Reversible Photoisomerization of the Isolated Green Fluorescent

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Reversible Photoisomerization of the Isolated Green Fluorescent Protein Chromophore Eduardo Carrascosa, James N Bull, Michael S. Scholz, Neville J. A. Coughlan, Seth Olsen, Uta Wille, and Evan John Bieske J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.8b01201 • Publication Date (Web): 03 May 2018 Downloaded from http://pubs.acs.org on May 6, 2018

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The Journal of Physical Chemistry Letters

Reversible Photoisomerization of the Isolated Green Fluorescent Protein Chromophore Eduardo Carrascosa,† James N. Bull,† Michael S. Scholz,† Neville J. A. Coughlan,† Seth Olsen,‡,¶ Uta Wille,† and Evan J. Bieske∗,† †School of Chemistry, The University of Melbourne, Parkville, Victoria 3010, Australia ‡School of Chemistry, Monash University, Clayton, Victoria 3800, Australia ¶deceased: 28 January, 2018 E-mail: [email protected] Phone: +61 3-8344-7082

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Abstract Fluorescent proteins have revolutionised the visualisation of biological processes, prompting efforts to understand and control their intrinsic photophysics. Here we investigate the photoisomerization of deprotonated p-hydroxybenzylidene-2,3-dimethylimidazolinone anion (HBDI− ), the chromophore in green fluorescent protein and in Dronpa protein, where it plays a role in switching between fluorescent and non-fluorescent states. In the present work, isolated HBDI− molecules are switched between the Z and E forms in the gas phase in a tandem ion mobility-mass spectrometer outfitted for selecting the initial and final isomers. Excitation of the S1 ←S0 transition provokes both Z →E and E →Z photoisomerization, with a maximum response for both processes at 480 nm. Photodetachment is a minor channel at low light intensity. At higher light intensities, absorption of several photons in the drift region drives photofragmentation, through channels involving CH3 loss, and concerted CO and CH3 CN loss, although isomerization remains the dominant process.

Graphical TOC Entry

Keywords isomerization, green fluorescent protein, ion mobility, action spectroscopy, anion

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Green fluorescent protein (GFP) and similar biomolecules such as the Dronpa protein are commonly employed for monitoring and reversibly switching biological systems including light harvesting materials and photocatalysts. 1–5 The optical absorption and emission of GFP (and mutants) relies on the strong S1 ←S0 transition of a chromophore based on the phydroxybenzylidene-2,3-dimethylimidazolinone anion (HBDI− ) residing inside the protein’s folded β-barrel structure. 6,7 The photochemistry and function of proteins containing HBDI− are intrinsically linked to isomerization of the HBDI− core following photoexcitation. As shown in Figure 1, the HBDI− chromophore has Z and E isomers, corresponding to rotation of the linking C=C bond, with the Z isomer being the more stable isomer in solution and in the gas phase. 8 For GFP, which has a high fluorescence quantum yield (φf =0.8; ref. 9), the HBDI− chromophore is locked in the Z isomeric form in the S0 and S1 states through hydrogen bonds with surrounding amino acid residues. 5 On the other hand, in the Dronpa protein, the HBDI− chromophore can be photoswitched between a fluorescent state (Z-HBDI− anion chromophore core) and a non-fluorescent state (E-HBDI neutral chromophore core) formed following proton transfer. 10–13 In solution at room temperature, the isolated HBDI− chromophore fluoresces very weakly because of internal conversion associated with rapid passage through conical intersections involving twisted structures corresponding to internal rotation of either the imidazolinone moiety (τI ) or phenol moiety. 14,15 The fluorescence quantum yield in solution increases as the temperature is lowered due to frictional inhibition of twisting on the S1 surface. 16 The intricate nature of the HBDI− chromophore’s photophysics in proteins and in solution has prompted a series of spectroscopic and dynamical investigations in the gas phase, where comparisons between theory and experiment should be more straightforward. Photodestruction and photodissociation action spectra measured for HBDI− in a fast ion beam 17–22 and in ion traps 23,24 exhibit a prominent S1 ←S0 band in the visible region, extending from 510 nm down to 420 nm with a peak at 480 nm. The main deactivation pathways from the S1 state are summarized in Figure 1 and include internal conversion (IC) and electron autodetach-

