic forms, [START_REF] Olsen | Bond Selection in the Photoisomerization Reaction of Anionic Green Fluorescent Protein and Kindling Fluorescent Protein Chromophore Models[END_REF] while other authors reported computed or experimental differences of only a few degrees between the transition dipole moment directions of the neutral and anionic cis forms. [START_REF] Ansbacher | Calculation of Transition Dipole Moment in Fluorescent Proteins-Towards Efficient Energy Transfer[END_REF][START_REF] Usman | Excited-State Structure Determination of the Green Fluorescent Protein Chromophore[END_REF] Assuming that the transition dipole moment directions of the cis and trans neutral forms are within ± 5° of those calculated by Olsen et al. for the cis and trans anionic forms, we obtained a theoretical range of 30 to 50° for the reorientation expected for the cis neutral form of Dronpa. The corresponding anisotropy range, calculated using Eq. 4, is given in Table 2 (last column). The experimental anisotropies and reorientation angles of the photoactivation intermediates of Dronpa and Dronpa-2 lie within the predicted ranges, as illustrated in Table 2. We conclude that full trans→cis isomerization of the chromophore takes place upon excit-ed-state decay of the OFF state, in a few tens of picoseconds (2.1-ps, 10-ps and 64-ps lifetimes in Dronpa; see §3.2).

The above result contradicts the conclusions of Lukacs et al., who did not detect trans→cis isomerization in their femtosecond IR experiments on Dronpa-2. [START_REF] Lukacs | Protein Photochromism Observed by Ultrafast Vibrational Spectroscopy[END_REF] Based on our experience, we suggest that at least two factors could explain why. First, IR experiments were carried out with very high excitation densities, of a few mJ cm -2 , [START_REF] Warren | Ground-State Proton Transfer in the Photoswitching Reactions of the Fluorescent Protein Dronpa[END_REF][START_REF] Lukacs | Protein Photochromism Observed by Ultrafast Vibrational Spectroscopy[END_REF] whereas we used less than 70 µJ cm -2 . The IR data could therefore have been contaminated by signals resulting from two-photon processes (see §3.3 and SI §2). In addition, we found that the photoactivation quantum yields of Dronpa and Dronpa-2 are significantly lower than previously thought (see §3.1 and Table 1). The resulting lowamplitude isomerization signature could therefore have escaped detection, especially in the presence of interfering multi-photon photochemistry.

Investigation of the deprotonation step by nanosecond UV-visible spectroscopy

Since the femtosecond data indicated that the chromophore is still protonated at 1.5 ns, we looked for the deprotonation step on longer timescales (5 ns to 10 ms) using a different transient absorption spectroscopy setup with nanosecond time resolution (see §2.5). We first probed the appearance of the final ON state at 500 nm, following 425-nm excitation with 5 ns laser pulses (Figure 5A). It appears that the ΔA signal remains close to zero until about 1 µs and then grows on the timescale of 10 µs to reach a plateau that lasts at least 10 ms. The microsecond rise could be fitted with a single exponential of characteristic time 12.5 ± 1.0 µs, in aqueous Tris buffer of pH 8.0. This lifetime increased to 43 ± 3 µs in deuterated Tris buffer, indicating a slowing down by a factor of about 3.5. Such a large kinetic isotope effect is in good agreement with a proton transfer reaction. We also measured the spectrum of the plateau at 1 ms, by tuning the probe wavelength in the 460 to 530 nm range (Figure 5C, symbols). It was found to be superimposable to the difference spectrum associated with OFF→ON photoswitching (cisphenolate minus trans-phenol; line in Figure 5C), confirming the assignment of the final signal to the ON state. The present 12.5-µs kinetics constitutes the first direct observation of the deprotonation step involved in Dronpa photoactivation.

In order to check the precursor-successor relationship between the X intermediate identified by femtosecond spectroscopy and the ON state, we then tuned the probe to 380 nm (Figure 5B). At this wavelength, the signal is initially positive and decays to a plateau with negative amplitude on the same timescale as the 500-nm rise. The kinetic isotope effect upon H/D exchange is moreover similar to that at 500 nm. We attribute the initial positive signal, which persists until about 1 µs, to X. The negative signal is due to the bleaching of the OFF state, which dominates at this wavelength once X has deprotonated. The fact that the kinetics at 380 nm and 500 nm are the same indicates that X and the ON state are directly connected, and that no other intermediate is involved in the photoactivation of Dronpa. Nanosecond measurements carried out on Dronpa-2 led to the same conclusion, with a deprotonation kinetics of 19.2 ± 0.4 µs (Figure S8).

Model for the photoactivation of Dronpa and Dronpa-2

The data presented in the previous sections finally allow us to propose a common model for the photoactivation mechanisms of Dronpa and Dronpa-2 (Scheme 3). Excitation of the OFF state (trans-phenol) produces the initial excited-state, OFF*. Subtle reshaping of the excited-state spectrum then occurs on the timescale of a few picoseconds, evoking some relaxation (dynamic solvation and/or vibrational cooling). The preservation of the excited population during the initial ~200 fs phase, present in both Dronpa and Dronpa-2, in particular suggests that this ultrafast component is entirely due to such a process. We thus propose that it leads to a relaxed excited-state, OFF*'.

