Photoisomerization and Proton Transfer in the Forward and Reverse

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Photoisomerisation and Proton Transfer in the Forward and Reverse Photoswitching of the Fast-Switching M159T Mutant of the Dronpa Fluorescent Protein Marius Kaucikas, Martijn Tros, and Jasper J. van Thor J. Phys. Chem. B, Just Accepted Manuscript • Publication Date (Web): 04 Nov 2014 Downloaded from http://pubs.acs.org on November 5, 2014

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The Journal of Physical Chemistry

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

Marius Kaucikas, Martijn Tros†, Jasper J. van Thor*

Imperial College London, South Kensington Campus, SW7 2AZ London, United Kingdom, †University of Amsterdam, Faculteit der Natuurwetenschappen, Wiskunde en Informatica (FNWI), Science Park 904, 1098 XH Amsterdam, The Netherlands *To whom correspondence should be addressed: email [email protected]

KEYWORDS: Dronpa, fluorescent protein, ultrafast infrared spectroscopy, TR-IR, photoisomerisation, proton transfer, photoswitching

ACS Paragon Plus Environment

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ABSTRACT

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


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closely with the on-minus-off FTIR difference spectrum, indicating thermal deprotonation and rearrangement of the Arg66 sidechain to be complete.

1. Introduction

The ‘Dronpa’ protein from the coral Pectiniidae 1 is one of the most commonly used reversibly photoconvertable fluorescent proteins. The p-hydroxybenzylideneimidazolinone chromophore of Dronpa is derived from the Cys-Tyr-Gly tripeptide and is present in a cis conformation in the ‘On’ state 2,3, as in the Aequorea victoria Green Fluorescent Protein (avGFP) 4,5. The X-ray structures of the highly fluorescent ‘On’ state were reported by Stiel et al 2 and Wilmann et al 3. Andresen et al

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able to photoaccumulate and cryo-trap a partially occupied off structure, which supported the trans configuration of the chromophore in the blue-absorbing off state (λmax = 390 nm for both Dronpa and M159T-Dronpa). In addition to cis-trans photoisomerisation, the most notable difference included the reorientation of the side chains of Arg 66 and His 193 6. The blue-shifted absorption maximum at 390nm indicates protonation of the hydroxyphenyl oxygen in the off state. In addition to these local structural differences, NMR spectroscopy indicated significant differences with the on state structure of a number of other residues from 1H-15N heteronuclear single-quantum coherence (HSQC) spectra 7. Specifically, chemical shift differences were seen for seven residues Gly36 (β3), Cys62(β3), Met93(central helix), Ala160(β8), Cys171(β9), Asp172(β9), Phe173(β9). In addition, 25 further residues were not observed in the off state due to exchange broadening in the intermediate regime, indicating substantial structural fluctuations affecting sites in the


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β4, β7, β8, β10 and β11 strands7. It is therefore clear that on-off photoswitching globally affects the equilibrium structure as well as the magnitude and frequencies of structural fluctuations, which are roughly localized to one half of the protein, contained within the A-C dimmer interface7. Interestingly, the dimerisation binding constants are additionally affected and have been exploited for optogenetics experiments 8. Several fast-switching mutants have been reported for Dronpa, particularly improving on the low photochemical quantum yield of on-off switching, which was estimated to be 3.2*10-4 for the wild type Dronpa1,9. One of those mutants, Dronpa-M159T, was selected for showing both an increased quantum yield for on-off photoswitching as well as for the off-on reaction 2. Stiel et al report a 1143-fold increase of the rate of the on-off reaction at room temperature for the M159T mutant, implying an absolute quantum yield of 0.37 based on the 3.2*10-4 value for the wild type 1,2,9. In addition, the off-on reaction was found to have increased 2-fold, suggesting a quantum yield of 0.72 relative to the 0.36 value given for the wild type 9. The fundamental mechanisms and sequence of events were investigated previously for the reversible photoswitching reactions of the wild type Dronpa fluorescent protein using visible-pump infrared-probe spectroscopy 10. Previous proposals for the off-on switching invoked excited state proton transfer (ESPT) in addition to trans-cis photoisomerisation 9,11,12. Furthermore, others proposed a twisted intramolecular charge transfer state in the photoisomerisation reaction, favoring a mechanism that included a concerted excited state proton transfer and trans-cis photoisomerisation process 13. Additionally, in the very similar asFP595 reversible photoswitchable fluorescent protein from the sea anemone Anemonia sulcata (which has a Met-TyrGly derived chromophore) it was proposed that the chromophore is present as a


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zwitterion in the trans form and that excited state proton transfer proceeds from the imidazolinone nitrogen 14,15. FTIR difference spectroscopy identified the on and off states as the cis anion and the trans neutral chromophore, respectively 10. Considering the high structural and spectroscopic similarities with Dronpa, these assignments could possibly also apply to the asFP595 on and off states. Blue excitation of the photoaccumulated off state results in a dominant 9ps excited state decay component, with formation of a primary photoproduct that was assigned to the cis neutral chromophore on the basis of TR-IR measurements 10. The 9 ps time constant, measured in 2H2O at pD 7.8, was also in agreement with fluorescence measurements, which gave a 14 ps time constant in 1H2O at pH 7.4 with excitation at 390nm and detection at 440 nm 9. The IR difference spectrum of the primary photoproduct prominently lacks phenolate modes, thus excluding the possibility of excited state proton transfer, and was stable up to 100ps of the delays reported 10. A recent study reported TR-IR measurements of the off state of the M159T mutant of Dronpa and came to very different conclusions 16. Firstly, Lukacs et al assign the primary photoproduct to an electronic ground state of the trans neutral chromophore. Second, pump-probe delays beyond 100ps showed an additional phase with a 459ps time constant and spectral features which were assigned to a relaxation process. Lukacs et al proposed ground state trans-cis isomerisation to follow the primary photoproduct thermally on longer time scales, eventually forming the on state. This interpretation relied on the apparent absence of a frequency upshift for the C=O stretching mode in the infrared spectrum of the primary photoproduct, which was expected to result from trans-cis isomeristion 16. Due to the very low quantum yield of on-to-off switching in the wild type Dronpa, green excitation resulted in observation of radiative decay only, as the


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amplitude of the photoproduct is too small to be resolved within the available signal to noise of the TR-IR measurements. However, the excited state vibrational response was found to include transient absorption features belonging to protein groups, which were proposed to belong to Arg66 10. The suggested quantum yield of 0.37 for the M159T mutant 2 would certainly allow the photoproduct to be resolved, at least on nanosecond time scale as the product of excited state decay. However, a recent TR-IR study of the M159T mutant reported only three decay time constants 3ps, 30ps and 300ps with decay associated spectra which represent only radiative decay processes 16