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Figure 1: Photoinduced processes for HBDI− . Molecules in the S1 state can undergo internal conversion (IC) mediated by passage through a conical intersection (CI) accompanied either by photoisomerization (PI) or return to the original structure. Statistical isomerization can occur by passing over a barrier on the S0 surface (TS). IC to the S0 state can precede fragmentation (IC+PF, mostly CH3 loss). Photodetachment can occur through autodetachment (AD) from the S1 state, direct detachment (DD), or IC followed by thermionic electron emission (IC+TE). ment (AD) to give the neutral HBDI radical in the D0 state. Because of their low intrinsic cross sections, direct electron detachment (DD) transitions are likely to be less important in the region of the S1 ←S0 transition. Thermionic emission (TE) and photodissociation (PD) are both relatively slow processes following absorption of a single photon and internal conversion. 19,25 Competition between electron detachment, photodissociation and fluorescence for HBDI− in the S1 state has been explored using ultrafast pump-probe photoelectron spectroscopy 19,25–30 and photodissociation spectroscopy. 21,31 Theoretical investigations suggest that HBDI− molecules excited to the S1 state access two separate conical intersections associated with internal rotation of either the imidazole ring (see Figure 1) or the phenolate ring. 8,32–34 The relative importance of pathways leading to the two CIs depends on details of the excited state potential energy surfaces (existence of barriers on the S1 surface) and possibly also on the excitation wavelength. Whereas rotation about the methine bond connected to the imidazole may cause Z-E isomerization, rotation about the bond connected to the phenolate leaves the structure unchanged. However, even 4


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in the latter case, IC could conceivably be followed by internal rotation of the imidazole ring, with crossing of a 1.24 eV Z →E barrier on the S0 surface (see SI). Previous gas-phase studies have been insensitive to the HBDI− anion’s isomeric form and to its photoisomerization dynamics. Further information on these aspects is desirable because torsion around the linking methine bonds has been implicated in non-radiative deactivation of HBDI− from the S1 state and in photoswitching of the Dronpa protein. 12,32,35 Furthermore, it is conceivable that in previous gas phase studies mixtures of Z -HBDI− and E -HBDI− have been probed inadvertantly, potentially complicating interpretation of the data. Here we explore the photochemistry of HBDI− isomers in the gas phase using a combination of tandem ion mobility-mass spectrometry and laser spectroscopy, whereby ions drifting through N2 buffer gas under the influence of an electric field are exposed to a pulse of tunable radiation, which, depending on wavelength and intensity, causes photoisomerization, electron detachment or photodissociation. There are several questions we seek to address. Do both the Z -HBDI− and E -HBDI− isomers exist in the gas phase? Is photoisomerization following excitation of the S1 ←S0 transition an intrinsic response of the isolated HBDI− molecule? Do the S1 ←S0 photoisomerization action spectra of Z -HBDI− and E -HBDI− resemble previously recorded photodissociation and photodestruction action spectra? Finally, what are the relative importances of photoisomerization, photodissociation and electron photodetachment following excitation of the S1 ←S0 transition? Photoisomerization, photodetachment and photodissociation of the Z and E isomers of HBDI – were probed in a tandem ion mobility mass spectrometer illustrated in Figure 2. 36 HBDI− ions were generated via electrospray ionization and collected by a radio frequency (RF) driven ion funnel from which they were injected into the first stage of a drift region, where they were propelled by an electric field (44 V/cm) through N2 buffer gas at pressure ≈6 Torr. The Z and E isomers were separated spatially and temporally due to their different collision cross sections with the N2 buffer gas. The arrival time distribution (ATD) for the

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Figure 2: Tandem drift tube ion mobility-mass spectrometer for studying photoisomerizaton of HBDI− anions. Ions from an electrospray source pass through a desolvation capillary, are collected by an ion funnel (IF1) and injected into the drift region through an ion gate (IG1). Charged isomers separate in the first stage of the drift region, with the target isomer ions selected by ion gate IG2. Selected ions are exposed to tunable radiation from an OPO with resulting photoproducts separated in the second stage of the drift region. Finally, the ions are collected by a second ion funnel (IF2), transmitted by octupole guide, mass-selected by a quadrupole mass filter and detected by a channeltron. HBDI – ions shows evidence for a single isomer at low collision energy conditions in the ion funnel (low RF drive voltage), with a second faster isomer appearing at higher collision energies (high RF drive voltage). Although the arrival times of the two isomers differed by ≤0.5 % with pure N2 buffer gas, addition of ≈1 % 2-propanol (C3 H7 OH) to the drift gas increased the separation to ≈2 % (see Figure 3a and Figure S2 in the SI). Electronic structure calculations at the CCSD(T)/aug-cc-pVTZ//ωB97X-D/aug-cc-pVTZ level of theory (see SI) predict the E isomer lies 0.11 eV higher in energy than the Z isomer, suggesting the earlier, weaker peak in Figure 3a is associated with E -HBDI− and the later, stronger peak is connected with Z -HBDI− . These assignments are consistent with collision cross sections calculated using the MOBCAL package, 37 which predicted a slightly larger cross section for the Z isomer (Ωc =156.2 Å2 ) than for the E isomer (Ωc =155.7 Å2 ). For comparison, the measured cross sections are Ωm = 161 ±4 Å2 (Z isomer) and Ωm = 160 ±4 Å2 (E isomer). Photoisomerization of HBDI− was probed by operating the apparatus as a tandem IMS. An electrostatic ion gate (IG2) midway along the drift region was opened for 100 µs at an appropriate delay to select either Z -HBDI− or E -HBDI− anions, immediately after which