The OFF*' state then decays multiexponentially in 2.1 ps, 10 ps and 64 ps for Dronpa (2.6 ps, 15 ps, 160 ps and 280 ps for Dronpa-2) to a ground-state species, X, that we unambiguously identified as a cis-phenol form. Trans→cis isomerization therefore takes place upon excited-state decay, in a few tens of picoseconds. Following the epitome of photoisomerization, trans-stilbene, [START_REF] Waldeck | Photoisomerization Dynamics of Stilbenes[END_REF] we tentatively suggest in Scheme 3 that the decay of OFF*' proceeds via an excited-state energy barrier, followed by ultrafast crossing with the ground-state around the perpendicular geometry of the double-bond, possibly at a conical intersection. Since the yield of photoactivation is only 7 % for Dronpa (14 % for Dronpa-2), we propose in addition that the rest of the initially excited population returns to the OFF state at this stage of the reaction. We attribute the multiexponential character of the photoisomerization kinetics to the reported conformational heterogeneity of the OFF ground-state of Dronpa [START_REF] Andresen | Structural basis for reversible photoswitching in Dronpa[END_REF][START_REF] Mizuno | Light-Dependent Regulation of Structural Flexibility in a Photochromic Fluorescent Protein[END_REF] . The slight reshaping of the excited-state spectrum on the timescale of the 2.1 ps photoisomerization component of Dronpa (2.6 ps component of Dronpa-2) moreover suggests that this component still contains some solvation or cooling contribution. The lifetimes and quantum yields indicated for the different reaction steps are those of Dronpa.

The ON state (cis-phenolate) is finally formed from the neutral cis isomer (X) in 12.5 µs in Dronpa (19.2 µs in Dronpa-2), by ground-state deprotonation of the phenol group. This final step is probably determined by the 5 pH units decrease in pK a of the phenol group induced by trans→cis isomerization. A reason for the absence of ESPT in OFF-state Dronpa, as opposed to neutral GFP, [START_REF] Chattoraj | Ultra-Fast Excited State Dynamics in Green Fluorescent Protein: Multiple States and Proton Transfer[END_REF][START_REF] Lossau | Time-Resolved Spectroscopy of Wild-Type and Mutant Green Fluorescent Proteins Reveals Excited State Deprotonation Consistent with Fluorophore-Protein Interactions[END_REF] could be the larger flexibility of the chromophore and the β-barrel in OFF-state Dronpa. [START_REF] Mizuno | Light-Dependent Regulation of Structural Flexibility in a Photochromic Fluorescent Protein[END_REF] Weaker geometrical constraints are indeed expected to favor torsion coordinates over ESPT, as attested by the excited-state dynamics of the free GFP chromophore in solution. [START_REF] Conyard | Chemically Modulating the Photophysics of the GFP Chromophore[END_REF][START_REF] Usman | Excited-State Structure Determination of the Green Fluorescent Protein Chromophore[END_REF][START_REF] Vengris | Ultrafast Excited and Ground-State Dynamics of the Green Fluorescent Protein Chromophore in Solution[END_REF]

CONCLUSION

We followed for the first time the entire photoactivation process of Dronpa, by transient absorption spectroscopy from 100 fs to milliseconds. The Dronpa-2 variant was also studied for comparison, and showed the same mechanism. Thanks to a careful control of the excitation energy in femtosecond spectroscopy, we obtained both the spectrum and the anisotropy of the first ground-state photoactivation intermediate. This intermediate exhibits a spectral shift of a few nanometers to the blue with respect to the OFF state, which we showed to be characteristic for the neutral cis form of the chromophore. In addition, its anisotropy is significantly smaller than that of the OFF state, in agreement with the large reorientation of the transition dipole moment expected upon isomerization. These data constitute the first clear evidence that trans→cis isomerization of the chromophore precedes its deprotonation and occurs on the picosecond timescale, concomitantly to the excitedstate decay. We also monitored the deprotonation step in real time. It was found to occur in ~10 μs and lead directly from the neutral cis isomer to the final ON state, without any other detectable intermediate.

ASSOCIATED CONTENT

Supporting information. Dronpa-2 steady-state and timeresolved spectroscopy data, DADS of Dronpa, excitation energy dependence of the 600-ps transient spectrum, concentration of excited molecules in fs spectroscopy, determination of the photoactivation quantum yields. This material is available free of charge via the Internet at http://pubs.acs.org.

Scheme 1 .

 1 Scheme 1. Chromophore Structure in the ON and OFF States of Dronpa.

Figure 2 .

 2 Figure 2. Transient absorption spectra of OFF-state Dronpa in Tris buffer at pH 8.0 measured after excitation at 388 nm, for pump-probe delays ranging from A) 0.125 ps to 0.85 ps, B) 0.85 ps to 3 ps and C) 3 ps to 300 ps. The data were corrected for the chirp of the probe. The grey dashed lines in C represent the steady-state absorption and fluorescence spectra of OFF-state Dronpa.

Figure 3 .

