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Supplementary Information for
Probing the early stages of photoreception in photoactive yellow protein
with ultrafast time-domain Raman spectroscopy
Hikaru Kuramochi1,2, Satoshi Takeuchi1,2*, Kento Yonezawa3, Hironari Kamikubo3, Mikio Kataoka3,
Tahei Tahara1,2*
1Molecular Spectroscopy Laboratory, RIKEN, 2-1 Hirosawa, Wako 351-0198, Japan
2Ultrafast Spectroscopy Research Team, RIKEN Center for Advanced Photonics (RAP), 2-1 Hirosawa,
Wako 351-0198, Japan
3Graduate School of Materials Science, Nara Institute of Science and Technology, 8916-5 Takayama,
Ikoma, Nara 630-0192, Japan
*Correspondence to: stake@riken.jp, tahei@riken.jp
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2717
NATURE CHEMISTRY | www.nature.com/naturechemistry 1
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Table of Contents
1. Computational details
2. Stimulated Raman spectrum of the pG state
3. One-color pump-probe spectroscopy with 30-fs time resolution (465-nm pump and probe):
Monitoring low-frequency vibrations of the pG state
4. Comparison of the Raman spectra of the pG, I0, and pR states
5. Comparison of the I0 spectra obtained by TR-ISRS and FSRS
6. Two-color pump-probe spectroscopy with 20-fs time resolution (460-nm pump and 500-680 nm
probe): Monitoring the initial dynamics and vibration of the pG* state
7. Effect of the actinic pump pulse duration (290 fs) on the temporal profile of the 135-cm-1 band
8. Analysis of the phase of the 135 cm-1 mode
9. Comparison of the I0 spectrum with the calculated Raman spectra of the pR0 and IT states
10. Dihedral angle dependence of the Raman intensity of the HOOP mode
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1. Computational details
Geometry optimization and normal mode calculation of the model pG, pR, pR0, and IT states were
performed with Gaussian 091. We employed the crystal structures of these states (PDB ID: 2ZOI, 4BBU,
4B9O, and 4I38) to prepare the initial guess for the geometry optimization. To reduce the computational
time, we restricted the computational area to the only chromophore pocket that consists of pCA and the
three key amino acid residues (Tyr42, Glu46 and Cys69). In the actual calculation, we modified the
structure of the three amino acid residues as follows: Tyr42 and Glu46 were modelled by p-cresol and
acetic acid, respectively. The terminus hydroxyl group and α-amino group of Cys69 were replaced by a
methyl group and hydrogen atom, respectively. The resultant model consists of 56 atoms in total
(Supplementary Fig. 1). All the geometry optimizations were carried out at the B3LYP level of theory
by using the 6-311+G** basis set. During the optimization process, the coordinates of the atoms
marked with asterisks in Supplementary Fig. 1 were fixed to avoid fragmentation of the model structure.
Furthermore, three dihedral angles (φ(C5-C4-C7-C8), φ(C4-C7-C8-C9), and φ(C7-C8-C9-O2)) were fixed at
the values of the original crystal structures to represent structural characteristics of the pG, pR, pR0 and
IT states. This model is very similar to that previously used by Unno et al., whereby they reasonably
reproduced a spontaneous Raman spectrum and Raman optical activity of PYP in the pG state2, 3.
Except for the case of the pR0 state, normal mode calculations showed one imaginary frequency for each
model, which was assigned to the translational motion of the geometrically fixed amino acids. However
this result does not affect the discussion on the high-frequency localized modes. The calculated
frequencies are scaled according to the wavenumber-linear scaling method4.
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Supplementary Figure 1. Chemical structure of the model chromophore pocket used for the theoretical
calculations. Chromophore conformation is trans in this Figure, corresponding to the pG state of the
photocycle.