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(a) N2 + C3H7OH, light-off

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light-off diff.(x 15)

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Figure 3: (a) ATDs for HBDI – for low (black) and high (orange) RF drive voltage conditions in IF1. Peaks corresponding to Z - and E -HBDI− are indicated. The peak at later arrival times corresponds to a methanol-HBDI− cluster that traverses the drift region intact and then dissociates to yield bare HBDI− in the second ion funnel prior to mass selection (b) light-off ATD (black) and light on-light off difference ATD (red) for Z -HBDI− obtained at low light intensity (LI) in N2 +C3 H7 OH buffer gas showing the photoisomerization peak (PI), (c) light-off ATD (black) and light on-light off difference ATD (red) for Z -HBDI− obtained at high light intensity (HI) in N2 +C3 H7 OH buffer gas, (d) light-off ATD (black) and light on-light off difference ATD (red) for Z -HBDI− obtained at low and high light intensity in N2 +SF6 buffer gas, (e) light-off ATD (black) and light on-light off difference ATD (red) for Z -HBDI− obtained at low and high light intensity in N2 +SF6 buffer gas. The excitation wavelength was 445 nm for all measurements.

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the selected ion packet was intercepted with a light pulse from a tunable wavelength optical parametric oscillator (OPO). Resulting photoisomers were separated from the original isomers in the second drift region. Figure 3 (b) presents a light-off ATD for mobility selected Z -HBDI− anions and a light-on/off difference ATD with λ=445 nm, clearly showing formation of faster E -HBDI− anions. Similarly, selection of the E -HBDI− anions and exposure to visible light over the 410-500 nm range leads to E →Z photoisomerization (see Figure S2 in the SI), proving that HBDI – can be reversibly photoisomerized in the gas phase. As shown in Figures 4 (a) and (b), the photoisomerization action (PISA) spectra of Z -HBDI – and E -HBDI – are very similar over the 425–500 nm range, suggesting that the S1 ←S0 transition energy is relatively insensitive to the isomeric form. However, it should be remembered that the ions are warm - collisions with buffer gas molecules in the drift region are predicted to raise the ions’ temperature slightly above the buffer gas temperature (Teff ≈300 K). Spectra of cryogenically cooled Z -HBDI – and E -HBDI – ions may exhibit subtle variations, reflecting differences in their geometries and vibrational frequencies in the S0 and S1 states. (a)

PI (Z E)

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Figure 4: Z→E (a) and E→Z (b) photoisomerization (PI) action spectra measured at low intensity (0.1–0.2 mJ/cm2 /pulse). Both spectra are normalized with respect to light intensity. (c) Photoisomerization (PI, blue), total photofragmentation (PF, purple) and photodetachment (PDet, olive) action spectra of the Z -isomer of HBDI− at high light intensity (>1 mJ/cm2 /pulse).

