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

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Article

Photoisomerisation and Proton Transfer in the Forwardand Reverse Photoswitching of the Fast-SwitchingM159T Mutant of the Dronpa Fluorescent Protein

Marius Kaucikas, Martijn Tros, and Jasper J. van ThorJ. Phys. Chem. B, Just Accepted Manuscript • Publication Date (Web): 04 Nov 2014

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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†, 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

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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 visible-

pump, 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-minus-

on 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-hydroxybenzylidene-

imidazolinone 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

6 were

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-Tyr-

Gly 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 pRESTb-

Dronpa, 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 cm

2 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 cross-

sections 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-2,3-

dimethylimidazolinone (HBDI) in 1H2O

22, which showed a particular pattern of

isotope shifts. While 5-13

C labeling resulted in a 15 cm-1

downshift of this mode, 13

C

labeling at positions 1-13

C, 4-15

N, 3-13

C, and α-13

C did not result in significant

frequency shifts22

. Contrary to the mode character obtained from harmonic frequency

calculations, He et al 22

concluded that the 5-13

C 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

2H2O and the

1H2O on-minus-off difference spectra showed a distinct band at 1695cm

-

1 in 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 pump-

probe 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 10-

4 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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19

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 1750-

1670 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

1H2O in the on-minus-off FTIR difference spectrum of Dronpa

10, and is insensitive to

1H/

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/6-

311+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 Dronpa-

M159T 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 on-

to-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 Dronpa-

M159T (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 Dronpa-

M159T. 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 Dronpa-

M159T. SVD was carried out according to ∆A=UD(s)VT. (A) Scaled D(s)UI-IV basis

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38

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 out-

of-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

1H2O (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

1H2O 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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Documents

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