2. Stimulated Raman spectrum of the pG state
To compare with the time-resolved vibrational spectra obtained by TR-ISRS, we measured a steady-
state Raman spectrum of pG by stimulated Raman spectroscopy. The stimulated Raman measurement
was performed on the Stokes side under the pre-resonance condition, in which the picosecond Raman
pump pulse at 490 nm and femtosecond white-light probe pulse were used. The obtained stimulated
Raman spectrum is shown in Supplementary Fig. 2b, and it is compared with the spontaneous resonance
Raman spectrum of pG reported by Unno et. al. (λexc = 413 nm, Supplementary Fig. 2a)2, 5. These
stimulated and spontaneous Raman spectra accord excellently with each other in both the peak
frequency and spectral pattern. Therefore, the observed stimulated Raman bands are assignable to pG,
reflecting the vibrational structure of the dark state of PYP. This pG spectrum is distinctly different
from the time-resolved vibrational spectra obtained by TR-ISRS (Fig. 3c), which ensures that the
contribution due to the pG state is negligible in the TR-ISRS data.
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Supplementary Figure 2. Comparison of the resonance Raman spectra of pG, I0 and pR. a, Reported
spontaneous resonance Raman spectrum of pG. The spectra in the high- and low-frequency regions are
adapted with permission from ref. 2 and 5 (Copyright 2007 and 2004 American Chemical Society),
respectively. The spectra in the two frequency regions are shown after intensity matching for the 766-
cm-1 band. b, Stimulated Raman spectrum of pG measured in the present study with 490-nm Raman
pump. c, Time-resolved Raman spectrum of I0 obtained at ΔT = 60 ps, as the Fourier transform power
spectrum of the oscillatory component of the TR-ISRS signal (the same as Fig. 5d). This spectrum is
not corrected for the frequency-dependent sensitivity due to the time-domain measurement. d, Reported
spontaneous resonance Raman spectrum of pR, which is adapted with permission from ref. 5 (Copyright
2004 American Chemical Society). The low- and high-frequency parts of this pR spectrum are taken
from different data sets and stitched at 800 cm-1. Therefore, their relative intensities cannot be directly
compared.
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3. One-color pump-probe spectroscopy with 30-fs time resolution (465-nm pump and probe):
Monitoring low-frequency vibrations of the pG state
To obtain Raman spectrum of the pG state in the low frequency (<100 cm-1) region, we carried out one-
color pump-probe spectroscopy using 20-fs pulses at 465 nm, which is in resonance with S1←S0
absorption of pG (Supplementary Fig. 3a). This measurement is complementary to the stimulated
Raman measurement described in the previous section. Supplementary Fig. 3b represents the obtained
pump-probe signal of wild-type PYP. The signal shows rapid recovery of the ground-state bleach, as
well as the oscillatory feature due to coherent nuclear wavepacket motions. Fourier transform power
spectrum of the oscillatory component is shown in Supplementary Fig. 3c. The peak frequencies of the
observed bands in the 350-1000 cm-1 region are in good agreement with those of the pG spectrum
obtained by the stimulated Raman measurement (Supplementary Fig. 2b). This indicates that the
wavepacket motion in pG, which is created through the resonance impulsive stimulated Raman
scattering, is predominantly probed in this experiment. Noticeable differences in the spectral pattern
were recognized in the 100-350 cm-1 region of the spectra obtained by the one-color pump-probe and
stimulated Raman methods. We attribute this difference to contributions of the pG* Raman bands,
because the spectrum of the pump and probe pulses has some overlap with the stimulated emission band
of pG* (see Supplementary Fig. 3a for the spectral condition).
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Supplementary Figure 3. a, Spectrum of the pulses used for the one-color pump-probe measurement.
Steady-state absorption / emission spectra and transient absorption spectrum at 200 fs are shown for
comparison. ESA and SE denote excited-state absorption and stimulated emission, respectively. b,
One-color pump-probe signal of wild-type PYP measured with 20-fs pulses at 465 nm. The oscillatory
component is extracted and shown at the top. Broken line denotes the best fit to the population
component of the pump-probe signal. c, Fourier transform power spectrum of the oscillatory component.