. Here, we present a TR-IR, FTIR and DFT study of the fast switching M159T

mutant of Dronpa, which additionally addresses both the experimental differences as well as the different assignments made by Lukacs et al. 16 relative to the previously reported study of the wild type Dronpa 10. Comparison with wild type Dronpa measurements should reveal if the fundamental processes are conserved after the M159T mutation. A number of observations were made that differ from to the previous M159T TR-IR study16, in addition to new data revealing intermediate products of the on and off states. Firstly, TR-IR measurements of the on state with extended delays reveal spectral evolution occurring with a 185ps rise time and a 1.1 ns decay time constant, signaling modification of the excited state geometry but may have contributions of induced absorption belonging to the primary photoproduct. Second, negative pump-probe time delays of the on state reveals the intermediate product spectrum at 1 ms which clearly signals protonation of the chromophore, confirming a previous proposal for the wild type Dronpa which was primarily based on the absence of anionic phenolate modes on picosecond time scale10, while in this study we were able to directly reveal the millisecond time resolved protonated


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reaction product. Third, Singular Value Decomposition (SVD) of TR-IR of the off state provides statistical significance for the conclusion that the data collected at 1 KHz repetition rate can not support a 500 ps time constant from our measurements, in contrast to those reported by Lukacs et al. 16, possibly due to the transient background difference at 10KHz and 1 KHz pump-probe repetition rate. Fourth, a 1 ms measurement of the off state agrees closely with the static FTIR on-minus-off difference spectrum, showing that deprotonation and structural rearrangements are completed in this time interval. Finally, the assignment of the primary photoproduct of the off state is addressed in view of the distorted chromophore geometry, using redundant coordinates for geometry optimization and frequency calculation using Density Functional Theory (DFT). These calculations indicate that the experimental TRIR spectrum of the primary photoproduct of the off state formed with 0.6 and 14ps time constants can be supported on the basis of the frequency shifts caused by geometry distortions of the trans neutral chromophore in the ground state relative to in vacuo optimized coordinates.

2. Materials and Methods

The M159T mutation was introduced into the original expression construct pRESTbDronpa, and was expressed in E.coli, purified by Ni-NTA affinity chromatography and gel filtration chromatography as previously described 10. For photoswitching kinetics measurements the sample was in 1H2O, 50 mM This/HCl pH 7.8. The macroscopic on-off switching kinetics of Dronpa and M159T-Dronpa were measured using a continuous wave diode pumped solid state Nd:YAG laser at 473 nm and 15 mW/cm2 power and approximately 1 cm2 diameter beam diameter using an expander.


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The off-on switching was measured by illumination with an LED array at 400 nm (700mA, 3.9V, 5W, Mouser Electronics 897-LZ110UA00-U8) and 26.7 mW/cm2 power. For both FTIR spectroscopy and TR-IR spectroscopy the samples were concentrated to approximately 2mM concentration in 2H2O, 10mM Tris/HCl pD 7.8, in a Harrick cell with a 12 µm spacer, and had an absolute absorption of ~0.8 at 1650 cm-1 and ~ 1.0 at the maximum at 1626 cm-1. FTIR measurements were recorded on a Bio-Rad FTS 175C FT-IR spectrophotometer equipped with a mercury cadmium telluride (MCT) detector, and collected at 2 cm-1 resolution

TR-IR The femtosecond time resolved pump-probe mid-infrared spectrometer was described previously 10,17. In brief, the output of Ti:Sapphire regenerative amplifier (Spitfire PRO, Spectra Physics, 4W, 70 fs) was divided between two optical parametric amplifiers (Topas-C, Light Conversion). One of the parametric amplifiers was equipped with non-colinear difference frequency generation module that produced mid IR probe pulses. The other used additional frequency mixing stages to generate UV and visible pump pulses. Detection system consisted of spectrometers (Triax190, Horiba) with mercury cadmium telluride (MCT) array detectors (128 pixel arrays, Infrared Systems Development Corp) attached to them. The measurements were performed at 6.1 µm centre probe wavelength and a spectrometer resolution of 3.3 cm-1. The pump beam intensity was adjusted using reflective neutral density filters and the polarization was rotated to a ‘magic angle’ (54.7 deg) relative to probe beam. The beam was focused on the sample to 300 µm spot FWHM. This corresponded to an average power density of 1 W/cm2 for


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femtosecond excitation at 503 nm (on state) and 2.8 W/cm2 for pumping at 400 nm (off state). The sample cell was translated orthogonally to the beam in Lissajous patterns with average speed of 50 µm ms-1. 473nm and 400nm background illumination was provided by the diode laser and LEDs to maintain the off and on states, respectively. Singular Value Decomposition (SVD) and global analysis was performed as previously described 18.

3. Results

3.1 Quantum yield of reversible photoswitching of Dronpa-M159T

Considering the previous report of an increase of the on-off switching rate by three orders of magnitude of the M159T mutant relative to the wild type, Stiel et al used an Hg lamp with a 10nm band pass filter at 488 nm and 300mW/cm2 power and observed 263s and 0.23s half-time constants from fluorescence traces on live recombinant bacteria for wild type Dronpa and Dronpa-M159T constructs. These whole-cell measurements therefore correspond to an 1143-fold increased switching rate in the M159T mutant. In addition, the absorption maximum of Dronpa-M159T is blue-shifted at 489nm relative to the 503nm value for the wild type, and the extinction coefficient was reduced from 95,000 M-1 cm-1 to 61,732 M-1 cm-1 2. The crosssections of Dronpa and Dronpa-M159T at 488nm are comparable, which would suggest an absolute quantum yield of 0.37 for the on-off photoswitching (1143 multiplied with 3.2*10-4) 2, based on the 3.2 x 10-4 value for the wild type 1,9. Using continuous illumination at 473nm (with the Dronpa-M159T mutant having ~ 10%


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higher cross section at this wavelength compared to Dronpa) and 15 mW/cm2 we observed 529s and 8.2s time constants for wild type and mutant, at room temperature. The observed macroscopic kinetics were seen to follow first order behavior with respect to the incident flux. Considering the much lower illumination power used here, the observation of only two-fold slower on-to-off conversion suggests that in purified form the wild type Dronpa switches considerably faster by orders of magnitude as compared to the in-vivo kinetics previously reported 2. In contrast the Dronpa-M159T mutant on-to-off switching kinetics observed here are in reasonable agreement with the faster kinetics seen in-vivo under more intense illumination 2. Since the thermal on-state recovery half times are 840 and 0.5 minutes for wild type and mutant, both our measurements of the on-off photoswitching rates and those of Stiel et al 2 should reflect the ratios of the absolute quantum yields. Thus, in contrast to Stiel et al, including also the 0.91 ratio of optical cross sections we find a 59-fold acceleration (0.91*529s/8.2s) of the on-off switching. This different result is cause mostly from recording a more efficient rate for the wild type, relative to Stiel et al. Therefore, based on the value of 3.2x10-4 for the on-off photoswitching quantum yield of the wild type 1,9, we estimate a value of 0.02 for the M159T mutant. For the off-on switching reaction Stiel et al reported half times of 100 ms and 50 ms for wild type and mutant, using UV light source at 405 nm and 10 nm band pass at 200mW cm-2. Under 400 nm and 26.7 mW/cm2 power illumination we recorded time constants of 450 ms and 415 ms, for wild type and mutant. Normalised to the reported power, we thus find a 33-fold and 16-fold relative increased efficiency under our conditions. Mostly, our measurements indicate only an 8% increase in the off-on switching rate for the mutant relative to the wild type. Based on the TR-IR measurements which supported an approximate 30% quantum yield for the off-on


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switching 10, amplitudes for the mutant may be expected to be similar to the wild type data.

