Enlightening the photoactive site of channelrhodopsin-2
by DNP-enhanced solid-state NMR spectroscopy
Johanna Becker-Baldusa,b, Christian Bamannc, Krishna Saxenab,d, Henrik Gustmanne, Lynda J. Brownf,
Richard C. D. Brownf, Christian Reiterg, Ernst Bambergc, Josef Wachtveitle, Harald Schwalbeb,d,
and Clemens Glaubitza,b,1
a
Institute of Biophysical Chemistry, Goethe University Frankfurt, 60438 Frankfurt, Germany; bCentre for Biomolecular Magnetic Resonance, Goethe
University Frankfurt, 60438 Frankfurt, Germany; cMax-Planck-Institute of Biophysics, 60438 Frankfurt, Germany; dInstitute of Organic Chemistry and
Chemical Biology, Goethe University Frankfurt, 60438 Frankfurt, Germany; eInstitute of Physical and Theoretical Chemistry, Goethe University Frankfurt,
60438 Frankfurt, Germany; fDepartment of Chemistry, University of Southampton, Southampton SO17 1BJ, United Kingdom; and gBruker Biospin GmbH,
76287 Rheinstetten, Germany
Edited by G. Marius Clore, National Institutes of Health, Bethesda, MD, and approved June 25, 2015 (received for review April 21, 2015)
Channelrhodopsin-2 from Chlamydomonas reinhardtii is a light-
gated ion channel. Over recent years, this ion channel has attracted
considerable interest because of its unparalleled role in optogenetic
applications. However, despite considerable efforts, an understand-
ing of how molecular events during the photocycle, including the
retinal trans-cis isomerization and the deprotonation/reprotonation
of the Schiff base, are coupled to the channel-opening mechanism
remains elusive. To elucidate this question, changes of conformation
and configuration of several photocycle and conducting/nonconduct-
ing states need to be determined at atomic resolution. Here, we
show that such data can be obtained by solid-state NMR enhanced
by dynamic nuclear polarization applied to 15N-labeled channelrho-
dopsin-2 carrying 14,15-13C2 retinal reconstituted into lipid bilayers. In
its dark state, a pure all-trans retinal conformation with a stretched
C14-C15 bond and a significant out-of-plane twist of the H-C14-C15-H
dihedral angle could be observed. Using a combination of illumina-
tion, freezing, and thermal relaxation procedures, a number of in-
termediate states was generated and analyzed by DNP-enhanced
solid-state NMR. Three distinct intermediates could be analyzed
with high structural resolution: the early P500
1 K-like state, the slowly
decaying late intermediate P4804 , and a third intermediate populated
only under continuous illumination conditions. Our data provide
novel insight into the photoactive site of channelrhodopsin-2 during
the photocycle. They further show that DNP-enhanced solid-state
NMR fills the gap for challenging membrane proteins between
functional studies and X-ray–based structure analysis, which is re-
quired for resolving molecular mechanisms.
channelrhodopsin | retinal | solid-state NMR | DNP | freeze trapping
S ince their discovery (1), channelrhodopsins (ChRs) have
generated enormous interest because of the rapid development
of their applications in optogenetics (2–7). Commonly, ChR2 from
Chlamydomonas reinhardtii (8) and its variants are used thanks to
their favorable expression levels. They are the only proteins known
today functioning as light-gated ion channels (Fig. 1A). Like other
microbial retinal proteins, they undergo a periodic photocycle. In
ChRs, this photocycle is coupled to channel opening and closing as
revealed in electrophysiological recordings (8). A chimera of ChR1
and ChR2 has been crystallized to yield a structure at 2.3-Å reso-
lution (9). However, little is known on how this coupling functions
on a molecular level, and a large number of studies based on visible
(10–13), IR (11, 14–19), resonance Raman spectroscopy (20, 21),
and EPR spectroscopy (22, 23) has been performed to address
this question.
The photocycles of microbial rhodopsins are usually compared
with bacteriorhodopsin, the first discovered and most studied light-
driven proton pump (24). Without any illumination, microbial
retinal proteins thermally equilibrate into a dark state (25). In the
case of bacteriorhodopsin, for example, this state contains a mixture
of two species termed bacteriorhodopsin568 (all-trans,15-anti retinal
www.pnas.org/cgi/doi/10.1073/pnas.1507713112
Schiff base) and bacteriorhodopsin548 (13-cis,15-syn conformation)
(26, 27). On illumination, light adaption occurs from the dark state
to the ground state, which contains only the all-trans,15-anti con-
former as the photocycle starting point (28). A similar light–dark
adaption has been found in halorhodopsin from Halobacterium
salinarium (29). However, such a light/dark adaption in conjunction
with a conformer mixture does not seem to be a general property of
COMPUTATIONAL BIOLOGY
microbial membrane proteins. Other systems have been described
BIOPHYSICS AND
where the ground state contains only an all-trans,15-anti retinal Schiff
base chromophore [e.g., green proteorhodopsin (30), Anabaena
sensory rhodopsin (31), Oxyrrhis marina proteorhodopsin (32),
sensory rhodopsin I from H. salinarum (33) and Salinibacter ruber
(34), and sensory rhodopsin II from Natronobacterium pharaonis
(35, 36) and H. salinarum (37)].
In ChR2, the retinal is covalently bound to the lysine residue
257 conserved in all retinal proteins through a Schiff base linkage
(38). The X-ray structure of the ChR chimera shows the retinal
in an all-trans configuration (9), although other conformations
cannot be excluded at the obtained resolution. Results of retinal
extraction in conjunction with resonance Raman studies were
interpreted as an isomer mixture containing 30% of a 13-cis
retinal in dark- and light-adapted ChR2 (20). In addition,
nanosecond IR spectroscopy on the E123T mutant of ChR2
indicated the presence of some 13-cis retinal in the dark state
Significance
Channelrhodopsin-2 is a dimeric membrane protein functioning
as a light-gated ion channel, which has triggered numerous
optogenetic applications. We present the first NMR study, to
our knowledge, by which structural details of the retinal co-
factor could be resolved. This study was only possible by en-
hancing the detection sensitivity 60-fold through dynamic
nuclear polarization (DNP), a highly promising hybrid method
linking EPR with solid-state NMR spectroscopy. Our data show
that ground-state channelrhodopsin-2 contains the retinal co-
factor in its all-trans configuration with a slightly perturbed
polyene chain. Three different photointermediates could be
trapped and analyzed. Our study shows that DNP-enhanced
solid-state NMR is a key method for bridging the gap between
X-ray–based structure analysis and functional studies toward a
highly resolved molecular picture.
Author contributions: J.B.-B., E.B., J.W., H.S., and C.G. designed research; J.B.-B., C.B., K.S.,
and H.G. performed research; C.B., K.S., L.J.B., R.C.D.B., C.R., E.B., and H.S. contributed
new reagents/analytic tools; J.B.-B., H.G., and C.G. analyzed data; and J.B.-B. and C.G.
wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1
To whom correspondence should be addressed. Email: glaubitz@em.uni-frankfurt.de.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1507713112/-/DCSupplemental.
PNAS Early Edition | 1 of 6
A B C B0 Px to some extent can convert directly to the ground state, which
ht
K+ lig
gh
t Na+ ne
l
ChR2470
al u e is indicated by dashed lines in Fig. 1B.
li Ca+ a n ing l-
13 tran bl
ue H+ c h los -c s All of the above-described states were detected by visible and
bl c is