 3 Figure 3. A) Spectral signatures of the first ground-state photoactivation intermediates of Dronpa and Dronpa-2. The spectra shown are the DADS (decay-associated difference spectra) of the plateaus used to fit the long-lived transient absorption signals in our global kinetic analysis of femtosecond data. These data were noise reduced by SVD (singular value decomposition; see §2.3 of Methods). B) Steady-state difference spectra associated with trans→cis isomerization of phenol chromophores in Dronpa and Dronpa-2. The data were smoothed over 11 points using Savitzky-Golay algorithm with a second order polynomial. All spectra have been normalized to 1 at 380 nm to allow easier comparison.

  Scheme 2. Alignment of the X-ray Structures of the ON and OFF States of Dronpa Showing the Reorientation of the Chromophore upon Cis/Trans Isomerization a

Figure 4 .

 4 Figure 4. A) Decomposition of the long-lived isotropic spectrum of Dronpa (DADS of the plateau) into ground-state bleaching and intermediate absorption. B) Total long-lived anisotropy, OFF-state anisotropy and anisotropy of the intermediate deduced from the spectral decomposition of the isotropic data. These data were noise reduced by SVD (see §2.3 of Methods).

Figure 5 .

 5 Figure 5. Nano-to millisecond transient absorption spectroscopy of OFF-state Dronpa. The samples were excited at 425 nm. A) Kinetics measured at 500 nm in aqueous and deuterated Tris buffers of pH/pD 8.0. The symbols correspond to the raw data and the lines to monoexponential fits. B) Kinetics measured at 380 nm in the same buffers. C) 1-ms transient absorption spectrum in the 460 to 530 nm spectral range overlapped with the ON minus OFF difference spectrum (cis-phenolate minus trans-phenol).

Scheme 3 .

 3 Scheme 3. Model for the Photoactivation Mechanisms of Dronpa and Dronpa-2 b

Table 2 . Anisotropy and Reorientation Angle of the First Photoactivation Intermediates of Dronpa and Dronpa-2, Compared with Theoretical Predictions for a Cis Isomer

 2

		Experimental	Predicted a
	Species	X Dronpa	X Dronpa-2	Cis isomer
	Intrinsic anisotropy	0.24 ± 0.03	0.16 ± 0.04	0.04-0.25
	Reorientation angle α (°)	30 ± 3	39 ± 4	30-50

a Prediction based on Dronpa ON-and OFF-state structures and on the transition dipole moment directions reported in

Ref 44.

ACKNOWLEDGMENT

This work was supported by the P.-G. de Gennes foundation (grant FPGG 033). The authors thank Valérie Derrien for the construction of the expression plasmid for Dronpa and Fabienne Mérola for fruitful discussions. F. R. acknowledges financial support from CNRS and the "Initiative d'Excellence" program from the French state (grant "DYNAMO", ANR-11-LABX-0011-01).

AUTHOR INFORMATION

Corresponding author agathe.espagne@ens.fr

Notes

The authors declare no competing financial interest.


Fabien Lacombat

Pascal Plaza

Marie-Aude Plamont

Agathe Espagne
email: agathe.espagne@ens.fr

Photoinduced Chromophore Hydration in the Fluorescent Protein Dreiklang Is Triggered by Ultrafast Excited-State Proton Transfer Coupled to a Low-Frequency Vibration

Abstract :

Due to growing applications in advanced fluorescence imaging, the mechanisms and dynamics of photoinduced reactions in reversibly photoswitchable fluorescent proteins are currently attracting much interest. We report the first time-resolved study of the photoswitching of Dreiklang, so far the only fluorescent protein to undergo reversible photoinduced chromophore hydration. Using broadband femtosecond transient absorption spectroscopy, we show that the reaction is triggered by an ultrafast deprotonation of the chromophore phenol group in the excited state, in 100 fs. This primary step is accompanied by coherent oscillations that we assign to its coupling with a low-frequency mode, possibly a deformation of the chromophore hydrogen bond network. A ground-state intermediate is formed in the ps-ns regime, that we tentatively assign to the deprotonated water adduct. We suggest that proton ejection from the phenol group leads to a charge transfer from the phenol to the imidazolinone ring, which triggers imidazolinone protonation by nearby Glu222 and catalyzes the addition of the water molecule.

Table of Contents Graphic :