4. Comparison of the Raman spectra of the pG, I0, and pR states
Supplementary Fig. 2 compares the Raman spectrum of I0 with that of pG, as well as the spontaneous
resonance Raman spectrum of pR reported by Chosrowjan et. al.5. As one can clearly see in this Figure,
the Raman spectrum of I0 is distinctly different from those of pG or pR in the fingerprint region. We
also note that the I0 spectrum shows an intense band at ~45 cm-1, although such a remarkable vibrational
signature is not found in the low-frequency Raman spectrum of pG (Supplementary Fig. 3c). These
results strongly suggest the unique structural characteristics of I0 as discussed in the main text.
5. Comparison of the I0 spectra obtained by TR-ISRS and FSRS
Creelman et al. recently reported the stimulated Raman spectrum of I0 in the 400-1700-cm-1 region using
FSRS6. As shown in Supplementary Fig. 4, the I0 spectra obtained by TR-ISRS and FSRS agree with
each other in terms of the frequencies of characteristic bands, such as the most intense hydrogen-out-of-
plane bands around 630 cm-1 and the C=O stretch band around 1630 cm-1, although the overall intensity
pattern looks different probably due to a difference in the resonance condition. Remarkably, each band
in the FSRS spectrum looks significantly broadened, which makes it difficult to examine its spectral
similarity to the spectra calculated for different structures of the ground-state intermediate (pR0 and IT).
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The higher frequency resolution and signal-to-noise ratio achieved by our TR-ISRS measurement reveal
fine vibrational features that were not clearly resolved in the FSRS spectrum, allowing the detailed
comparison with the calculated spectra, as discussed in the main text.
Supplementary Figure 4. Comparison of the I0 Raman spectra obtained by TR-ISRS and FSRS. The
FSRS spectrum is adapted with permission from ref. 6 (Copyright 2013 American Chemical Society).
6. Two-color pump-probe spectroscopy with 20-fs time resolution (460-nm pump and 500-680 nm
probe): Monitoring the initial dynamics and vibration of the pG* state
Two-color pump-probe measurements for wild-type PYP and its E46Q mutant were carried out to
observe their initial population dynamics and vibrational structures in the Franck–Condon state. In this
experiment, we used sub-20-fs pulses at 460 nm for photoexcitation and probed transient absorbance
changes by sub-7-fs pulses at 500-680 nm, which overlap with the red side of the pG* stimulated
emission (SE) (see Supplementary Fig. 5a for the spectral condition). Supplementary Fig. 5b shows the
obtained pump-probe signals of wild-type PYP and E46Q. The overall temporal profile of the pump-
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probe signal of wild-type PYP is essentially the same as the trace presented in Fig. 3a, but the higher
time resolution revealed a gradual growth of the SE signal on the sub-100-fs time scale (see the inset of
Supplementary Fig. 5b). Since the probe spectrum overlaps with the red side of the SE band, this
gradual growth of the SE signal suggests a temporal red shift of the SE band, which is reminiscent of the
fluorescence dynamic Stokes shift observed in solution. In photoreceptor proteins, the corresponding
dynamics would arise from the rearrangements of the amino acid residues surrounding the chromophore.
The sub-100-fs dynamics observed in the present pump-probe measurement can be highly likely
associated with the initial hydrogen-bond rearrangement discussed in the main text.
In the pump-probe traces, the oscillatory feature due to the coherent nuclear wavepacket motion,
created in the Franck–Condon state by photoexcitation, is also evident. Extracted oscillatory
components and corresponding Fourier transform power spectra are shown in Supplementary Fig. 5c
and d, respectively. The Fourier transform power spectrum of wild-type PYP is almost identical to the
pG* spectrum obtained with TR-ISRS (Fig. 3c). (Note that the visibility of the high frequency modes is
reduced in the pump-probe measurement due to the lower time resolution.) The data clearly
demonstrate that the 135-cm-1 band shows much higher intensity with respect to the others, supporting
that the mode has the significant Franck–Condon activity around the Franck–Condon region, as we
discussed in the main text. Importantly, this band showed substantial intensity decrease upon E46Q
mutation (Supplementary Fig. 5d), and it also showed ~1-cm-1 downshift upon hydrogen / deuterium
exchange of the buffer solution (Supplementary Fig. 5e), whose reproducibility was rigorously checked.