Px P4480 (desensitized state) FTIR spectroscopy, and assignments of spectroscopic signatures

out P500
1 (K-like) e to conformational and configurational states of the retinal were
av
ow
icr based on analogous data previously studied. However, detailed
m

P520
3
in
P390
2b
H+
Na+ Ca+
(M-like)
K+
Fig. 1. (A) Visualization of dimeric ChR2 reconstituted into the lipid bilayer
as used in this study [cartoon based on the crystal structure of the ChR1/2
chimera (data from ref. 9)]. Blue light illumination activates ChR2. (B) Single
turnover (black arrows) and continuous illumination photocycle (blue ar-
rows) (14, 40). (C) Schematic view of the experimental setup for generating
and measuring different photointermediates. (D) The DNP enhancement is
generated by magnetization transfer from the biradical AMUPOL to ChR2.
using a similar spectroscopic assignment as in the resonance
Raman study (39). In contrast to bacteriorhodopsin, no light
adaption was observed using resonance Raman techniques (20)
or visual spectroscopy (12). The occurrence of a conformer
mixture in the ground state without light adaption would make
ChR2 unique among the microbial retinal proteins, but addi-
tional data are needed to confirm these observations more directly
at improved atomic resolution.
The current model of the ChR2 photocycle is shown in Fig.
1B (14, 40). According to this model, blue light excitation leads
to a retinal all-trans to 13-cis isomerization, resulting in a
red-shifted first intermediate P500 1 (12) resembling a K-like
state, which most likely contains a 13-cis,15-anti retinal Schiff
base chromophore similar to Bacteriorhodopsin (27). To our
knowledge, such red-shifted K-like intermediates occur in all
microbial retinal proteins (38). Schiff base deprotonation leads
to the M-like state P390
2 (10, 11). This state is followed by the red-
shifted intermediate P5203 , which has previously been correlated
with the open state (10). However, later data confirmed that
channel opening occurs before P520 3 formation and might happen
during a spectroscopically silent transition between P390 2a and
P390
2b states (41). The last photocycle intermediate is the long-
lived intermediate P480 state (τ = 24 s), which is referred to as
4 13
P390
2a D O O information on bond lengths or torsion angles that would also
l O O link to quantum chemical calculation is still missing. To fill this
ne N O N
an ing gap between static crystallographic data on the one hand and
ch en
p N N
o O O
H
kinetic and functional data based on optical spectroscopy and
O
4
electrophysiology on the other hand, we applied solid-state
magic angle spinning NMR on isotope-labeled ChR2 and retinal
to obtain site-resolved structural data directly in a membrane
environment under various experimental conditions. In this way,
fine details of the chromophore conformation during the pho-
tocycle could be resolved, which will be important to understand
the link between channel and photocycle activity in ChR2. A
limitation using proteoliposomes is the amount of sample that
can be studied, because the protein-to-lipid ratio cannot be in-
creased too much without compromising protein integrity. In
addition, trapping photointermediates works best using sam-
ples with low optical density, which reduces further the usable
amount of protein, resulting in a poor NMR signal-to-noise ratio.
Therefore, cross-effect dynamic nuclear polarization (DNP) en-
hanced magic angle spinning (MAS) NMR [review in the work
by Maly et al. (44)] was indispensable in overcoming these sen-
sitivity problems (Fig. 1C). This technique requires temperatures
around 100 K that are also compatible with trapping of photo-
intermediates as outlined below. DNP-enhanced MAS NMR is
not yet a routine method but is applied increasingly to complex,
mechanistic studies on retinal proteins (45–48) and other mem-
brane proteins (49–51).
Here, DNP-enhanced solid-state NMR spectroscopy has been
applied to 15N-labeled ChR2 carrying 14,15-13C2 retinal recon-
stituted into lipid bilayers and incubated with the DNP polarizing
agent AMUPOL (52) in a glycerol–water mixture. The labeling
scheme adopted here is shown in Fig. 2A. The 13C14 chemical
shift is sensitive to the configuration of the C13-C14 bond. To-
gether with the neighboring 13C15 atom, the two 13C-labeled
A 13
C15
Lys257
C experimental data
15 +
N D = 2100 Hz / 1.53 Å
C14
intesnity a.u.