Reversibly photoswitchable fluorescent proteins (RSFPs) are Green Fluorescent Protein (GFP) homologues that can be switched back and forth between a fluorescent (ON) state and a nonfluorescent (OFF) state by irradiation at two wavelengths. Over the last decade, they have become essential tools for super-resolution microscopy and other advanced fluorescence imaging techniques. 1 X-ray crystallography studies of the ON and OFF states of several RSFPs revealed that in most cases, photoswitching is based on cis/trans isomerization of the chromophore coupled with proton transfer. [2][3][4] The photoswitching pathways of this class of RSFPs, the prototype of which is Dronpa, 5 have been further investigated by time-resolved spectroscopy with visible [6][7][8][9] and IR 10,11 probes, quantum chemistry 12,13 and low-temperature X-ray crystallography, 14 in order to establish the sequence in time of the two events, their characteristic timescales and detailed mechanisms. Although questions remain, a clear picture of chromophore transformations involved in the OFF→ON photoswitching of Dronpa has recently emerged: the first step consists of picosecond trans→cis isomerization of the chromophore in the excited-state, while proton transfer follows much later in the ground state, on the microsecond timescale. 7,11 In Dronpa-like RSFPs, the wavelength used to excite and image the fluorescence of the ON state under a microscope is identical to one of the wavelengths driving the ON-OFF transitions (e.g. ON→OFF for Dronpa 5 and OFF→ON for Padron 4 ), which prevents a perfect control of the ON or OFF state of the probe during data acquisition. Brakemann et al. recently tackled this problem by developing Dreiklang, 15 a green-emitting RSFP in which fluorescence excitation is decoupled from photoswitching. Interestingly, X-ray crystallography combined with mass spectrometry 15 showed that Dreiklang exhibits an entirely new photoswitching mechanism, based on the reversible covalent addition of a water molecule across the C=N bond of the chromophore imidazolinone ring, as illustrated in Scheme 1. The reaction reduces the electronic delocalization within the chromophore, causing a strong blue-shift of the absorption of the OFF state, 15 which is key for disentangling fluorescence from photoswitching. Dreiklang is still today the only reported example of a fluorescent protein displaying such photochemistry. The thermal formation of a hydrated structure similar to Dreiklang's OFF-state has however been proposed to occur in some fluorescent proteins during chromophore maturation, [16][17][18] suggesting that photoinduced hydration might be transferrable to other members of the GFP family. In spite of its originality and high potential for the development of advanced RSFPs, the dynamics and mechanistic details of Dreiklang photoswitching are still completely unknown. We report here the first time-resolved study of Dreiklang photoswitching. The ON→OFF photoconversion was investigated from ~100 fs to 10 ns using broadband femtosecond UV-visible transient absorption (TA) spectroscopy, in order to establish the events immediately following the absorption of a photon and the onset of the hydration process.

Figure 1 shows the absorption spectra of Dreiklang at pH 7.5, in the equilibrium ON state and after irradiation at ~410 nm (in agreement with the report of Brakemann et al. 15 ). In the ON state (black line), the two bands peaking at 413 nm and 511 nm correspond to the neutral (A) and anionic (B) forms of the chromophore, respectively. 15 Irradiation in the A band switches the protein to the OFF state, characterized by a strongly blue-shifted band, peaking at 339 nm (C; Figure 1, red line). The ON state can be regenerated by irradiation of the OFF state in the C band.

It also recovers spontaneously in the dark with a half-life of ~10 min at room temperature, which explains the residual A and B bands observed after 410-nm irradiation. Excitation of the B band (ON state) elicits a strong green fluorescence with a maximum at 525 nm (Figure 2A, grey dashdotted line). Interestingly, the weak steady-state fluorescence spectrum of form A is identical to that of form B (see Supporting Information (SI), Figure S4), which suggests that the phenol group of the neutral chromophore deprotonates very fast in the excited state.

Form A (ON state) was excited at 405 nm with 110-fs pulses, to initiate the ON→OFF switching. The experiment was performed at pH 6.3 in order to increase the amount of neutral chromophore (pK a = 7.2; 15 Figure S3). Homologous measurements were performed in D 2 O (Figures S1 andS2) to characterize proton transfer steps. Photoinduced spectral changes were recorded between 300 and 700 nm, as shown in Figure 2. The initial spectrum at 100 fs (black line in Figure 2A) exhibits several characteristic bands. The negative one extending from 360 to 430 nm matches the steady-state absorption of form A (grey dotted line) and is attributed to ground-state bleaching (GSB). The other negative band, ranging from 465 to 630 nm, is due to stimulated emission (SE). The shoulder at ~560 nm evokes the fluorescence spectrum of form B (grey dash-dotted line) and suggests that the red wing of the band includes the contribution of some readily deprotonated population. The blue wing (below 490 nm) has however no equivalent in the fluorescence spectrum of form B. It proves that a population of excited neutral chromophores (noted A * ) is also present at 100 fs. GFP-like fluorescent proteins containing neutral chromophores have indeed been reported to emit in the 400 to 600 nm spectral range, with maxima at 470-480 nm. 7,[19][20][21] We show as an example the fluorescence spectrum of the A * state of GFP, measured by Jaye et al. 21 before excited-state deprotonation of the chromophore, by time-resolved fluorescence spectroscopy (grey dashed line). Finally, the three positive bands (below 360 nm, from 430 to 465 nm and above 630 nm) correspond to spectral regions where excited-state absorption (ESA) dominates the other contributions. Let us note that a significant contribution of SE from A * is expected in the region of the central ESA band.

The transient spectra evolve then in three steps, highlighted in sections A-C of Figure 2.