3.2 Static FTIR on-minus-off difference spectroscopy of the M159T mutant

A first approach to evaluating possible differences in the switching mechanisms of the wild type and the mutant records and compares the static on minus off FTIR difference spectra. Figure 1 shows a comparison of the previously reported result for the wild type Dronpa 10 and that for the M159T mutant in 2H2O, pD 7.8. Generally, nearly all features for the on and off states are conserved, with only small differences in frequency of local maxima and minima. Based on the frequency positions at 1688 and 1655 cm-1 of the best characterized mode assignment, which is the chromophore C=O mode, in the M159T mutant spectrum (Figure 1) the off and on states are likewise assigned to the trans neutral and cis anion chromophore, as for the wild type previously 10,19-21. Following the proposed mode assignments for the wild type Dronpa FTIR difference spectra 10, the on state local maxima at 1622, 1577, 1545, 1497 and 1150 cm-1 have approximate mode characters ν(C=C), Phenol-1, ν(C=N/C=C), Phenol-3 and phenolate δ(CH). For the off state 1639, 1615, 1557, 1514 and 1176 cm-1 have approximate mode characters ν(C=C), Phenol-1, ν(C=N/C=C), Phenol-3 and phenol δ(CH). One clear difference between wild type and mutant is the 1280 – 1380 cm-1 fingerprint region where the amplitudes of phenolate modes of the on state are visibly modified in the mutant. Whereas the wild type shows local maxima at 1389, 1364, 1349 and 1323 cm-1 all with approximately equal amplitude, the mutant has a


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prominent maximum at 1345 cm-1, with smaller peaks at 1398 and 1322 cm-1 (Figure 1). Interestingly, the 1345/1349 (Dronpa-M159T/Dronpa) on-state bands correspond to a 1371 cm-1 mode seen in anionic 4’-hydroxybenzylidene-2,3dimethylimidazolinone (HBDI) in 1H2O 22, which showed a particular pattern of isotope shifts. While 5-13C labeling resulted in a 15 cm-1 downshift of this mode, 13C labeling at positions 1-13C, 4-15N, 3-13C, and α-13C did not result in significant frequency shifts22. Contrary to the mode character obtained from harmonic frequency calculations, He et al 22 concluded that the 5-13C sensitive mode is more delocalized. The assignment is thus likely to include skeletal deformations that includes the phenol ring and displacement of 5-C 22. The differences observed between the wild type and M159T samples may indicate a minor equilibrium conformation difference of the on state, potentially dominated by the non-planar configuration at C5 Bands at 1674/1655 cm-1 and 1609/1594 cm-1 (1H2O/2H2O) belonging to protein in the on state were suggested to arise from arginine νasym(CN3H5+) and νsym(CN3H5+) modes10. Considering the altered position of the Arg66 sidechain in the on and off states 6, a specific assignment to Arg66 was suggested. In the 2H2O spectrum of the M159T spectrum a 1592 cm-1 peak has become a shoulder on the intense phenolate-1 mode, but is otherwise conserved in the mutant spectrum. Lukacs et al. 16 assign both the 1688 and 1677 cm-1 off state bands to the chromophore C=O, whereas Warren et al assigned only the 1688 cm-1 off state band to the ν(C=O) mode 10. An apparent double-bleach feature at 1688 and 1677 cm-1 is more pronounced in the Dronpa-M159T sample recorded at 2 cm-1 as compared to the wild type Dronpa recorded at 4 cm-1 resolution (Figure 1). On the basis of the 1H/2H isotope shift patterns wild type Dronpa FTIR difference spectra, it was seen that the 1674 cm-1 (Arg νasym(CN3H5+)) and 1665 cm-1 ν(C=O)


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bands in 1H2O combine at 1655 cm-1 for the on state in 2H2O, judged from the frequency shift and intensity of the local maximum. Furthermore, subtracting the 2

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H2O and the 1H2O on-minus-off difference spectra showed a distinct band at 1695cmin the double difference spectrum. Therefore, two modes contribute to the 1700-

1655 cm-1 spectral region, of which one is sensitive to 1H/2H exchange and the combination of which results in two local minima at 1687 and 1677 cm-1 in 2H2O. The main conclusion from the comparison of the Dronpa and Dronpa-M159T FTIR on-minus-off difference spectra in 2H2O is that both protein and chromophore modes have very similar frequency positions and intensities and, with the exception of the fingerprint region containing phenolate modes, the on and off states are structurally highly similar.

3.3 TR-IR measurements of the on state of Dronpa-M159T

In order to evaluate possible differences in the vibrational response of the on state of the Dronpa-M159T with the wild type Dronpa, pump-probe TR-IR measurements were made, including an extended spectral window 1480-1750 cm-1, comparable to that reported by Lukacs et al 16. Furthermore, measurements were done for pumpprobe delays up to 1800 ps, observing the majority of population decay to ground state. In addition, a -100 ps negative time delay was collected extensively in order to resolve the 1 ms transient absorption, which was also subtracted from the positive delays. SVD analysis of the TR-IR data up to 1800 ps showed one dominant spectral contribution having a singular vector describing 88 % of the data. A further two statistically significant components account for the remaining 12% of the amplitudes (Figure 2).


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Within the time resolution of the instrument and fitting of the response, the shortest life time found was 1.9 ps. Assuming intramolecular vibrational energy redistribution (IVR) to be complete within the ~ 150fs instrument response time, the 1.9 ps component is seen to be dominated by the excited state decay process, having approximately 4.5% amplitude of the total decay. While the alternative assignment would assume a 1.9 ps ‘dwell’ time, assignment to dominating for excited state decay is also developed independently from the spectral data analysis. To illustrate, the SVD decomposition shows that the 1.9ps phase is dominated by the most significant left singular vector, which has the majority of the spectral differences belonging to the S1 minus S0 contribution (Figure 2). Specifically, the first left singular D(s)U vector represents 44% of the 1.9ps component within the data matrix ∆A=UD(s)VT. Since also D(s)UI has approximately 50 times higher peak amplitudes than D(s)UII, both the 1.9 ps and 185 ps phases are represented primarily by the spectrum seen in D(s)UI (Figure 2A, top,blue). Furthermore, a heterogeneous global fit of the on state measurements separates the spectrum belonging to the 1.9 ps component (Figure S6). The largest amplitude of the 1.9 ps spectrum, approximately 0.3 mOD, corresponds to a re-filling of the 1495 cm-1 band, signalling ground state recovery. It is therefore concluded that the 1.9 ps phase is dominated by the S1 minus S0 difference spectrum including also re-filling of the 1570 and 1650 cm-1 bands at the 1 x 10-4 OD level (in agreement with the SVD results), perhaps with the exception of the induced absorption band at 1581 cm-1, which is seen at 1589 cm-1 in the 185 ps spectrum (Figure S6). The latter may be interpreted to correspond to vibrational cooling of ‘hot’ excited state 23,24. However, it should be noted that this observation addresses only 104