H D = 2200 Hz / 1.51 Å
the desensitized state with a spectral characteristic similar to the lipid D = 2300 Hz / 1.49 Å

ground state (11, 42). In addition, time-resolved FTIR spec-

C15 lipid C14
troscopy indicated that P520 3 could partially convert directly to B C=C

the ground state (14). C’

The situation becomes more complicated under continuous CP at RT
glycerol 0.0 0.5 1.0 1.5
DQ build-up time in ms
2.0

light illumination (40, 43). Under these conditions, a high tran- D

sient current is observed first that is quickly reduced to a much experimental data
154°
intensity a.u.

lower steady-state current. After turning off the irradiation, the DQF + DNP 156°
158°
steady-state current decays biexponentially. This observation can aromatic
160°
162°
only be explained by a branching of the photocycle. Two open CP + DNP

states and two closed states are required to quantitatively de- CP

scribe the observed behavior under continuous light conditions. 200 150
13
100 50 0
0.0 50.0 100.0 150.0 200.0 250.0
C chemical shift (ppm)
The two closed states are most likely the ground state and the HCCH evolution time in µs

desensitized state P480

4 that accumulates under continuous illu- Fig. 2. (A) DNP-enhanced MAS NMR has been applied to U-15N-ChR2 con-
mination and is identical to the same intermediate from a single taining 14,15-13C all-trans–retinal. (B) A 62-fold signal enhancement is
turnover (18). One of the open states is probably the open state achieved for 13C cross-polarization (CP; CP vs. CP + DNP). The 13C natural
P520
3 observed in single-turnover experiments. However, little is abundance background can be efficiently suppressed by a double quantum
known about the identity of the second open state, which only filter (DQF; DQF + DNP), resulting in a spectrum with only the resonances of
C14 and C15. As a control, one additional CP spectrum was acquired at 850
occurs under continuous light conditions. It might be an M-like
MHz close to room temperature (CP at RT). The gray bar indicates where the
P390
2 state, another P520
3 state, or another unknown state. Light 13
C14 signal would be expected for a chromophore in the 13-cis,15-syn con-
excitation of probably P480
4 creates this additional state. This state formation. (C) 13C14-13C15 double-quantum (DQ) build-up curve. (D) HCCH
or group of states here is referred to as Px containing at least one dephasing curves for the C14-C15 spin system in ChR2 during two rotor
open state (Fig. 1B). It is also likely that the open states P520
3 and periods reporting on the HCCH dihedral angle.

2 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1507713112 Becker-Baldus et al.

spins can be used for double quantum filtering of this spin pair
against the natural abundance background and at the same time,
offer the possibility to study the length and the dihedral angle of
the C14-C15 bond. Furthermore, the chemical shift of the Schiff
base nitrogen is also sensitive to the chromophore conformation,
reports on the protonation state of the Schiff base, and reflects
counterion interactions. Using this approach, we were able to
provide a first analysis, to our knowledge, of the retinal–Schiff
base chromophore in ChR2 in its ground state as well as three
different photointermediate states at atomic resolution.
Results and Discussion
13
Dark State—Ground State. Fig. 2B shows the C-DNP–enhanced
MAS NMR spectrum of ChR2. Using AMUPOL as the polarizing
agent, a 62-fold signal enhancement was achieved under our ex-
perimental conditions. The observed resonances mainly stem from
the 13C natural abundance background of protein, glycerol, and
lipid. To suppress these signals and identify the 13C-labeled retinal
sites, a double quantum filter has been applied, revealing just two
peaks from retinal carbons C14 and C15 at 126.3 and 166.5 ppm,
respectively (Fig. 2B). The C14 chemical shift is very sensitive to
the conformation of the chromophore (26), and the value of 126.3
ppm is, therefore, a very strong indicator for an all-trans,15-anti
chromophore conformation as observed for bacteriorhodopsin
and proteorhodopsin (SI Appendix, Table S1). By contrast, the
C14 signal of the 13-cis,15-syn chromophore in dark-adapted
bacteriorhodopsin appears at 111 ppm. A signal resonating at a
similar chemical shift has been observed for a 13-cis,15-syn sub-
population in the A178R mutant of green proteorhodopsin (SI
Appendix, Table S1). In ChR2, no signal at or near 111 ppm could
be observed (Fig. 2B, gray area). We, therefore, conclude that the
chromophore in ChR2 is present in a single all-trans,15-anti con-
formation. To exclude that this result is an artifact of the sample
conditions required for DNP, a 13C-CP spectrum of ChR2 without
the addition of radicals or cryoprotectants was recorded at am-
bient temperature using 832 times the number of scans compared
with the DNP experiment (Fig. 2B, CP at RT vs. CP + DNP). In
both cases, the retinal resonances compare well. Only the C14
signal is shifted slightly upfield, which can be attributed to tem-
perature effects. The small additional intensities observable in the
ambient temperature spectrum result from spinning side bands
and can be moved by changing the MAS frequency (SI Appendix,
Fig. S1). In addition, keeping the sample in the dark at 4 °C for
24 h did not change the appearance of the DNP NMR spectra (SI
Appendix, Fig. S2).
To further study the conformation of the chromophore, the
C14-C15 retinal distance has been determined using double-
quantum build-up experiments (Fig. 2C). The obtained value of
1.51 ± 0.02 Å is significantly longer than the 1.42 Å observed for
green proteorhodopsin (47). This increase in bond length cor-
responds well with a lower double-bond character of the bond as
expected from the blue shift of the absorption maximum com-
pared with green proteorhodopsin. Measurements of the H-C14-
C15-H retinal torsional angle revealed a significant out-of-plane
twist with an angle of 158° ± 2°. A similar out-of-plane twist has
also been observed for bacteriorhodopsin (164°) (53) and green
proteorhodopsin (161°) (47), indicating that this out-of-plane
twist is a general property of microbial retinal rhodopsins and
might help to provide a favorable orientation of the Schiff base
during the subsequent photocycle steps.
The 15N chemical shift of the protonated Schiff base (pSB) was
detected as single resonance at 196.5 ppm using 1H-13C/13C-15N
double cross-polarization magnetization transfer from the termi-
nal retinal carbon (C15) to the directly bonded Schiff base ni-
trogen (Fig. 3). Hu et al. (54) established a relationship between
the Schiff base chemical shift and the wavelength of the absorp-
tion maximum using Schiff base–counterion model complexes,
which is shown in Fig. 3, Inset together with the values for
Becker-Baldus et al.
amide
22
ds
1/λmax (103 cm-1)
un
po
ChR2
om
20
lc
pSB
de
mo
DCP+DNP
GPR
18
CP+DNP
His
Arg Lys BR568
16
200 150 100 50 0 165 175 185 195
15 15
N chemical shift (ppm) N (ppm)
Fig. 3. DNP-enhanced N cross-polarization (CP) spectrum (CP + DNP) and
15
double cross-polarization (DCP) –filtered 15N spectrum (DCP + DNP) of ChR2.
(Inset) Comparison of the correlation of the absorption maximum with the
15
N Schiff base chemical shift for ChR2, green proteorhodopsin (GPR), bac-
teriorhodopsin (BR), and model compounds. Modified from ref. 54.
bacteriorhodopsin, green proteorhodopsin, and ChR2. ChR2
(470 nm/196.5 ppm) agrees well with the predicted trend but de-
viates more strongly from model behavior than bacteriorhodopsin.
Based on the model compound geometries, it can be concluded
COMPUTATIONAL BIOLOGY
that the distance from the Schiff base to its counterion in ChR2 is
BIOPHYSICS AND
shorter than in bacteriorhodopsin. As for C14- or C15-retinal
atoms, no peak doubling or splitting is observed, confirming that
retinal is present in only one conformation in the ChR2 ground
state. The chemical shift difference of the all-trans,15-anti and the
13-cis,15-syn 15N signals observed in dark-adapted bacteriorho-
dopsin is 8.3 ppm (46), which would be well-resolved under the
experimental conditions applied here.
The observation of a 100% all-trans,15-anti conformation of
the retinal cofactor in ground-state ChR2 is in line with many
other microbial rhodopsins but in contrast to previous reports on
ChR2. Previously, a population of 30% 13-cis retinal based on
retinal extraction with subsequent HPLC analysis and resonance
Raman experiments has been reported (20).
The reason for this discrepancy is the invasive character of
retinal extraction in the previously reported work (20). This
process required breaking the Schiff base linker by subjecting the
protein to EtOH followed by extraction of retinals into hexanes
and HPLC purification (55). For bacteriorhodopsin, the results
obtained in this way (56) agree well with data from other methods
(26, 27). In other cases, however, this protein treatment led to less
consistent results. For example, retinal extraction studies of green
proteorhodopsin suggested values between 5% and 20% cis-reti-
nal (57, 58). Later, resonance Raman (59) and solid-state MAS
NMR (30) studies confirmed that the ground state contains very
little (if any) cis-retinal. Similarly, for Anabaena sensory rhodop-
sin, retinal extraction experiments showed 24% of the 13-cis iso-
mer in the dark-adapted state (60), whereas solid-state MAS
NMR experiments showed a purely all-trans configuration (31).
Resonance Raman experiments are noninvasive and should give
reliable information on the retinal chromophore. However, as-
signment of the vibrational bands is very challenging and cannot
easily be transferred between different systems. In the case of
bacteriorhodopsin, assignment was based on differently isotope-
labeled retinals (27). Such data are missing for ChR2, resulting in
ambiguous interpretation of Raman data (20) (more detailed
discussion is in SI Appendix).
Our data, therefore, show unambiguously that ground-state
ChR2 contains an all-trans,15-anti chromophore, which also ex-
plains the previously reported monoexponential decay of the
photoexcited state (12) and the absence of a light adaption step
as observed in bacteriorhodopsin, because the amount of this
conformer is already close to 100% in the dark.
PNAS Early Edition | 3 of 6
 A C15 C14 pSB 480 nm (ground-state bleaching) and positive absorption with a
maximum at 500 nm (Fig. 4C). These values are characteristic
for the K-like intermediate P5001 , which strongly suggests that this
state has been trapped and observed by DNP-enhanced MAS
NMR in agreement with earlier UV-visible and FTIR spectro-
scopic studies (11).
dark
The NMR data are also in good agreement with the thermally
trapped K state of bacteriorhodopsin, which was accumulated by
P1500 illumination at 532 nm at 90 K (45, 46). The authors in refs. 45 and
B 46 reported upfield shifts for both 13C14-retinal and 15N pSB signals
with respect to the ground state by 4.9 and 8.7 ppm, respectively.
P1500 The same trend is observed here, and the newly generated state is,
blue light therefore, assigned to the K-like intermediate P500 1 containing a
at 110 K 13-cis,15-anti chromophore. Because P500 1 is also photoactive and
can be converted to the ground state by light, only a mixture of
180 160 140 120 100 200 100 states can be obtained as reported for bacteriorhodospin (65).
13 15
C chemical shift (ppm) N chemical shift (ppm) To reach other intermediates during the photocycle, ChR2 was
C now subjected to a thermal relaxation and a thermal trapping
protocol (Fig. 5A). In the first case, the P500
1 state was created as
Δ abs.