During the first 500 fs (Figure 2A), one observes mainly a rise of the central ESA band, a decay of SE below 514 nm and a rise of SE emission above 514 nm (with temporary isosbestic point at 514 nm). We assign these spectral changes to the deprotonation of A * , leading to an excited anionic species noted I * . At 500 fs (blue spectrum), the characteristic SE of A * below 490 nm has indeed entirely disappeared and the new SE band peaks at 523 nm, close to the fluorescence maximum of form B. The inset of Figure 2A shows the double difference spectrum ΔΔA = ΔA(500 fs) -ΔA(100 fs) between 440 and 580 nm (black line), which illustrates the early changes. We could satisfactorily simulate ΔΔA by a weighted difference of anionic and neutral SE (inset of Figure 2A, red line; see Methods in SI for details). This demonstrates that the rise of the central ESA band around 460 nm is mainly due to the disappearance of the underlying SE of A * . GSB shows little evolution on this ultrashort timescale, suggesting a high yield of A * deprotonation. Finally, the partial decays of ESA below 360 nm and above 630 nm point to a lower absorption of I * in these regions, compared to A * . It is well-known that an excited-state proton transfer (ESPT) also occurs in GFP upon excitation of its neutral form, in a few picoseconds. 22,23 Remarkably, the dynamics is here more than one order of magnitude faster. The spectral signature is however qualitatively similar. 22 From 500 fs to 3 ps (Figure 2B), the ESA and GSB bands below 490 nm decay significantly, while the SE band remains essentially stable. To rationalize these apparently asynchronous evolutions of ESA and SE from the same species, we hypothesized that a slight reshaping of the ESA band was "amplified" by the overlap with GSB. The contribution of GSB was estimated by reference to the TA signal of a solution of [Ru(bpy) 3 ] 2+ (see Methods in SI for more details), and subtracted from the data. GSB-free spectra are displayed in the inset of Figure 2B. They indeed show a slight narrowing and blue-shift of the ESA band, in line with the slight narrowing of SE (Figure 2B, main frame). We assign these spectral changes to the vibrational cooling of I * .

Finally, the ps-to-ns timescale (Figure 2C) is characterized by a simultaneous decay of all bands, reflecting the decay of I * . Excited-state contributions are still present at 2.6 ns (pink spectrum), as evidenced by the remaining SE, and obscure the detection of potential ground-state photoproducts. An additional TA spectrum recorded at 10 ns (inset of Figure 2C) is however fully devoid of SE, showing that the decay of I * is complete at this delay. The spectrum consists of GSB and of the absorption band of a ground-state photoproduct (apparent maximum at 450 nm), which we named X. The signal is zero below 365 nm, where OFF-state absorption is expected (see Figure 1). We conclude that the switching reaction is not over at 10 ns.

A major result at this stage is that the ON→OFF photoconversion of Dreiklang involves as intermediate an excited anionic species (I * ) formed by excited-state deprotonation of form A. In these circumstances, one may wonder why the OFF state cannot be produced by direct excitation of the anionic B form. To clarify this point, we measured the spectral changes triggered by femtosecond excitation (80-fs pulses) of form B at 505 nm, at pH 10.0 (see Figure S3). As can be seen in Figure 3, the recorded TA spectra are made up of two bands: a positive ESA band below 465 nm and a negative band from 465 nm to ~650 nm, resulting from overlapping GSB and SE contributions (see the grey dotted steady-state absorption and fluorescence spectra). They decay simultaneously on the nanosecond timescale, as the excited state (noted B * ) decays. Comparison of Figure 2C with Figure 3 shows that the lifetime of I * is much shorter than that of B * . The TA signal half life is ~30 ps in the first case (I * ), and ~1 ns in the second case (B * ), thereby demonstrating that the two species are different. Moreover, the TA spectrum measured 20 ns after excitation of B shows no significant absorption around 450 nm (inset of Figure 3; the small negative signal seen at ~515 nm is likely due to some minor long component of the excited-state decay). This is in sharp contrast with the formation of X upon I * decay (inset of Figure 2C). We suggest that the peculiar reactivity of species I * is due to the fact that it combines the charge distribution of the excited anionic chromophore with the protein conformation of the neutral ground state.

We next proceeded to kinetic analysis of the TA data by a standard multiexponential global fitting procedure. Inspection of the TA kinetics of both the A and B forms however revealed lowfrequency oscillations occurring during the first picosecond, as shown at representative wavelengths in Figure 4, panels A and C. The modulation is particularly obvious at 520 nm for form B (Figure 4C, green line) and at 505 nm for form A (Figure 4A, dark blue trace).

Oscillations are also observed in the TA kinetics of form A in D 2 O (Figure S2A). These oscillations were taken into account by adding a damped cosine with wavelength-dependent phase to our fit function (see Methods in SI for details). The frequencies, periods and damping times of the oscillatory components of the different samples are given in Table 1 and The strong wavelength-dependence of the phases points to coherent oscillations resulting from the excitation of vibrational wave packets by the spectrally broad femtosecond pump pulses. [24][25][26] Vibrational wave packets can be created in the excited state, but also in the ground state, through impulsive stimulated Raman scattering. 27 Ground-state wave packets modulate GSB, while excited-state wave packets modulate ESA and SE. Photoproduct bands may also be concerned, when the vibrational coherence persists throughout a photochemical reaction. 26,28 The affected band shifts back and forth as a result of wave packet motion about the corresponding excited-or ground-state potential energy surface (PES) minimum, leading to out-of-phase oscillations of the kinetics in its blue and red sides. 25,27 When a single electronic transition is probed, the maximum of the band is expected to feature both a minimum in the amplitude of the modulation and a phase jump of π. 25,27 The oscillatory pattern may however get more complicated when several contributions overlap.