OD level signals, whereas the 0.3 mOD re-filling of the 1495 cm-1 band should be

taken as the most significant spectral feature at this level of Signal to Noise Ratio. In


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conclusion, the 1.9 ps phase is seen to be dominated by excited state decay, representing ~ 4.5% recovery of the total ground state bleach amplitude, and shows smaller, additional, spectral differences of chromophore modes compared to the 185 ps phase. A global fit of the three D(s)V(t) time traces with a sum of exponentials determines the fundamental time constants that describe the full dataset with better accuracy than a global fit of all data with free fitting of both amplitudes and time constants 25. Subsequent global fitting using these three time constants was done with a homogeneous model 18. The sequential scheme was chosen in order to describe the spectral evolution and did no assume a physical connectivity scheme. The global fit minimized the sum of square root of differences to the same level when the time constants determined from SVD were fixed or when the time constant values were left free in the optimization, in which case no significant modification of the fitted time constants was seen. Figure 3 presents the species associated spectra and the corresponding time traces. The spectra belonging to τ1=1.9 ps and τ2=185 ps are very similar and are assigned to radiative decay (Figure S6). The spectra of the wild type Dronpa are characterized by local minima at 1494, 1535, 1574, 1628-1637 and 1666 cm-1, belonging to phenol-3, ν(C=N/C=C), Phenol-1, ν(C=C), and ν(C=O) and Arg66. For the wild type Dronpa, the ν(C=C) mode is spectrally broad compared to the FTIR on-minus-off difference spectrum, which consists of a dominant bleach at 1623 cm-1 with a minor shoulder at ~ 1632 cm-1. A difference feature 1593(-)/1586(+) cm-1 assigned to Arg66 νsym(CN3H5+) in the wild type Dronpa with 16ps and 2 ns time constants 10, is also seen in both 1.9 ps and 185 ps spectra of the Dronpa-M159T measurements. A complex signal in the 1640-1670 cm-1 likely contains contributions from the chromophore v(C=O) as well as Arg66 νasym(CN3H5+), also seen for the wild


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type Dronpa 10. A small but reproducibly observed difference signal in the wild type at 1684(-)/1679(+) cm-1 is seen at 1694(-)/1688(+) cm-1 in Dronpa-M159T (Figure 3, S3), likely belonging to protein carbonyl stretching mode. A comparison of Dronpa and Dronpa-M159T species associated spectra for the on state of the same spectral regions is shown in Figure S3 (supplementary information). As noted previously, the frequency positions and intensities for the cis anion in the on state correspond well with those observed for the Aquorea Victoria Green Fluorescent Protein (GFP) 10,19-21. While both 1.9 ps and 185 ps spectra are in agreement with Lucaks et al. 16, spectral evolution is observed with longer delays. Specifically, the spectrum belonging to the third species which has a 1.1 ns decay time constant is associated with small frequency shifts (Figure 3). The bleach at 1574 cm-1 belonging to anion Phenol-1 at early time shows a minor shift to 1576 cm-1 in the 1.1 ns decay spectrum. Interestingly the feature at 1648-1652 cm-1, having contribution from Arg66 νsym(CN3H5+) is also slightly shifted to 1652 cm-1 (Figure 3). The spectral evolution at long delays is further supported by the growing contributions of the minor SVD components II and III (Figure 2). It should be noted that the shape of the spectrum that decays with 1.1 ns time constant is determined by fitting to a reaction model which assumes full recovery to the ground state, thus disregarding the transient absorption belonging to the primary photoproduct, which can not be reliably retrieved with observations to 1800 ps because these are still dominated by radiative state decay. By including a final product state with ‘infinite’ lifetime and using a homogeneous model, a spectrum with very small peak amplitudes (below 10-4 ∆OD) was retrieved (Figure S5) which resembles the 1 ms spectrum more than the excited state decay spectra (Figures 3, S5). Accurate pump-probe measurements with longer delays would be needed to better resolve the


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product state spectrum. In summary, the most likely interpretation of the spectral evolution assumes geometrical modification of both the chromophore and Arg66 within the excited state lifetime and associated with the observed 1.1 ns decay time constant. Measurements with a -100 ps negative pump-probe time delay resolved small but reproducible transient absorption (Figure 4). While the sample was rapidly moved during data acquisition (average speed of 50 µm ms-1, probe beam size 75 µm, pump beam size 300 µm), some overlap with the previous measurements was generally seen. The measurement therefore also included some contribution of 2, 3 and 4 ms, but estimation of the overlap taking into account the Gaussian profiles indicates the measurement to be dominated by the 1 ms pump-probe spectrum. Comparison with the static off-minus-on FTIR difference spectra shows reasonable agreement, with negative signals belonging to the on state present at the same frequencies. The amplitude of the bleach feature at 1622 cm-1 represents ~ 5% of the instantaneous signal after excitation, in reasonable agreement with the estimated quantum yield of 0.02, considering additionally that the 1 ms measurement only resolved a portion of the population. It was unclear whether product absorption at 1687 cm-1 was already developed in this spectrum due to insufficient signal-to-noise (Figure 4), but the observation of features at 1622 and 1652 cm-1 indicates the likely ground state bleaching of characteristic phenolate modes 10, thus suggesting thermal protonation within the 1ms time delay. These observations are thus in agreement with the previously proposed time scale of thermal proton transfer 10.