(Our capability to detect such small frequency shifts, which is based on the high S/N and precision of
the interferometrically calibrated delay stage, has been reported elsewhere7.) These data indicate that the
mode is not a simple intramolecular vibration of pCA, but has an intermolecular character that involves
the motion of the residues that are hydrogen-bonded to pCA.
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Supplementary Figure 5. a, Spectra of the pulses used for the two-color pump-probe measurement.
Steady-state absorption / emission spectra and transient absorption spectrum at 200 fs are shown for
comparison. b, Pump-probe signals of wild-type PYP and E46Q measured with sub-20-fs, 460-nm
pump / sub-7-fs, 500-650 nm probe pulses. The magnified view of the early time region is also shown
in the inset. The broken line denotes the best fit to the population component of the pump-probe signal.
c, Oscillatory components of the pump-probe data. d, Fourier transform power spectra of the oscillatory
components of wild-type PYP and E46Q. e, Comparison of the Fourier transform power spectra of
wild-type PYP in H2O (red solid lines) and D2O (green broken lines) buffer solutions. Expanded view
around the 135-cm-1 band is shown in the inset.
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7. Effect of the actinic pump pulse duration (290 fs) on the temporal profile of the 135-cm-1 band
In the TR-ISRS experiment of wild-type PYP, a rapid intensity drop was observed for the 135-cm-1 band
as discussed in the main text. Although the timescale of this intensity drop (~100 fs) is close to the
temporal overlap between the actinic pump (P1) and Raman pump (P2) pulses, we are able to rule out a
possibility of the coherent artifact as the origin of this observation on the basis of the following two sets
of TR-ISRS experiments. All the results support that the rapid dynamics of the 135-cm-1 band shown in
the main text is not due to the experimental artifact but originated from the structural dynamics of PYP.
a. TR-ISRS measurements of PYP with various actinic pump pulse durations
We performed the TR-ISRS experiments of PYP with various actinic pump pulse durations to confirm
that the temporal behavior of pG* Raman bands does not depend on the actinic pump pulse duration and
the absence of any artifact during the P1-P2 temporal overlap. Supplementary Fig. 6 (the same as the
inset of Fig. 3d) shows temporal profiles of the Fourier amplitude of the 135-cm-1 band obtained by the
TR-ISRS measurements performed with 35- and 75-fs actinic pump pulses. Incident energy of the all
three pulses (P1, P2 and P3), as well as the spectrum and pulse duration of the P2 and P3 (probe) pulses
were kept the same as the measurement presented in the main text. It is evident from this data that the
early temporal profiles measured with these two different P1 pulse durations are essentially the same and
that they do not show any specific change on the time scale of the actinic pump duration (i.e., 35 and 75
fs), although the early temporal profiles are superimposed by a substantial oscillatory feature. This
result indicates that the temporal profile of the 135-cm-1 band does not depend on the actinic pump
duration (except for the oscillatory feature observed with short actinic pump pulses), which assures that
the rapid dynamics shown in the main text does not arise from the artifact due to the temporal overlap
between the P1 and P2 pulses.