The oscillations in the TA kinetics of form B are mainly localized in the 480-550 nm region, where both GSB and SE contribute (inset of Figure 4D). Their amplitude goes through a minimum and their phase jumps simultaneously by ~π at 525 nm, the maximum of the SE contribution (see steady-state fluorescence spectrum in Figure 3). We conclude that the observed modulation originates mainly from vibrational wave packet motion on the excited-state PES. In the case of form A, the oscillatory behavior is observed over a broad spectral range extending from 350 to 600 nm (inset of Figure 4B). Since GSB does not contribute above 480 nm (see steady-state absorption spectrum in Figure 2A), the oscillations in the red part of the TA spectrum indicate that an excited-state wave packet is again involved. The amplitude of the modulation is the highest between 425 and 525 nm, the region where the main spectral changes associated to the ESPT reaction are observed (Figure 2A). Interestingly, a sharp phase jump of ~π and a minimum in the amplitude are seen at 525 nm, peak of the anionic SE, but not at ~475 nm, peak of the neutral SE. 7,[19][20][21] We propose that the ESPT step is too fast compared to the 630-fs vibration period (see Table 1) to allow the wave packet to oscillate in the reactant well (A * species), but that the vibrational coherence persists throughout the reaction and is observed in the longer-lived photoproduct I * . This scenario is supported by the kinetic analysis, which sets the time constant for ESPT to 100 fs (see below). The ~π phase jump and amplitude minimum found at 425 nm could then originate from oscillations in the ESA contribution of I * , expected in this very region (see the inset of Figure 2B). Similar low-frequency oscillations have been observed in the femtosecond transient grating and stimulated Raman data of GFP 29 and one of its mutants 30 , albeit with higher frequencies of 95 and 120 cm -1 . They have been assigned to the coherent excitation of intramolecular deformation modes of the excited chromophore. 29,30 The lower frequencies of the oscillations observed here in the femtosecond TA data of Dreiklang (30-90 cm -1 ; see Table 1) suggest that they are rather due to the excitation of intermolecular modes involving the chromophore and its environment.

Moreover, the sensitivity of the low-frequency mode of form A to deuteration (31 cm -1 in D 2 O compared to 53 cm -1 in H 2 O) suggests that it involves proton motion. One could think of a mode implying a deformation of the hydrogen bond network around the chromophore. The excited electronic transition, of charge transfer character, has indeed been reported to involve a decrease of the electron density on the phenol oxygen, 29,31 which is expected to displace the equilibrium geometry of the hydrogen bond network in which it participates.

In addition to the oscillatory component, three independent exponentials plus a biexponential decay (i.e. fixed ratio of two exponentials with a single amplitude spectrum) were needed to achieve a good fit of the TA data of form A, while a biexponential decay was sufficient to fit those of form B. The time constants are gathered in Table 1, and the corresponding amplitude spectra, or DADS (decay associated difference spectra) are shown in Figures 4B, 4D andS2B.

The 100-fs component of form A (Table 1) corresponds to ESPT, which is hence ~50 times faster than in neutral GFP. 22,23 As previously mentioned, this lifetime is much shorter than the 630-fs period of the coherent oscillations, which explains why the vibrational coherence is mainly observed in I * . Moreover, the ESPT dynamics in Dreiklang is significantly less sensitive to H/D exchange than in GFP (slowing down by a factor of 1.5 instead of ~522,23 ; Table 1).

Interestingly, the isotope effect is comparable to that on the period of the low-frequency vibration (slowing down by a factor of 1.7), suggesting that both phenomena are intimately related. We tentatively propose that ESPT in Dreiklang is gated by the coherently excited low-frequency vibration, which allows the protein to reach, after 100 fs, a nuclear configuration much more favorable to proton transfer than the Franck-Condon configuration. This idea is illustrated in the contour diagram of the excited-state PES at the top of Scheme 2A.

The 750-fs lifetime of form A (1.3 ps in D 2 O; Table 1) is assigned to the vibrational cooling of I * , and the three longer lifetimes (19 ps, 108 ps and 1.8 ns) to its multiexponential decay to X (Scheme 2A). Multiexponential kinetics in proteins are often attributed to structural heterogeneity. 7,8,10,32 The X-ray structure of ON-state Dreiklang at 100 K shows only a single conformation, 15 but the three decay times could be due to some conformational flexibility of the protein at room temperature, on timescales longer than the probed dynamics, as has been reported for other GFP variants. 33 The fact that two of the decay components can be fitted with a single biexponential decay is in agreement with this interpretation. No satisfactory fit could however be obtained replacing all three decay components by a triexponential decay, presumably because the 19-ps component still reflects some vibrational cooling of I * mixed to the excited-state decay, as suggested by its comparatively larger amplitude in the 350-500 nm region (Figure 4B). These fits confirm that the lifetime of I * is much shorter than that of the anion formed by direct excitation: the average lifetimes at 525 nm are 219 ps (I * ) and 2.3 ns (A * ). In addition, H/D exchange results in a slowing down of I * decay by a factor of ~2 (average lifetime of 469 ps in D 2 O), suggesting the involvement of proton motion in the quenching mechanism.