3.4 TR-IR measurements of the off state of Dronpa-M159T


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The off state was photo-accumulated under continuous illumination with a 473nm laser source (see Materials and Methods). Compared to the wild type Dronpa, the Dronpa-M159T sample converted more readily and the resulting photoequilibrium from the defocused 473nm illumination and the scanned pump was ensured to occupy the off state fully. Subsequent excitation with 400nm femtosecond pulses allow measurement of the TR-IR spectra in the same frequency range as collected for the on state, in 2H2O. Figure S2 shows selected spectra for delays up to 1800 ps. The broader frequency range and the extended delays were chosen to evaluate the possibility of relaxation processes slower than the maximal 100 ps pump-probe delay which were previously reported for the wild type Dronpa 10. Furthermore, Lukacs et al (2013) 16 reported that their measurements of the same sample under the same conditions required global fitting with three time constants 2.3, 22 and 458ps, for data collected up to 1000 ps. These measurements were done under similar conditions of optical excitation except at 10 KHz repetition rate, and 2H2O solvent, although the pH and experimental temperature was not explicitly mentioned 16. Comparison of the raw data shows the off state TR-IR to be generally in agreement with Lukacs et al 16 except a relatively more intense bleach amplitude at 1688 cm-1 seen in Figure 2 of Lukacs et al 16

compared to our data (Figure 6, S2). An SDV analysis was performed for our

measurements in order to evaluate the number of time constants needed and their statistical significance. Figure 5 shows the resulting four significant components. A global fit of the scaled time traces D(s)V(t) required three time constants τ1=0.6 ps, τ2=14 ps and τ3=17ns, the latter time constant being poorly determined with measurements up to 1800 ps and modeling a subsequent decay to ground state. It was noted that no ~ 458 ps could be resolved from the D(s)V(t) time traces. Specifically, forcing a fixed value at 500 ps increased the sum of residuals from 5.8071 x 10-6 to


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8.3499 x 10-6 and visibly resulted in unsatisfactory fit results. Global fitting of the data with a homogeneous model was done to describe the spectral evolution without assuming a physical model. The spectra for the 0.6 ps and 14 ps components were highly similar (Figure 6). The associated time constants could be assigned to excited state decay in agreement with Warren et al 10, Habuchi et al. 9, Lucaks et al. 16 and Fron et al. 11. The primary photoproduct is characterised by a distinct spectrum with upshifted induced absorption relative to ground state bands, with local maxima and minima 1654(+)/1639(-) cm-1 and 1625(+)/1617(-) cm-1 and 1595(+) cm-1 (Figure 6). Only a very small, but reproducibly observed, minimum at 1685(-) cm-1 was observed, in agreement with Warren et al 10 and Lucaks et al. 16, for Dronpa and Dronpa-M159T, respectively. Figure 6 presents the basis spectra applying a homogeneous global fit of the on state TR-IR data, which identifies the primary photoproduct with a 14 ps rise time. Pump-dump-probe measurements evaluating amplitudes belonging to S1 and photoproduct with sub-ps and few-ps pump-dump delays would be required to support further ‘target’ analysis evaluating possible reaction models, which would provide the branching ratios of the excited state decay phases. The pump-probe data presented here are therefore exclusively analysed by applying a model-free homogenous global fit (Figure 6). A minus 100 ps negative (probe-pump) time delay measurements resolved the 1 ms spectrum of the off state, which had amplitudes comparable to the primary photoproduct. It is noted that the positive pump-probe data shown used subtraction of the negative time delay, which significantly altered the spectra. The 17 ns time constant is an estimate of some decay amplitude observed for delays up to 1800 ps (Figure 5). It is possible that spatial overlap was partially lost with long delays, causing a small reduction of the transient absorption signals, in which case the primary photoproduct formed with 0.6 and 14 ps


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would be seen to be stable over this time. However, a similar effect was not seen for the on state measurements (Figure 2). In case this represented genuine decay, this was modelled by a return to ground state as no data for longer delays was obtained. Clearly, considering the 1 ms transient absorption (Figure 7), this assumption cannot be correct, but the resulting amplitude errors are considered to be very small from the small decay amplitude (Figure 5).

3.5 Geometry optimization and frequency calculation for distorted neutral trans HBDI using DFT

In order to address the different assignments made to the primary photoproduct spectrum by Warren et al 10 and Lucaks et al. 16 DFT geometry optimisations and frequency calculations were performed for chromophore geometries as observed in the X-ray structure of the off state by Andresen et al 6. Geometry optimization and frequency calculation was performed using redundant internal coordinates 26 using Gaussian 09 27, with removal of internal coordinates including either the dihedral angle 5-C/α-C/1’-C/2’-C, corresponding to rotation of the phenol ring out of plane, or the linear bend 3-C/5-C/O/4’-C, corresponding to an out-of-plane bending of the phenol and imidazolinone rings (Figure 1, 8, 9 ). The resulting effects on the force constant that determines the frequency position of the chromophore C=O stretching mode is evaluated from subsequent harmonic frequency calculations which remove the same redundant coordinates as done for the geometry optimization. Since the C=O stretching mode must contain also C=C stretching character due to ring deformation in the mode displacement, both the C=O


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and C=C equilibrium bond lengths were evaluated, in addition to the frequency positions. For out-of-plane bending (while keeping the phenol ring torsion fixed to 140° as observed in 2POX pdb 6), decreasing of the bending angle (Φ) from 32° as observed in 2POX pdb increased the C=O equilibrium bond length, while decreasing the C=C bond length (Figure 8A). The subsequent C=O/C=C frequencies were however seen to increase, indicating a dominant contribution of the increased force constant in the C=C displacement when moving to a more planar geometry. For rotation of the phenol ring by decreasing dihedral torsion angle (Ψ) to a more planar geometry, while keeping the out-of-plane bending angle fixed to 32°, increased both the equilibrium bond lengths for the C=O and C=C bonds, and concurrently reduced the frequency considerably (Figure 8B). It was thus concluded that the main effect of the distorted geometry seen in the X-ray structure of the off state is a frequency upshift of the C=O/C=C mode as compared to that calculated for a planar geometry for neutral HBDI in vacuum. This was already noted previously, on the basis of a single geometry restrained calculation by Warren et al 10. Inspection of the Fo-Fc difference electron density maps for the off state structure indicates negative electron density on the phenol ring, which is modeled in all four chains in a distorted geometry as shown in Figure 8. This density is indicative of partial occupancy of the off state, having some remaining on state present, but furthermore indicates that the dihedral torsional angle (Ψ) is not very precisely determined from the X-ray data, which could also support a 5° or 10° larger value. For example, assuming a -130º value (10º larger than the X-ray coordinates), the frequency difference between the distorted neutral trans chromophore and the neutral cis chromophore would be very small as a result (Figure 9). Additionally, frequency positions of ν(C=C) and phenol-1 and those further reported previously for the distorted neutral trans chromophore Warren et al


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from calculations are positioned close to those calculated for the cis neutral structure (Figure 9 and reference 10). The calculated absorption in Figure 9 is given as the band integrated intensity (km / Mole = 1000 m / Mole).