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The substantial oscillatory feature recognized in Supplementary Fig. 6 is due to the coherent fifth-
order (χ(5)) processes, which actually hampers us to extract the intrinsic dynamics of the low-frequency
modes in the femtosecond ΔT delay region. In the TR-ISRS measurement, the femtosecond actinic
excitation pulse (P1) is used to populate the excited state, but the P1 pulse can also generate the
vibrational coherence, particularly for low-frequency modes. Then, the Raman excitation pulse (P2) can
deplete (or transfer) this P1-induced vibrational coherence8, and its effect appears in the raw TR-ISRS
signal because we measure the P2-induced differential absorbance. The amplitude and phase of this
fifth-order component change with the P1-P2 delay (ΔT), and hence the resultant Fourier amplitude of the
P1-induced vibrational coherence exhibits a substantial oscillation against ΔT, as shown in
Supplementary Fig. 6. This fifth-order process obscures the signal due to the P2-induced vibrational
coherence that we intend to observe in TR-ISRS experiments. Thus, in the present study, we
deliberately stretched the P1 pulse to 290 fs so that it only generates the pG* population without
inducing any vibrational coherence, at least, for the modes in the >100-cm-1 region. This assures that the
Fourier spectra in the >100 cm-1 region shown in Fig. 3c and 4b only represent the intrinsic dynamics
that is not contaminated by the fifth-order component. Nevertheless, even with such a stretched P1
pulse, the modes at <100 cm-1 can be coherently excited by the actinic excitation, and hence the
dynamics of the <100 cm-1 vibration can be distorted by the fifth-order process. Therefore we did not
show the < 100 cm-1 region of the Fourier spectra in Fig. 3c and Fig. 4b. In contrast, the measurement
for the I0 state was performed at the ΔT delay as long as 60 ps when the P1-induced vibrational
coherence is completely vanished. Therefore, the entire frequency region of the I0 spectrum, including
the very low frequency part (<100 cm-1), is free from the fifth-order artifacts, which allows us to safely
discuss the low-frequency Raman activity of the I0 state. More detailed description of these fifth-order
processes and their contribution to TR-ISRS measurements will be reported elsewhere.
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Supplementary Figure 6. Temporal profiles of the Fourier transform amplitude of the 135-cm-1 band,
as obtained by the TR-ISRS experiments with short actinic pump pulses of 35-fs and 75-fs durations.
Oscillatory feature represents the coherent fifth-order process.
b. TR-ISRS measurements of the standard dye molecule with 290-fs actinic pump pulse
We carried out TR-ISRS experiments of a non-reactive, highly fluorescent dye molecule, N,N’-bis(2,6-
dimethylphenyl)perylene-3,4,9,10-tetracarboxylic diamide (PERY, Supplementary Fig. 7a), with the
290-fs actinic pump to confirm that the actinic pump pulse duration does not affect the temporal
behavior of particular Raman bands. Its high photo-stability, rigid structure, and negligibly small Stokes
shift9 allow us to examine the effect of the pulse duration on the temporal profiles of low- to high-
frequency Raman bands without considering solute structural (or solvent) dynamics. In fact, the absence
of noticeable dynamics for excited-state PERY on the femtosecond-to-picosecond timescale has been
already demonstrated in the previous high-time resolution transient absorption spectroscopy study10. It
should be also pointed out that PERY has an excited-state Raman band at 136 cm-1, which is comparable
frequency to that of the pG* Raman band of PYP that we extensively discussed in the main text (135
cm-1). Therefore, the TR-ISRS experiment of PERY allows us to investigate the effect of the 290-fs
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actinic pump pulse duration on the temporal profile of particular low-frequency bands having a
comparable vibrational period, as well as other higher frequency bands.
Supplementary Figs. 7b-d summarize the results of the pump-probe spectroscopy and TR-ISRS of
PERY in a CCl4 solution. For the measurements, the actinic pump pulse duration was set to 290-fs (525
nm), which is the same as that employed in the TR-ISRS experiment for PYP. The 7-fs Raman pump
and probe pulses were tuned at 550-700 nm, which were resonant with the excited-state absorption and
stimulated-emission bands of PERY10. The pump-probe data (Supplementary Fig. 7b) shows a gradual
growth of the transient absorption signal, which is limited by the instrumental response (290 fs), and the
subsequent plateau confirms the absence of significant dynamics on this timescale. Supplementary Fig.