The present work establishes ultrafast excited-state deprotonation of the chromophore phenol group as the primary step of Dreiklang's ON→OFF photoconversion. The connection with the final addition of a water molecule to the imidazolinone ring is however not obvious. We give below a few directions. Inspection of the ON-state X-ray structure in the vicinity of the chromophore hydroxyl shows that it is hydrogen-bonded to a water molecule, which itself interacts with a histidine residue (His145; Scheme 2B). We propose that the photoejected proton is accepted by this water molecule, which in turn protonates His145 (Scheme 2B, step 1).

Deprotonation is then expected to trigger a charge redistribution in the chromophore, leading to an increase of the electron density on the imidazolinone ring (Scheme 2B, step 2). This is due to the π-conjugated and electron donor-acceptor nature of the chromophore structure. Interestingly, the hydrogen-bond network on the imidazolinone side comprises a glutamic acid residue (Glu222) which is suitably located to catalyze the addition of the water molecule (Scheme 2B).

Mutagenesis studies by Brakemann et al. 15 suggest that Glu222 is crucial for the photoswitching properties of Dreiklang. We propose that Glu222 is protonated in form A and transfers its proton to nitrogen N a in response to the increase in electron density on the imidazolinone ring (Scheme 2B, step 3). Protonation of N a would indeed activate the C b =N a bond and presumably catalyze the nucleophilic addition of the water molecule, which is only 3.5 Å away from electrophilic C b .

Glu222 could in addition increase the nucleophilicity of the water molecule by abstracting a proton from it.

The fact that X is formed following excitation of the A state, but not of the B state suggests that it plays the role of an intermediate in the photoswitching reaction. Within the above framework, one could imagine that X corresponds to the zwitterionic species formed upon imidazolinone protonation. This hypothesis seems however unlikely, because quantum chemistry calculations carried out on RSFPs predict that the chromophore zwitterion absorbs at longer wavelengths than the anion. 34,35 This has also been experimentally verified in the case of a methylated derivative of the GFP chromophore in solution. 36 X could more likely be the anionic water adduct formed from I * by the concerted mechanism illustrated in Scheme 2B (step 3).

Further evolution of X to the final OFF state would then involve phenolate protonation and conformational relaxations of the chromophore and its environment. This would be in line with the strongly blue-shifted absorption of the OFF state, since phenolate protonation is expected to reduce electronic delocalization within the chromophore.

In conclusion, we studied the photoswitching dynamics of Dreiklang for the first time by timeresolved spectroscopy. We found that the ON→OFF photoconversion, which ultimately results in chromophore hydration, 15 is triggered by an ultrafast deprotonation of the chromophore phenol group in the excited state, with a time constant of 100 fs. This primary reaction is accompanied by coherent oscillations that we assign to the coupling of the reactive motion with a lowfrequency mode, possibly a deformation of the chromophore hydrogen bond network. Decay of the deprotonated species (I * ) on ps-ns time scales leads to the formation of a ground-state intermediate (X). We suggest that charge transfer from the phenol to the imidazolinone ring in I * triggers protonation of the C=N bond nitrogen by nearby Glu222, thereby catalyzing the addition of the water molecule in X. Conversion of X to the final OFF state would require phenolate reprotonation, on a timescale longer than our observation window (10 ns).

  their amplitude and phase spectra are shown in the insets of Figures 4B, 4D and S2B (amplitude in black, phase in red). The insets of Figures 4A and S2A display the oscillatory parts of the fits for form A in H 2 O and D 2 O.

Figure 1 :Figure 1 :

 11 Figure 1:

Figure 2 :

 2 Figure 2: Femtosecond transient absorption spectra of Dreiklang's neutral ON state (form A)

Figure 3 :

 3 Figure 3: Femtosecond transient absorption spectra of Dreiklang's anionic ON state (form B)

Figure 4 :

 4 Figure 4: Global fits of Dreiklang's femtosecond transient absorption data. (A) and (B): Neutral

Table 1 :

 1 Global Kinetic Analysis of the Transient Absorption Data of ON State Dreiklang in its Neutral (A) and Anionic (B) Forms. Frequencies, Periods and Damping Times of the Oscillatory Parts. Time Constants of the Multiexponential Parts.

				Oscillatory part		Multiexponential part
			ν (cm -1 )	T (ps)	τ 0 (ps)	τ 1 (ps)	τ 2 (ps)	τ 3 (ps)	τ 4 (ps)	τ 5 (ps)
	A	H 2 O	53 ± 5	0.63 ± 0.06	0.24 ± 0.04	0.10 ± 0.01 0.75 ± 0.03	19 ± 1	108 ± 8	1800 ± 200
		D 2 O	31 ± 3	1.1 ± 0.1	0.29 ± 0.03	0.15 ± 0.01	1.3 ± 0.1	10 ± 2	144 ± 14	1100 ± 90
	B	H 2 O	86 ± 2	0.39 ± 0.01	0.36 ± 0.05	368 ± 61	2700 ± 450

Acknowledgments

We acknowledge financial support from CNRS, Ecole Normale Supérieure, P. and M. Curie University and the P.-G. de Gennes foundation (grant FPGG 033).