described above followed by thermal relaxation and equilibration

at temperatures between 194 and 273 K, after which the sample
300 350 400 450 500 550 600
was again cooled to 110 K for DNP detection (Fig. 5A). In this way,
the thermal energy should allow ChR2 to overcome the energy
wavelength (nm)
barrier between its P500
1 state and subsequent photocycle inter-
Fig. 4. 13C double quantum filter- and 15N double cross-polarization–fil- mediates. The second protocol involved continuous illumination
tered, DNP-enhanced spectra of ChR2 in a sapphire rotor (A) in the dark and at temperatures between 234 and 254 K followed by freezing (Fig.
(B) after illumination with blue light at 110 K. (C) Optical difference spec- 5B). In addition, a sample was illuminated at room temperature
trum of ChR2 at 150 K of dark- and blue light-illuminated ChR2 (Δ abs refers and then quickly frozen in liquid nitrogen. The second procedure
to the absorption difference). should make especially those photointermediates accessible, which
only occur under continuous illumination (Fig. 1B).
The thermal relaxation approach results in altered line shapes
Observing ChR2 Photointermediates by DNP-Enhanced MAS NMR. For
of the 13C14- and 13C15-retinal resonances (Fig. 5A). With in-
the analysis of photointermediates, sample illumination has been
creasing relaxation temperatures, the 13C14-P500 1 signal broadens
used in the past [e.g., combined with solution-state NMR to
access, for example, the photokinetics of rhodopsin (61, 62) and
solid-state NMR for thermal trapping of bacteriorhodopsin or
rhodopsin intermediates (46)]. Special consideration has to be A C14 B thermal relaxation
given to an efficient illumination setup, because light penetration RT
C15 P 500
of the sample significantly decreases with sample thickness (62). 1
ChR2 470 blue light
The amount of ChR2 proteoliposomes within the optically
transparent sapphire rotor used for DNP NMR was, therefore, 110 K
P 480
4
reduced to about 20% and evenly distributed across the inner (a)
blue light detection
rotor surface by short sample rotation at room temperature. In 273 K
RT thermal
principle, sample illumination can be done in two ways. One trapping
possibility is to illuminate the sample outside of the NMR magnet 265 K
and then, trap the generated state by quickly freezing it and
110 K
inserting it in the cold probe. However, temperature control using 255 K
this method can be difficult. Another option is to equip the DNP (b)
C14 detection
solid-state probe with a light guide and illuminate the sample Px
245 K C15
directly while it is spinning in the stator. Such an approach has
been shown by a number of laboratories using different designs ChR2 470
P 480
4
with DNP (46) or standard MAS probes (62–64). The latter ap- 227 K
blue light
proach offers better temperature control, but quick freezing of RT
the spinning samples is not possible. In this study, both methods 217 K fast freeze
have been used and yielded similar results as discussed below.
blue light
Significant spectral changes are observed on illumination with 254 K
207 K
blue light at 110 K (Fig. 4). For the retinal 13C14 signal, one ad-
ditional peak at 124.2 ppm is observed in addition to the ground- blue light
state signal, whereas the 13C15 resonance broadens slightly. Sim- 194 K 245 K
ilarly, a new 15N signal upfield of the pSB ground-state resonance
is detected at 181 ppm. It can be concluded that illumination at 110 K blue light
234 K
low temperatures leads to a mixture of two states, one of which
corresponds to the ground state. 180 140 100 180 140 100
13 13
For assigning the second state to one of the photointermediates C chemical shift (ppm) C chemical shift (ppm)
(Fig. 1B), optical spectra on thermally trapped samples prepared Fig. 5. (A) 13C double quantum filter (DQF) DNP-enhanced spectra of ChR2
under conditions very close to those used for DNP have been obtained by thermal relaxation. (B) 13C DQF DNP-enhanced spectra of ChR2
recorded. The difference between spectra of a dark sample and obtained by thermal trapping. Dashed lines indicate the observed peak
those of an illuminated sample shows negative intensities below position. RT, room temperature.

4 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1507713112 Becker-Baldus et al.