7c shows the oscillatory component of the TR-ISRS signals of PERY obtained at various ΔT delay times,
and the corresponding Fourier spectra (Supplementary Fig. 7d), i.e. time-resolved Raman spectra, show
gradual growth of the excited-state Raman bands at 136, 277, 538, 727, 1278, and 1596 cm-1. (The
bands at 136 and 277 cm-1 were observed and characterized in detail previously10.) Temporal profiles of
the Fourier amplitude of these Raman bands are plotted against the ΔT delay in Supplementary Fig. 8.
All the temporal profiles show the comparable amplitude growth with the instrumental response (290 fs),
and they almost perfectly follow the temporal profile of the pump-probe signal that represents the
population dynamics of the excited state of PERY. It should be emphasized that the 136-cm-1 band of
PERY, whose vibrational period (260 fs) is comparable to the actinic pump pulse duration, also
perfectly follows the pump-probe kinetics. These data demonstrate that the temporal profile of a
particular band is irrelevant to the actinic pump pulse duration in TR-ISRS experiments, regardless of
whether or not its vibrational period is comparable to the actinic pump pulse duration, and that the
significant dynamics observed for the 135-cm-1 band of PYP does not originate from the experimental
artifact but from its intrinsic structural dynamics.
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Supplementary Figure 7. a, Chemical structure of PERY. b, Pump-probe signal of PERY in a CCl4
solution, measured with 290-fs pump / 7-fs probe pulses. Blue broken line denotes the best fit to the
data using the step function convoluted with the 290-fs instrumental response. c, Oscillatory
components of the TR-ISRS signal of PERY at selected ΔT delay times. Non-resonant ISRS signal of
CCl4 is also shown at the bottom. d, Fourier transform power spectra of the oscillatory components of
TR-ISRS signals of PERY. Bands with the asterisk denote the CCl4 Raman bands.
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Supplementary Figure 8. Temporal profiles of the Fourier amplitude of the excited-state Raman bands
of PERY. Black broken lines denote the best fit to the data using the step function convoluted with the
290-fs instrumental response.
8. Analysis of the phase of the 135 cm-1 mode
Because the phase of the oscillatory component due to the wavepacket motion may provide additional
information on its origin, we analyzed the phase of the 135-cm-1 oscillation obtained at the early ΔT
delay times. For extracting the phase, the oscillatory components of the TR-ISRS data were fitted by the
following damped cosine function,
∆A ∆𝑇, 𝜏 = 𝐴!(∆𝑇)cos 2𝜋𝜈𝜏 + 𝜑(∆𝑇) exp −𝜏
𝑇!"#!,
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where 𝐴!(∆𝑇), 𝜈, 𝜑(∆𝑇), 𝑇!"#! represent the oscillation amplitude, frequency, phase and dephasing
time, respectively. In the fitting procedure, 𝐴!, 𝜑, and 𝑇!"#! were free parameters and 𝜈 was fixed to
135 cm-1.
Supplementary Fig. 9 shows the time evolution of the amplitude and phase of the 135-cm-1 oscillation
against the ΔT delay time. At the delay times later than 0.25 ps, the phase is almost 0 rad., indicating
that the oscillation is almost cosine-like, which is expected for resonance impulsive stimulated Raman
scattering (RISRS) signal11. On the other hand, the phase change by almost π is recognized within ∼0.2
ps after the actinic excitation. One possible reason to account for this observation may be the change of
the resonance condition in this early ΔT time window, i.e., a temporal shift of the transient absorption
band with respect to the probe spectrum. It is known that the phase of the RISRS signal shows steep
change around the electronic transition maximum and the phase flip by π is expected when the red and
blue sides of the electronic transition band are probed11. When the broadband single channel detection
scheme is employed, the observed phase is sensitive to the spectral coverage of the electronic transition
band by the broad probe spectrum, particularly when the probe spectrum is tuned over the electronic
transition maximum. Because our probe spectrum is tuned very close to the stimulated emission (SE)
maximum of pG*, it is likely that the slight spectral shift of the SE band due to some dynamic process,
such as structural change or dynamic Stokes shift, leads to the change in the spectral coverage of the SE
band by the probe pulse. In fact, the rapid rise of the SE signal was observed in the pump-probe
spectroscopy of PYP (Supplementary Fig. 5b), implying the rapid spectral shift of the SE band. Such a
change in the resonance condition would result in the change of the phase.