4. Discussion

4.1 Assignments of the carbonyl stretching region 1750- 1650 cm-1.

The on-minus-off FTIR difference spectra for the wild type Dronpa were reported in both 1H2O and 2H2O, which clearly supported the contributions of at least two modes in the off state and two modes in the on state in the carbonyl stretching region 17501670 cm-1 10 (Figure 10). A consistent interpretation of the 1H/2H isotope shifts in this region assigns two off-state bands at 1695 and 1690 cm-1 in 1H2O, of which the 1695 cm-1 shifts to 1674 cm-1 in 2H2O, and the local minimum at 1690 cm-1 in 1H2O is observed at 1688 cm-1 in 2H2O (Figure 10)10. This shows that the 1695/1677 cm-1 (1H2O/2H2O) bleaches in the off state do not belong to the chromophore C=O mode, and below we argue the alternative assignment to Arg66 νasym(CN3H5+). The high resolution FTIR difference spectrum of the Dronpa-M159T mutant more clearly separates the two minima in the off state (Figure 1). This assignment contrasts with the assignment of both 1688 and 1677 cm-1 bands to the chromophore C=O mode made by Lucaks et al. 16. The possible assignment of the high frequency, 1695 cm-1, to Arg νasym(CN3H5+) may correspond to an isolated side chain with no ionic interactions 28,29. This assignment is also supported by the X-ray structures 2IOV for the on state 2 and 2POX for the off state 6. In the on state the Arg66 side chain is hydrogen bonded to the C=O group of the


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chromophore, which may stabilise the anionic cis ground state from interaction with the charged (CN3H5+) group. For the on state, two isotope-sensitive induced absorption bands in to the on minus off FTIR difference measurements, which were identified as not originating from chromophore modes, were seen at 1674 and 1609 cm-1 and 1655 and 1594 cm-1 in 1H2O and 2H2O, respectively . An assignment of the 1674/1655 and 1609/1594 cm-1 (1H2O/ 2H2O) bands to arginine νasym(CN3H5+) and νsym(CN3H5+) are well supported considering the hydrogen bonded structural position in the on state, and additionally from the well resolved 15 cm-1 downshift for the arginine νsym(CN3H5+) mode 10. For the off state, corresponding bleach amplitude in the on minus off FTIR difference spectrum at 1695 cm-1 is additionally supported by observation of arginine 108 νasym(CN3H5+) at the high frequency position of 1695 cm-1 in halorhodopsin 29. These assignments contrast with those by Lukacs et al., who propose to assign both the 1688 and 1677 cm-1 off state bands in 2H2O to the chromophore C=O, discussed further in section 4.3 below. Lucaks et al. 16 however did not take the 1H/2H isotope shifts into account, specifically the additional bleach amplitude at seen at 1695/1677 cm-1 (1H2O /2H2O) (Figure 10) 10. In contrast, the chromophore C=O stretching frequency should have very small sensitivity to 1H/2H exchange, and is found at 1690/1688 cm-1 (1H2O /2H2O) for the off state.

4.2 On state TR-IR measurements

Two fundamentally new observations were made for TR-IR measurements of the on state of Dronpa-M159T. Firstly, spectral evolution is observed for a 1.1 ns decay time constant, which includes small shifts of both protein and chromophore modes.


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Second, a 1 ms spectrum was resolved which has bleach features corresponding to the static FTIR difference spectrum. For the 1.1 ns decay associated spectrum of the globally fitted TR-IR data of the on state, ground state bleach at 1648-1652 cm-1, having contribution from Arg66 νasym(CN3H5+) is slightly shifted to 1652 cm-1 (Figure 3). The bleach at 1574 cm-1 belonging to the chromophore ν(C=C) shows a small shift to 1576 cm-1 in the 1.1 ns spectrum. The globally fitted spectrum for the 1.1 ns decay component is expected to have contribution from the induced absorption of the primary photoproduct, with an estimated quantum yield of 0.02. However, on the basis of evaluating the amplitudes seen for the 1 ms measurement, and also the low quantum yield, these are expected to have minor contribution to the shifted features of the 1.1 ns decay spectrum shown in Figure 3 (see Figure S5C for comparison). The three statistically significant orthogonal left singular vectors resulting from SVD factorisation (Figure 2A) are evidence that the data matrix ∆A=UD(s)VT must consist of linear combinations of basis spectra which have different spectra. Further, considering the scaled D(s)VII and D(s)VIII time traces (Figure 2B), it is seen that their contributions maximize and subsequently decay at delays longer than 100 ps. Therefore, the SVD results (Figure 2) and global fitting (Figure 3) are in agreement to support with statistical significance the spectral changes which have a 185 ps rise time and a 1.1 ns decay time constant. This analysis shows that modes belonging to the protein as well as the chromophore are modified in the spectrum that decays with 1.1 ns time constant: The 1648(-)/1659(+) cm-1 band assigned to νasym(CN3H5+) of Arg66 10

changes to 1652(-)/1659(+) cm-1 and the 1574(-) cm-1 local minimum belonging to

Phenol-1 10 changes to 1576(-) cm-1 in the spectrum associated with the 1.1 ns decay time constant. It may be proposed that these modifications are required for the photoswitching which includes photoisomerisation and protonation in addition to


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rearrangements of aminoacid side chains Arg66 and His192 6. This process may be distinct to the M159T mutant and necessary for accelerated on-to-off photoswitching, although previous TR-IR measurements of wild type Dronpa 10 and Dronpa-M159T 16 were both reported for delays up to 1000ps. Compared to the 16 ps and 2 ns excited state decay time constants for the wild type Dronpa 10, the shorter 1.9 ps, 185 ps and 1.1 ns time constants reported for the M159T mutant here agree with the reduced 0.23 value for the fluorescence quantum yield of the mutant relative to the 0.85 value for the wild type 2.

4.3 Off state TR-IR measurements

SVD analysis of pump-probe measurements in the 1750-1575 cm-1 spectral window of the off state with delays up to 1800 ps supported 0.6ps, 14ps and 17ns time constants, and could not support a ~ 458 ps time constant which was reported on the basis of global analysis by Lukacs et al. 16 for the same Dronpa-M159T mutant. While the determination of the number of time constants and their values is better supported on the basis of SVD than for global analysis 18, there appears to be a genuine difference between the off state measurements. The main difference between the experimental conditions for the measurements reported here and by Lukacs et al. 16 concerns the repetition rate which were 1 KHz and 10 KHz , respectively. The possibility of optical pumping with 400 nm excitation of an intermediate may possibly explain the differences seen, and the observation of an additional time constant for the 10 KHz measurements. For those reported here, the -100 ps negative delays which represent the 1 ms spectrum, indicate from the very close correspondence with the FTIR difference spectrum that deprotonation was complete, and therefore the