Supporting Information

Experimental methods, femtosecond TA data of form A in D 2 O, steady-state absorption spectra of the ON state at different pH, steady-state fluorescence spectra of forms A and B. This material is available free of charge via the Internet at http://pubs.acs.org.


Clemence Muller

Melanie Lavancier

Jeyan Bichon

Theo Hannotte

Jean-Francois Lampin

Sophie Eliet

Romain Peretti

Beyond the Dynamic range approach In noise evaluation for Terahertz Time domain spectrometers

come   L'archive ouverte pluridisciplinaire

I. INTRODUCTION

HE goal of every spectroscopy is to extract information from the recorded data. This information lies in models fitting a transfer function from a measured reference to a measured sample [START_REF] Peretti | Modeling and parameter retrieving in time domain spectroscopy of material and metamaterial[END_REF][START_REF] Peretti | Retrieving material and metamaterial parameters directly from time-domain spectroscopy time trace[END_REF][START_REF] Peretti | Thz-tds time-trace analysis for the extraction of material and metamaterial parameters[END_REF]. This comparison is straightforward in frequency-domain spectroscopy since the transfer function is used in a multiplication. In addition, the noise is often an uncorrelated white noise of which envelop serves as weighing function for the fit.

However, as well explained in [START_REF] Mohtashemi | Maximum-likelihood parameter estimation in terahertz time-domain spectroscopy[END_REF][START_REF] Steven Dodge | A maximum-likelihood analysis framework for terahertz time-domain spectroscopy[END_REF], the specificity of THz-TDS, the data are in the time domain and the models are still the same: in the frequency domain. It follows that the multiplications by the transfer function in the frequency domain become convolutions by a transfer function in the time domain. Therefore, the non-correlated hypothesis for noise is never true. E.g. for a slab sample, the model transforms the reference pulse into several Fabry-Perrot echoes, each of them getting the same, consequently correlated, noise. Thus, the weighting function does not apply any more, but one needs to use the noise correlation matrix as weighting in the fit. It means that the knowledge of the noise both correlated and uncorrelated is extremely important.

Impact of the noise on THz-TDS measuremnts is a long haul effort [START_REF] Duvillaret | Influence of noise on the characterization of materials by terahertz time-domain spectroscopy[END_REF] and we propose here to use a very common but still not standardized methods based on statistics and repetition of the measurements.

II. RESULTS

The datasheet of the THz-TDS systems includes dynamic range data that compare the signal at the peak frequency to the noise at frequency where the system does not operate. This clearly help making the difference from a first generation system with the new ones. However, it is not a clear figure on how reliable will be your measurements. To go further towards this direction, we propose a statistical approach in measuring several time traces and compare their mean value (signal) and standard deviation (noise) in the time (fig. 1 ) and frequency domain (fig. 2).

As seen on fig. 2, the dynamic range vision tremendously underestimate the noise in the range of high signal to noise ratio. Here we plotted the raw and corrected data from measurements of five 100 ps time long traces (5000 average). One of the reason is that noise does not only come from the variation of current from the detector but as well from imprecision on the delay line [START_REF] Rehn | Periodic sampling errors in terahertz time-domain measurements[END_REF], parasitic low frequency emission and variation of the power of the excitation laser. To take it into account, we corrected the time traces from a fixed delay (mean square optimization) after a low frequency filtering (1 st correction), and further away using a correction term proportional to the signal and to its second derivative to take into account the variation of the laser (2 nd correction). This shows that first the noise cannot simply be considered as a dark noise uncorrelated one. In addition, it shows that after few corrections it is possible to decrease the noise tremendously even bellow the usual dynamic range vision.

Clémence Muller 1 , Mélanie Lavancier 1 , Jeyan Bichon 1 , Théo Hannotte, Jean-François. Lampin 1 , Sophie Eliet 1 and Romain Peretti

SUMMARY

THz-TDS is one of the major assets of the THz range thanks to the compactness, robustness, broadband and high signal to noise ratio of this system. Today the use of the dynamic range as standard for signal to noise ratio does not meet the requirement for a qualitative information extraction from the recorded data. We proposed here a classic statistical method to compare two systems and show that additional corrections can improve the signal to noise ratio by more than 50 dB.

1 1Fig. 1 .

 11 Fig. 1. Time domain signal (mean value) and noise (standard deviation) of TDS measurements without correction (no correction), with a delay shift correction after a high pass filter ( 1 st correction), with in addition a correction term proportional to the signal and the second derivative of the signal (2 nd correction).

Fig. 1 .

 1 Fig. 1. Time domain signal (mean value) and noise (standard deviation) of TDS measurements without correction (no correction), with a delay shift correction after a high pass filter ( 1 st correction), with in addition a correction term proportional to the signal and the second derivative of the signal (2 nd correction).

ACKNOWLEDGMENTS

The authors want to thanks the "Région Hauts-de-France" for the International Chair of Excellence "ThOTroV, the "TeraStoVe" pluridisciplinary project from I-site ULNE, the welcome talent grant "NeFiStoV" from the European Metropole of Lille (MEL); and the "Agence Nationale de la Recherche" for the STEPSON ANR-20-CE42-0002 grant?