Because the phase is not only sensitive to the structural dynamics of the molecule, but also sensitive to
the resonance condition of the experiment, further detailed discussion based on the phase will require
additional, extensive experiments such as pump-probe and TR-ISRS measurements with dispersed
18
detection scheme12, examination of the chirp dependence13, and numerical simulations11, 14, which are
beyond the scope of the present study.
Supplementary Figure 9. Temporal profiles of the amplitude and phase of the 135-cm-1 oscillation
against the ΔT delay times.
9. Comparison of the I0 spectrum with the calculated Raman spectra of the pR0 and IT states
Supplementary Fig. 10a shows the calculated Raman spectra of the model PYP systems based on the
pR0 and IT structures in the entire frequency range. As discussed in the main text, the calculated Raman
spectrum for the pR0 structure shows higher similarity to the experimental I0 spectrum than that for the IT
structure in the HOOP region. It is also noteworthy that the calculated C9=O2 stretch frequencies of the
model pG, pR0, and pR structures also reproduce the tendency of the frequency upshift upon the
transition from pG to pR (via I0). (In Supplementary Fig. 10b, these calculated C9=O2 stretch
frequencies are summarized and compared with the experimental values for pG, I0, and pR.) These
comparisons of the key spectral features indicate that the pR0 structure, rather than the IT structure,
resembles the structure of the I0 state in solution.
19
Despite their good agreement of the vibrational frequency in the fingerprint region, differences in
the intensity pattern are seen between the experimental I0 and calculated pR0 spectra; the I0 spectrum
shows exceptionally large intensities of the low-frequency band at 45 cm-1 and the HOOP bands at
around 628 cm-1, as well as the absence of the Cet=Cet stretch band in the 1500 – 1600 cm-1 region.
These differences in the band intensity are attributable to the fact that the calculated spectrum
corresponds to a non-resonance Raman spectrum, while the experimental spectrum was measured under
the rigorously resonant condition. Because of this difference in the resonance condition, we cannot
rigorously compare the intensity patterns of the experimental and calculated Raman spectra over the
whole frequency region. However, it has been established that the Raman intensity of the HOOP mode
of polyene systems reflects the local point symmetry and hence it can be a sensitive probe of the skeletal
distortion15-17. Therefore, the appearance of the large intensity bands in the HOOP region and their
intensity pattern can provide strong evidence that the I0 state has a largely distorted structure that highly
resembles the pR0 structure.
Supplementary Figure 10. a, Calculated Raman spectra of the model PYP systems based on the pR0
and IT structures in the entire frequency range. The Raman spectrum of I0 obtained by TR-ISRS is also
20
shown for comparison. b, Frequencies of the C9=O2 stretch mode of pG, I0, and pR in comparison to the
calculated frequencies for the model pG, pR0, IT, and pR systems.
10. Dihedral angle dependence of the Raman intensity of the HOOP mode
To demonstrate a correlation between the skeletal planarity of the chromophore and the Raman activity
of the HOOP modes, we examined the dihedral angle (φ(C7-C8-C9-O)) dependence of the HOOP
intensity for thiomethyl p-coumaric acid (Supplementary Fig. 11a), a model PYP chromophore, by DFT
calculations. Supplementary Fig. 11b shows the calculated Raman spectra in the HOOP region with
various dihedral angles. The data clearly show that the HOOP intensity increases as the dihedral angle
increases.
Supplementary Figure 11. a, Chemical structure of the model PYP chromophore (thiomethyl p-
coumaric acid) together with the label for the atoms. b, Comparison of the calculated Raman spectra
with various C7-C8-C9-O2 dihedral angles, shown with 8-cm-1 bandwidth. Calculation was performed
with φ(C4-C7-C8-C9) fixed at 30°, adapted from the pR0 structure.
21
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