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electronic absorption is expected to have shifted to 480 nm and consequently significantly reduced the cross section at 400nm. While Lucaks et al (2013) 16 did not report a 100 µs spectrum, or describe subtraction of negative time point measurements, the possibility exists that deprotonation was not yet complete. In this case, remaining cross section at 400nm might explain additional transient absorption signals seen on the ~500ps time scale by Lucaks et al. 16. Additionally, moving the sample by raster scanning at 10 KHz repetition rate would have increased subsequent spatial overlap as well. The assignment of both 1688 and 1677 cm-1 bands to the chromophore C=O mode made by Lucaks et al. 16 contrasts with the analysis of the 1H/2H shifts of the FTIR data discussed here. Lucaks et al argue that the X-ray structure of the off state supports the presence of disorder explaining the two frequencies they assign to the off state, citing Mizuno et al., 2008 7. Mizuno et al however reported P21, P21212, P212121 crystal forms of the on state of Dronpa and a P43 bright state structure of ‘22G’, the wild type precursor of Dronpa. The off state structure which was reported by Andresen et al 6 did indicate disorder but rather at the level of a mixed on and off state. It is therefore unclear which X-ray crystallographic observation of the off state Lucaks et al. 16 put forward to support their proposed assignment of the 1677 cm-1 off state band. Another assignment made by Lucaks et al. 16 to protein contributions concerns the bleach feature at ~ 1620 cm-1 in the early time spectra of the off state, on the basis of its absence in TR-IR measurements of HBDI. In contrast, Warren et al suggested an assignment to phenol-1, which appeared reasonable on the basis of frequency calculations and also comparison to FTIR measurements of the Aquorea Victoria Green Fluorescent Protein 10. One additional consideration includes the structural


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disorder of HBDI in solution, which modeled the optical absorption spectrum that has increased width 30. For the cis anion in the on state, geometry optimization using DFT which results in a planar structure may thus be more indicative for the frequency positions of the protein spectra relative to the spectra for free HBDI in solution. A critical point for the mechanistic interpretation concerns the assignment of the primary photoproduct spectrum which is formed in 14 ps (Figure 7). While Warren et al 10 assigned the product to the cis neutral chromophore, Lucaks et al. 16 assigned it to a trans chromophore ground state intermediate. In this contribution we have confirmed that there are no significant differences seen in the TR-IR of the off state of wild type and M159T mutant (Figure S4). Lucaks et al. 16 propose that ground state isomerisation is responsible for the formation of the final cis chromophore structure in the on state. Combined with the observation made here that both isomerisation and deprotonation is complete within 1 ms, this would require a small barrier to be created by the initial optical pump. The main argument put forward by Lucaks et al. 16 is the absence of a frequency upshifted C=O mode in this spectrum. Alternatively, an assignment of the primary photoproduct spectrum to the cis neutral ground state 10 may consider two possible reasons why this mode is not seen upshifted relative to the trans neutral chromophore in the off state. Firstly, hydrogen bonding to the C=O group may downshift the mode in the primary photoproduct, thereby compensating for a part of the expected frequency upshift. There may be evidence that this has indeed occurred. Induced absorption at 1655 cm-1, assigned to the Arg νasym(CN3H5+) in the on state is seen also in the primary photoproduct spectrum (Figure 6,7). As the on state C=O chromophore frequency is observed at 1665 cm-1 in 1

H2O in the on-minus-off FTIR difference spectrum of Dronpa 10, and is insensitive to

1

H/2H exchange, the assignment of 1655 cm-1 to Arg66 is warranted. This in turn


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indicates the hydrogen bonding to the chromophore C=O group in both the primary photoproduct and the on state. Second, DFT calculations presented here (Figure 8 and 9) and previously 10 indicate that the ν(C=O/C=C) frequency is particularly sensitive to out-of-plane distortions, which can be rationalized on the basis of equilibrium ν(C=O) and ν(C=C) force constants. While a highly specific geometry for the cis neutral chromophore cannot be proposed for the primary photoproduct of the off state, these arguments can explain why a frequency upshifted C=O mode is not included. A comparison of the primary photoproduct spectrum (Figure 7, top; 17ns) shows similarities as well as distinct differences relative to the static FTIR difference spectrum (Figure 7, bottom). The photoproduct spectrum has characteristic bleach features at 1640 and 1617 cm-1 that correspond to the local minima in the FTIR spectrum, but their amplitudes relative to induced absorption bands observed at 1655 and 1623 cm-1 are larger in the photoproduct spectrum (Figure 7A). Furthermore, induced absorption bands at 1595 and 1528 cm-1, belonging to the chromophore, are specific to the photoproduct (Figure 7A) and do not correspond to the main induced absorption bands in the FTIR or 1 ms spectra (Figure 7B, C). The significantly shifted center frequencies and cross sections support the structural rearrangement of the chromophore, rather than its protein environment as proposed by Lucaks et al.16, thus supporting photoisomerisation in the primary photoproduct instead. It is noted that the 1595 cm-1 positive band may belong to the phenol-1 mode of neutral cis HBDI, which could correspond to the calculation shown in Figure 9. A key observation is the absence of characteristic anion phenolate modes at 1577 and 1497 cm-1 in the product spectrum indicate that thermal deprotonation has not yet occurred on the nanosecond time scale. This observation also argues against rearrangement of hydrogen-bonding potential that would result in a charge transfer process developing


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partial anionic character, as proposed by Lucaks et al. 16. Rather, the characteristic phenolate bands are fully developed with 1 ms, as seen from the correspondence with the on-minus-off FTIR difference spectrum. Finally, there are thermodynamic considerations for the proposed ground state trans-cis isomerisation (which are then followed by another ground state cis-trans isomerisation later to recover the ground state) by Lucaks et al. 16. Here, we have shown that in the time interval between 1800 ps and 1 ms, the primary photoproduct completely transforms into the final on state product (Figure 7). DFT calculations that scan the dihedral C=C bond angle from trans configuration at the B3LYP/6311+g(d,p) level, estimated a value of 199 kJ/mol, or 2.06 eV, for the electronic ground state barrier. Given the rate of the reaction reported here, it is unclear how the thermal barrier crossing proposed 16 could be supported, considering additionally that the thermal ground state recovery takes place on the minute time scale (840 min and 0.5 min for Dronpa and Dronpa-M159T, respectively) 2. With regard to the isomerisation state of the primary photoproduct, the following summarises the available evidence. Firstly, the frequency positions of the ν(C=O/C=C) mode in the primary photoproduct with cis neutral chromophore may be close to that of the distorted trans neutral chromophore in the off state, as shown by DFT frequency calculations (Figure 9). Second, the complete absence of a 500 ps component for measurements of the off state conducted in the absence of cross-section from intermediates at 1 KHz repetition rate is in contrast to the spectral dynamics reported of the same sample with 10 KHz repetition rate16, thus allowing assignment on the basis of the primary photoproduct only (Figure 6A). Third, the photoproduct spectrum having dominant chromophore bleach modes and shifted product bands (Figure 6A) is characteristic of structural modification of the chromophore rather than being


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dominated by protein absorption changes, thus strongly supporting a chromophore isomerisation. Fourth, the very large, 2eV, ground state barrier between a cis neutral and trans neutral structure is unlikely to be overcome on microsecond timescales, driven by protein conformational changes as speculated by Lukacs et al.

16

. It is

therefore concluded from the available evidence that the primary photoproduct of the off state is assigned to the cis neutral chromophore, in agreement with Warren et al 10. The new millisecond measurements for both the on and the off state presented here for the first time have confirmed the occurrence and the relevant time scale for the thermal protonation and deprotonation reactions in the photocycle of Dronpa, as previously proposed 10.