initially, converts into a new signal at 119.3 ppm between 217 and
255 K, and disappears above 265 K. In principle, the 13C15 reso-
nance follows this trend, but changes are less pronounced,
because only some line broadening can be detected. At higher
temperatures, all additional signals vanish, and the sample re-
turns into the ground-state ChR2470.
The thermal trapping protocol results in a different spectral
characteristic of the chromophore as shown in Fig. 5B. Con-
tinuous illumination at room temperature is known to accu-
mulate the long-lived intermediate P480 4 (10), which can be
further stabilized for DNP NMR detection by rapid freezing.
Therefore, the two signals observed under these conditions for
the 13C14 site can be assigned to the ground state (126.0 ppm)
13
and P480
4 (119.3 ppm). The latter is identical to the C14 chemical
shift that was observed during the thermal relaxation experi-
ment. We, therefore, conclude that this peak belongs to a P480 4
population, which can also be generated when lowering the
illumination temperature. The ground-state subpopulation is
almost completely depopulated at 245 K, which proves that
the whole sample can be illuminated sufficiently with the applied
setup. At this temperature, an additional signal at 122.7 ppm
can be detected, which is also seen in the 234- and 254-K
spectra. Because this species is only generated under continu-
ous light, it is tentatively assigned to one of the P x states
(Fig. 1B).
The most pronounced photointermediates have been pre-
pared at 245 K in both thermal relaxation as well as trapping
approaches. We have, therefore, recorded 15N double cross-
polarization–filtered spectra at this temperature using both
protocols (SI Appendix, Fig. S3). Under thermal relaxation,
only one signal for the pSB is observed at a chemical shift
identical to the ground state but with slightly reduced intensity.
Furthermore, no evidence for a deprotonated Schiff base spe-
cies (i.e., an M-like state) has been found. A possible expla-
nation could be that the Schiff base signal of this photo state is
similar to the ground state or broadened beyond detection. The
experiment was repeated under thermal trapping conditions.
Here, the ground state is depleted at 245 K, which results in a
loss of the pSB signal at 196.5 ppm and shows that the P480 4 was
not hidden underneath. A new signal occurs at 185 ppm, which is
not visible under thermal relaxation conditions and therefore, is
assigned to the Px state.
Thermal relaxation and the thermal trapping protocols at
245 K were also compared using optical spectroscopy under
nearly the same experimental condition (SI Appendix, Fig. S4).
These data confirm that the thermal relaxation and the thermal
trapping protocols at 245 K result in a different population of
photointermediates.
In summary, three photointermediates (P500 480
1 , P4 , and Px)
could be generated using different illumination/relaxation pro-
tocols. This assignment is also in agreement with UV-visible
spectra, which were obtained under cryogenic conditions similar
to those used here for DNP on ChR2 samples and subjected to
the same illumination and freeze-trapping protocols. Similar to
the ground state, we have recorded double-quantum filter build-
up and HCCH torsion angle data for all of them (SI Appendix,
Fig. S5). The results are given in Table 1 together with the
ground-state data. Our data show that the twisting and stretching
of the C14-C15 bond observed in the ground state are conserved
in all three trapped states.
At first glance, it seems surprising that the retinal isomeriza-
tion in the K-like state does not have a strong effect on the C14-
C15 bond, because a hydrogen-out-of-plane band indicative of
bond twisting has been reported to occur from the K to the L
state of bacteriorhodopsin (66, 67). However, direct solid-state
NMR experiments on bacteriorhodopsin, as discussed above, have
shown that this bond is already twisted in the ground state, which
was observed here for ChR2, and its out-of-plane orientation
Becker-Baldus et al.
Table 1. Parameters obtained for the retinal C14-C15 bond in
ChR2 ground state and its photointermediates
State δ(C14)/ppm δ(pSB)/ppm R/Å Φ/°
ChR2 470
126.0 ± 0.5 196.5 ± 0.5 1.51 ± 0.02 158 ± 2
P1500 124.2 ± 0.5 181.0 ± 1.0 1.56 ± 0.04 156 ± 4
P4480 119.3 ± 0.5 — 1.53 ± 0.04 152 ± 4
Px 122.7 ± 0.5 185.0 ± 1.0 1.52 ± 0.02 156 ± 2
PHI = C14-C15 HCCH torsion angle; R = C14-C15 bond length.
increases in the M state (53). The latter effect could also be
expected for ChR2, for which the deprotonation of the chromo-
phore in the M-like state is accompanied by channel opening (53).
However, such a state could not be trapped in this study. The
stable conformation of the C14-C15 bond during the ChR2 pho-
tocycle shows a strong coupling between retinal and channelopsin-
2 and indicates that, most likely, other parts of the retinal cofactor
respond more strongly.
Our data show that the K-like state P500
1 is relatively stable and
that thermal relaxation only allows accumulating the long-lived
P480
4 intermediate. This finding is in contrast to bacteriorhodopsin,
COMPUTATIONAL BIOLOGY
for which K, L, and late M states could be generated in this way
BIOPHYSICS AND
(46). The observed differences imply that the energy barriers be-
tween these states are significantly lower in ChR2 compared with
bacteriorhodopsin. Furthermore, the detection of a new inter-
mediate Px under continuous illumination shows that this branch
of the photocycle indeed exists, because it is required to explain
the electrophysiological data for the WTs (40, 43) and mutants
with slow photocycles (17, 68).
Conclusion and Outlook
Here, we presented the first NMR study, to our knowledge, of
ChR2 using DNP-enhanced solid-state MAS NMR. Our data
show a pure all-trans,15-anti retinal Schiff base with a stretched
C14-C15 bond length and a significant out-of-plane twist of the
H-C14-C15-H dihedral angle in the ground state of ChR2. Three
different photostates could be generated using thermal relaxation/
trapping protocols, including a so-far unknown intermediate that
only occurs under continuous light conditions. Additional inter-
mediates will become accessible using ChR2 variants like C128T
and D156A with long-lived P520 3 and P390
2 states (68). Our data
provide novel insight into the photoactive site of ChR2 and show