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If photoisomerization were the only process that ensued following excitation of the S1 ←S0 transition, depletion of the Z -HBDI− isomer would be balanced by formation of the E -HBDI− isomer. However, even at low intensity, a minor fraction of the HBDI− ions is lost, either through photodetachment or photodissociation. At low laser intensity and with the QMF set to transmit all ions, we found no trace of charged photofragments. However, adding ≈5 % SF6 to the N2 buffer gas to capture low energy electrons yielded light on/off difference ATDs exhibiting an SF6 – peak (Figure 3d), proving that electron detachment is responsible for the additional depletion. As observed previously, the onset of photodetachment, following absorption of a single photon, occurs below the electron detachment threshold (2.73 eV; ref 29) with the energy deficit presumably provided by the ions’ internal vibrational energy (≈0.3 eV at 300 K). 18,24,25,28 Clearly, photoisomerization dominates electron detachment for both Z -HBDI− and E HBDI− over the 440-500 nm range in the drift tube environment. Measurements were performed as a function of light intensity at λ=445 nm (0.06 eV above the electron detachment threshold) for Z -HBDI− in N2 +C3 H7 OH buffer gas to determine relative yields of photodepletion, photoisomerization and photofragmentation, and in N2 +SF6 buffer gas to ascertain relative yields of photodetachment and photofragmentation. The data (presented in Figure S3 in the SI) indicate that for light intensities ≤0.5 mJ/cm2 /pulse the ratio of the photoisomerization and photodetachment yields is φP I /φAD ≈4. This should represent a lower limit for the ratio of IC and electron autodetachment yields (φIC /φAD ) because IC of the Z isomer can be associated with isomerization to the E isomer or recovery of the Z isomer. Our measurements are roughly consistent with theoretical estimates by Bochenkova and Andersen for kIC /kAD =2.5 at 450 nm, 34 and with ultrafast pump-probe photoelectron measurements for HBDI – anions at 300 K, which indicate that the internal conversion rate is at least an order of magnitude larger than the autodetachment rate (kIC /kAD ≈25 at 480 nm). 28 At higher light intensities (≥1 mJ/cm2 /pulse) HBDI− anions absorb two or more photons, raising their internal energy to the point where rapid photofragmentation and thermionic

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emission compete with collision induced vibrational relaxation [see Figure 3(c) and (e)]. The main m/z 200 and m/z 131 photofragments have been observed previously, 18,24,38,39 with the former fragment arising from methyl loss from the imidazole N atom. We propose that the m/z 131 photofragment is a substituted phenoxide radical anion produced through loss of CH3 CN and CO from the m/z 200 species (mechanism outlined in Figure S4 of the SI). The photoisomerization action (PISA) spectrum for Z-HBDI – recorded at high light intensity (>1 mJ/cm2 /pulse) [Figure 4(c)] has the same 500 nm onset and maximum at 480 nm as the low intensity PISA spectrum (Figure 4a), but extends further to shorter wavelength, presumably because stronger transitions in the S1 ←S0 band are saturated. The photofragmentation action spectrum (monitoring m/z 200 and m/z 131 photofragments) which results from multi-photon absorption is narrower than the PISA spectrum and resembles the photodissociation spectra reported by Jockusch and co-workers. 23 The total photodepletion action spectrum for Z -HBDI – at high light intensity is very similar to the photodepletion action spectrum measured in an ion storage ring by Andersen and co-workers (see Figure S5 of the SI). 20,21 In summary, we have shown that both the Z and E isomers of HBDI− exist in the gas phase and are efficiently interconverted through exposure to visible light, with a maximum response at 480 nm. The photoisomerization action spectra are similar for Z and E isomers and resemble previously recorded photodissociation and photodestruction action spectra for HBDI− . 21,23 At low light intensity, photoisomerization is the dominant process across the S1 ←S0 band with electron autodetachment occurring as a minor channel. At higher light intensities absorption of several photons drives photodissociation, primarily through the methyl loss channel. The current study highlights the importance of characterizing and controlling the isomeric constitution of molecular samples that are subject to spectroscopic and dynamical investigations. Depending on its preparation and treatment, a population of HBDI− ions in the gas phase contains both Z and E isomers, which will have distinct properties. Undoubtedly, future investigations will combine isomer-specific techniques with low

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temperature spectroscopic approaches to elucidate subtle differences between the properties of the Z - and E -HBDI− . It is interesting to note that recent pump-probe measurements for HBDI− suggest IC is suppressed at the expense of fluorescence at low temperature, 31 an observation consistent with a shallow well in the Franck-Condon region of the S1 state that traps electronically excited anions and prevents passage to the conical intersection seam. If this interpretation is correct, there should be a decrease in the photoisomerization and IC yields as the HBDI− temperature is lowered.

Acknowledgement We thank Professor R. Jockusch and Professor L. Andersen for sharing data from ref. 24 and ref. 20, respectively. The authors thank the Australian Research Council for financial support under the Discovery Project Scheme (DP150101427 and DP160100474). E.C. acknowledges support by the Austrian Science Fund (FWF) through a Schrödinger Fellowship (Nr. J4013N36). M.S.S. thanks the Australian government for an Australian Postgraduate Award scholarship. Supporting Information Available The supplementary information contains details on the experimental arrangement, comparisons of action spectra recorded in this investigation with photodissociation and photodepletion action spectra from refs. 24 and 20, respectively, and calculations of the geometry and energy for the transition state between Z - and E HBDI− .

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Reversible Photoisomerization of the Isolated Green Fluorescent

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