5. General conclusions A brief summary of the forward and reverse photoinduced reactions of DronpaM159T are given from the evidence presented. Pumping the on state results in dominant excited state decay of the anionic cis chromophore with 1.9 ps, 185ps and 1089 ps time constants and includes transient absorption assigned to Arg66, as also seen for the wild type Dronpa10. Further modification of modes belonging to protein, assigned to Arg66, and chromophore are observed on the nanosecond time scale (belonging to the third species having a 185 ps rise time and 1089 ps decay time constant), which may be of importance considering the 65-fold acceleration of the onto-off photoswitching of the M159T mutant relative to the wild type. A 1 ms observation shows bleach features corresponding to those in the off-minus-on FTIR difference spectrum, indicating isomerisation and likely also thermal protonation of the hydroxyphenyl oxygen, from the comparable bleach frequencies and amplitudes


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belonging to characteristic phenolate modes at 1652 and 1622 cm-1. The final product as observed in static FTIR difference spectroscopy is the trans neutral chromophore in the off state, corresponding to that in the Dronpa wild type previously reported 10. Blue excitation of the off state results in excited state decay with 0.6 ps and 14 ps time constants, generating a primary photoproduct with very similar amplitudes, frequency positions and relative yield as those observed for the wild type Dronpa (Figure 6A and Supplementary information Figure S4). At 1 KHz repetition rate, having a transient background of cis anion only (Figure 7, 1 ms spectrum (red)), statistical analysis of time resolved measurements could not support a ~458 ps time constant previously reported by Lucaks et al. 16 done at 10 kHz repetition rate. Instead we find the photoproduct to be stable on nanosecond time scale, yet a 1 ms measurement corresponds very closely with the on-minus-off FTIR difference spectrum, indicating a completion of the thermal deprotonation and protein rearrangements between nanoseconds and 1 ms. Careful analysis of the isotope dependence of the FTIR difference spectra indicates that bleach amplitude at 1677 cm-1 in 2H2O belonging to the off state is present at 1695 cm-1 in 1H2O, and is tentatively assigned to arginine 66 νasym(CN3H5+). The resulting conclusion is that only one band, at 1690/1688 cm-1 (1H2O/2H2O) is assigned to ν(C=O) of the chromophore in contrast to Lucaks et al. who assigned both the 1688 and 1677 cm-1 to the ν(C=O) mode 16. Finally, the assignment of the primary photoproduct infrared spectrum is supported from DFT modeling which assess the frequency shifts resulting from distorted geometry seen in the off state crystal structure. In addition, a contribution to the frequency position of the chromophore C=O mode is expected to result from hydrogen bonding between Arg66 and the chromophore C=O, which was independently argued on the basis of mode assignments to arginine 66 νasym(CN3H5+) and arginine 66 νsym(CN3H5+)..


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FIGURES

Figure 1. A) On-minus-off FTIR difference spectra of Dronpa (black) 10 and DronpaM159T (red) in 2H2O, pD 7.8 and room temperature. Local maxima and minima are indicated for both wild type and mutant measurements, corresponding to their on and off states. The wild type measurement was previously reported at 4 cm-1 resolution, whereas the Dronpa-M159T was recorded at 2 cm-1 resolution. B) Structures of the cis anionic chromophore, hydrogen bonded to arginine 66 in the on state (top) and in the trans neutral structure with disrupted hydrogen bonding to arginine 66 in the off state (bottom).


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Figure 2. Singular value decomposition (SVD) analysis of the on state of DronpaM159T. SVD was carried out according to ∆A=UD(s)VT. A) Scaled left singular vectors D(s)UI-III represent the orthogonal basis spectra weighted by their singular


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values. B) Scaled time traces D(s)VI-III(t), representing the concentration profiles of left singular vectors, were fitted with a sum of three exponentials resulting in τ1=1.9 ps, τ2=185 ps and τ3=1089ps.


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Figure 3. Global analysis of the on state of Dronpa-M159T using a homogeneous model with three time constants. A) Species associated difference spectra belonging to τ1=1.9 ps, τ2=185 ps and τ3=1089 ps. B) Concentration profiles for basis spectra 1,2,3 and ground state, up to 1800 ps after excitation.


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Figure 4. TR-IR difference spectrum of the on state of Dronpa-M159T at 1 ms. For comparison the static off-minus-on FTIR difference spectrum is included below.


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Figure 5. Singular value decomposition (SVD) analysis of the off state of DronpaM159T. SVD was carried out according to ∆A=UD(s)VT. (A) Scaled D(s)UI-IV basis


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spectra. B) Scaled time traces D(s)VI-IV(t) were fitted with a sum of three exponentials resulting in τ1=0.6 ps, τ2=14 ps and τ3=17ns. Singular values D(s) are given for each component.


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Figure 6. Global analysis of the off state of Dronpa-M159T using a homogeneous model with three time constants. A) Species associated difference spectra belonging to τ1=0.6 ps, τ2=14 ps and τ3=17 ns. B) Concentration profiles for basis spectra 1,2,3 and ground state, up to 1800 ps after excitation

Figure 7. Stack plot of the 1 ms spectrum, the 17ns TR-IR primary photoproduct and the static on-minus-off FTIR difference spectrum of the off state.


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Figure 8. Geometry optimization and frequency calculation of neutral HBDI with outof-plane bending angle (Φ) (A) and dihedral angle for phenol ring torsion (Ψ) (B). Geometry optimized bond angles for the chromophore C=O (squares) and C=C (triangle) modes are plotted on the left Y axis, and harmonic frequencies (circles) for the chromophore C=O/C=C mode from subsequent harmonic frequency calculations are plotted on the right Y-axis. Frequencies are unscaled 31.


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Figure 9. Synthetic difference spectrum for a distorted neutral trans chromophore Φ=32º ; Ψ =-130º) (negative, yellow) and a neutral cis (positive, green) HBDI product state. Frequencies were scaled by 0.968 31.


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Figure 10. Carbonyl stretching region of the on minus off FTIR difference spectra in 1

H2O (black) and 2H2O (red) of Dronpa 10. The double difference 2H2O minus 1H2O

(blue) spectrum shows bleach amplitude belonging to the off state at 1695 cm-1 in 1

H2O downshifts to 1671 cm-1 in 2H2O.


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Author information

Corresponding author Jasper J van Thor: email [email protected]

Author contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript

Funding sources This work was supported by EPSRC via award EP/I003304/1.

Acknowledgements We thank Jalal Thompson for preparing the M159T mutation.

ABBREVIATIONS ESPT, Excited State Proton Transfer; MCT, mercury cadmium telluride; FTIR Fourier Transform Infrared spectroscopy; GFP, Green Fluorescent Protein; DFT, Density Functional Theory


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Table of Content Graphic


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Photoisomerization and Proton Transfer in the Forward and Reverse

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