that DNP-enhanced solid-state NMR fills the gap between func-
tional and X-ray–based structure analysis, which is required to
resolve its molecular mechanism. Additional studies using exten-
sively labeled retinals incorporated into isotope-labeled opsin for
more structural insight during channel-opening and -closing events
will be reported in the future.
Methods
15
N-ChR2 was expressed in Pichia pastoris, generated with 14–15-13C2–all-
trans retinal, and after purification, reconstituted into liposomes. Solid-
state MAS NMR was performed under cryogenic conditions (100 K) using
cross-effect DNP enhancement provided by doping the proteoliposomes with
AMUPOL and applying high-power microwave irradiation to the sample.
Trapping of the different photointermediates was achieved using protocols
combining illumination, freezing, and thermal relaxation. Optical data were
recorded under conditions that resembled the NMR samples as closely as
possible. SI Appendix has a detailed description of the applied methods.
ACKNOWLEDGMENTS. Oliver Ouari and Paul Tordo are acknowledged for
providing the polarizing agent AMUPOL. The work was funded by Deutsche
Forschungsgemeinschaft/Sonderforschungsbereich 807 Transport and Com-
munications across Membranes. The dynamic nuclear polarization experi-
ments were enabled through DFG Equipment Grant GL 307/4-1 and the
Cluster of Excellence Frankfurt: Macromolecular Complexes Frankfurt. Work
at the Center for Biomolecular Magnetic Resonance is supported by the
State of Hesse.
PNAS Early Edition | 5 of 6
 1. Nagel G, et al. (2002) Channelrhodopsin-1: A light-gated proton channel in green
algae. Science 296(5577):2395–2398.
2. Fenno L, Yizhar O, Deisseroth K (2011) The development and application of opto-
genetics. Annu Rev Neurosci 34(1):389–412.
3. Nagel G, et al. (2005) Light activation of channelrhodopsin-2 in excitable cells
of Caenorhabditis elegans triggers rapid Behavioral responses. Curr Biol 15(24):
2279–2284.
4. Liewald JF, et al. (2008) Optogenetic analysis of synaptic function. Nat Methods 5(10):
895–902.
5. Zhang F, et al. (2007) Multimodal fast optical interrogation of neural circuitry. Nature
446(7136):633–639.
6. Hegemann P, Moglich A (2011) Channelrhodopsin engineering and exploration of
new optogenetic tools. Nat Methods 8(1):39–42.
7. Boyden ES, Zhang F, Bamberg E, Nagel G, Deisseroth K (2005) Millisecond-timescale,
genetically targeted optical control of neural activity. Nat Neurosci 8(9):1263–1268.
8. Nagel G, et al. (2003) Channelrhodopsin-2, a directly light-gated cation-selective
membrane channel. Proc Natl Acad Sci USA 100(24):13940–13945.
9. Kato HE, et al. (2012) Crystal structure of the channelrhodopsin light-gated cation
channel. Nature 482(7385):369–374.
10. Bamann C, Kirsch T, Nagel G, Bamberg E (2008) Spectral characteristics of the pho-
tocycle of channelrhodopsin-2 and its implication for channel function. J Mol Biol
375(3):686–694.
11. Ritter E, Stehfest K, Berndt A, Hegemann P, Bartl FJ (2008) Monitoring light-induced
structural changes of Channelrhodopsin-2 by UV-visible and Fourier transform in-
frared spectroscopy. J Biol Chem 283(50):35033–35041.
12. Verhoefen MK, et al. (2010) The photocycle of channelrhodopsin-2: Ultrafast reaction
dynamics and subsequent reaction steps. ChemPhysChem 11(14):3113–3122.
13. Stehfest K, Ritter E, Berndt A, Bartl F, Hegemann P (2010) The branched photocycle of
the slow-cycling channelrhodopsin-2 mutant C128T. J Mol Biol 398(5):690–702.
14. Lórenz-Fonfría VA, et al. (2013) Transient protonation changes in channelrhodopsin-2
and their relevance to channel gating. Proc Natl Acad Sci USA 110(14):E1273–E1281.
15. Neumann-Verhoefen MK, et al. (2013) Ultrafast infrared spectroscopy on channelr-
hodopsin-2 reveals efficient energy transfer from the retinal chromophore to the
protein. J Am Chem Soc 135(18):6968–6976.
16. Radu I, et al. (2009) Conformational changes of channelrhodopsin-2. J Am Chem Soc
131(21):7313–7319.
17. Ritter E, Piwowarski P, Hegemann P, Bartl FJ (2013) Light-dark adaptation of chan-
nelrhodopsin C128T mutant. J Biol Chem 288(15):10451–10458.
18. Eisenhauer K, et al. (2012) In channelrhodopsin-2 Glu-90 is crucial for ion selectivity
and is deprotonated during the photocycle. J Biol Chem 287(9):6904–6911.
19. Kuhne J, et al. (2015) Early formation of the ion-conducting pore in channelrhodopsin-2.
Angew Chem Int Ed Engl 54(16):4953–4957.
20. Nack M, Radu I, Bamann C, Bamberg E, Heberle J (2009) The retinal structure of
channelrhodopsin-2 assessed by resonance Raman spectroscopy. FEBS Lett 583(22):
3676–3680.
21. Bruun S, et al. (2011) The chromophore structure of the long-lived intermediate of
the C128T channelrhodopsin-2 variant. FEBS Lett 585(24):3998–4001.
22. Krause N, Engelhard C, Heberle J, Schlesinger R, Bittl R (2013) Structural differences
between the closed and open states of channelrhodopsin-2 as observed by EPR
spectroscopy. FEBS Lett 587(20):3309–3313.
23. Sattig T, Rickert C, Bamberg E, Steinhoff HJ, Bamann C (2013) Light-induced move-
ment of the transmembrane helix B in channelrhodopsin-2. Angew Chem Int Ed Engl
52(37):9705–9708.
24. Oesterhelt D, Stoeckenius W (1971) Rhodopsin-like protein from the purple mem-
brane of Halobacterium halobium. Nat New Biol 233(39):149–152.
25. Lozier RH, Bogomolni RA, Stoeckenius W (1975) Bacteriorhodopsin: A light-driven
proton pump in Halobacterium Halobium. Biophys J 15(9):955–962.
26. Harbison GS, et al. (1984) Dark-adapted bacteriorhodopsin contains 13-cis, 15-syn and
all-trans, 15-anti retinal Schiff bases. Proc Natl Acad Sci USA 81(6):1706–1709.
27. Smith SO, Lugtenburg J, Mathies RA (1985) Determination of retinal chromophore
structure in bacteriorhodopsin with resonance Raman spectroscopy. J Membr Biol
85(2):95–109.
28. Stoeckenius W, Bogomolni RA (1982) Bacteriorhodopsin and related pigments of
halobacteria. Annu Rev Biochem 51(1):587–616.
29. Kamo N, Hazemoto N, Kobatake Y, Mukohata Y (1985) Light and dark adaptation of
halorhodopsin. Arch Biochem Biophys 238(1):90–96.
30. Pfleger N, Lorch M, Woerner AC, Shastri S, Glaubitz C (2008) Characterisation of Schiff
base and chromophore in green proteorhodopsin by solid-state NMR. J Biomol NMR
40(1):15–21.
31. Wang S, et al. (2013) Solid-state NMR spectroscopy structure determination of a lipid-
embedded heptahelical membrane protein. Nat Methods 10(10):1007–1012.
32. Janke C, et al. (2013) Photocycle and vectorial proton transfer in a rhodopsin from the
eukaryote Oxyrrhis marina. Biochemistry 52(16):2750–2763.
33. Yan B, Nakanishi K, Spudich JL (1991) Mechanism of activation of sensory rhodopsin I:
Evidence for a steric trigger. Proc Natl Acad Sci USA 88(21):9412–9416.
34. Kitajima-Ihara T, et al. (2008) Salinibacter sensory rhodopsin: Sensory rhodopsin I-like
protein from a eubacterium. J Biol Chem 283(35):23533–23541.
35. Hirayma J, Kamo N, Imamoto Y, Shichida Y, Yoshizawa T (1995) Reason for the lack of
light-dark adaptation in pharaonis phoborhodopsin: Reconstitution with 13-cis-reti-
nal. FEBS Lett 364(2):168–170.
6 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1507713112
36. Imamoto Y, et al. (1992) Chromophore configuration of pharaonis phoborhodopsin
and its isomerization on photon absorption. Biochemistry 31(9):2523–2528.
37. Scharf B, Hess B, Engelhard M (1992) Chromophore of sensory rhodopsin II from
Halobacterium halobium. Biochemistry 31(49):12486–12492.
38. Ernst OP, et al. (2014) Microbial and animal rhodopsins: Structures, functions, and
molecular mechanisms. Chem Rev 114(1):126–163.
39. Lórenz-Fonfría VA, et al. (2015) Pre-gating conformational changes in the ChETA
variant of channelrhodopsin-2 monitored by nanosecond IR spectroscopy. J Am Chem
Soc 137(5):1850–1861.
40. Nikolic K, et al. (2009) Photocycles of channelrhodopsin-2. Photochem Photobiol
85(1):400–411.
41. Lórenz-Fonfría VA, Heberle J (2014) Channelrhodopsin unchained: Structure and
mechanism of a light-gated cation channel. Biochim Biophys Acta 1837(5):626–642.
42. Nack M, et al. (2012) Kinetics of proton release and uptake by channelrhodopsin-2.
FEBS Lett 586(9):1344–1348.
43. Hegemann P, Ehlenbeck S, Gradmann D (2005) Multiple photocycles of channelrho-
dopsin. Biophys J 89(6):3911–3918.
44. Maly T, et al. (2008) Dynamic nuclear polarization at high magnetic fields. J Chem
Phys 128(5):052211.
45. Bajaj VS, Mak-Jurkauskas ML, Belenky M, Herzfeld J, Griffin RG (2009) Functional and
shunt states of bacteriorhodopsin resolved by 250 GHz dynamic nuclear polarization-
enhanced solid-state NMR. Proc Natl Acad Sci USA 106(23):9244–9249.
46. Mak-Jurkauskas ML, et al. (2008) Energy transformations early in the bacteriorho-
dopsin photocycle revealed by DNP-enhanced solid-state NMR. Proc Natl Acad Sci USA
105(3):883–888.
47. Mao J, et al. (2014) Structural basis of the green-blue color switching in proteo-
rhodopsin as determined by NMR spectroscopy. J Am Chem Soc 136(50):17578–17590.
48. Mehler M, et al. (2013) The EF loop in green proteorhodopsin affects conformation
and photocycle dynamics. Biophys J 105(2):385–397.
49. Jacso T, et al. (2012) Characterization of membrane proteins in isolated native cellular
membranes by dynamic nuclear polarization solid-state NMR spectroscopy without
purification and reconstitution. Angew Chem Int Ed Engl 51(2):432–435.
50. Ong YS, Lakatos A, Becker-Baldus J, Pos KM, Glaubitz C (2013) Detecting substrates
bound to the secondary multidrug efflux pump EmrE by DNP-enhanced solid-state
NMR. J Am Chem Soc 135(42):15754–15762.
51. Reggie L, Lopez JJ, Collinson I, Glaubitz C, Lorch M (2011) Dynamic nuclear polari-
zation-enhanced solid-state NMR of a 13C-labeled signal peptide bound to lipid-
reconstituted Sec translocon. J Am Chem Soc 133(47):19084–19086.
52. Sauvée C, et al. (2013) Highly efficient, water-soluble polarizing agents for dynamic
nuclear polarization at high frequency. Angew Chem Int Ed Engl 52(41):10858–10861.
53. Lansing JC, et al. (2002) Chromophore distortions in the bacteriorhodopsin photo-
cycle: Evolution of the H-C14-C15-H dihedral angle measured by solid-state NMR.
Biochemistry 41(2):431–438.
54. Hu J, Griffin RG, Herzfeld J (1994) Synergy in the spectral tuning of retinal pigments:
Complete accounting of the opsin shift in bacteriorhodopsin. Proc Natl Acad Sci USA
91(19):8880–8884.
55. Scherrer P, Mathew MK, Sperling W, Stoeckenius W (1989) Retinal isomer ratio in
dark-adapted purple membrane and bacteriorhodopsin monomers. Biochemistry
28(2):829–834.
56. Pettei MJ, Yudd AP, Nakanishi K, Henselman R, Stoeckenius W (1977) Identification of
retinal isomers isolated from bacteriorhodopsin. Biochemistry 16(9):1955–1959.
57. Friedrich T, et al. (2002) Proteorhodopsin is a light-driven proton pump with variable
vectoriality. J Mol Biol 321(5):821–838.
58. Imasheva ES, et al. (2005) Formation of a long-lived photoproduct with a de-
protonated Schiff base in proteorhodopsin, and its enhancement by mutation of
Asp227. Biochemistry 44(32):10828–10838.
59. Dioumaev AK, et al. (2002) Proton transfers in the photochemical reaction cycle of
proteorhodopsin. Biochemistry 41(17):5348–5358.
60. Vogeley L, et al. (2004) Anabaena sensory rhodopsin: A photochromic color sensor at
2.0 A. Science 306(5700):1390–1393.
61. Stehle J, et al. (2014) Characterization of the simultaneous decay kinetics of meta-
rhodopsin states II and III in rhodopsin by solution-state NMR spectroscopy. Angew
Chem Int Ed Engl 53(8):2078–2084.
62. Concistrè M, et al. (2009) Light penetration and photoisomerization in rhodopsin
studied by numerical simulations and double-quantum solid-state NMR spectroscopy.
J Am Chem Soc 131(17):6133–6140.
63. Daviso E, Diller A, Alia A, Matysik J, Jeschke G (2008) Photo-CIDNP MAS NMR beyond
the T1 limit by fast cycles of polarization extinction and polarization generation.
J Magn Reson 190(1):43–51.
64. Tomonaga Y, et al. (2011) An active photoreceptor intermediate revealed by in situ
photoirradiated solid-state NMR spectroscopy. Biophys J 101(10):L50–L52.
65. Xie AH (1990) Quantum efficiencies of bacteriorhodopsin photochemical reactions.
Biophys J 58(5):1127–1132.
66. Ames JB, et al. (1989) Bacteriorhodopsin’s M412 intermediate contains a 13-cis, 14-s-trans,
15-anti-retinal Schiff base chromophore. Biochemistry 28(9):3681–3687.
67. Rödig C, Chizhov I, Weidlich O, Siebert F (1999) Time-resolved step-scan Fourier
transform infrared spectroscopy reveals differences between early and late M in-
termediates of bacteriorhodopsin. Biophys J 76(5):2687–2701.
68. Bamann C, Gueta R, Kleinlogel S, Nagel G, Bamberg E (2010) Structural guidance
of the photocycle of channelrhodopsin-2 by an interhelical hydrogen bond.
Biochemistry 49(2):267–278.
Becker-Baldus et al.
 SUPPORTING
 INFORMATION
 
Enlightening
 the
 photoactive
 Site
 of
 channelrhodopsin-­‐2
 by
 DNP-­‐enhanced
 
 
solid-­‐state
 NMR
 spectroscopy
 
Johanna
 Becker-­‐Baldusa,b,
 Christian
 Bamannc,
 Krishna
 Saxenab,d,
 
 

Henrik  Gustmanne,  Lynda  J.  Brownf,  Richard  C.  D.  Brownf,  Christian  Reiterg,    

Ernst  Bambergc,  Josef  Wachtveitle,  Harald  Schwalbeb,d    

and  Clemens  Glaubitza,b*  

a)  Institute  of  Biophysical  Chemistry,  Goethe  University  Frankfurt,  Max-­‐von-­‐Laue-­‐Str.  9,  

60438  Frankfurt,  Germany.  
b)  Centre  for  Biomolecular  Magnetic  Resonance,  Goethe  University  Frankfurt,  Max-­‐von-­‐
Laue-­‐Str.  9,  60438  Frankfurt,  Germany.  
c)  Max-­‐Planck-­‐Institute  of  Biophysics,  Max-­‐von-­‐Laue-­‐Str.  3,  60438  Frankfurt,  Germany.  
d)  Institute  of  Organic  Chemistry  and  Chemical  Biology,  Goethe  University  Frankfurt,  Max-­‐
von-­‐Laue-­‐Str.  7,  60438  Frankfurt,  Germany.  
e)  Institute  of  Physical  and  Theoretical  Chemistry,  Goethe  University  Frankfurt,  Max-­‐von-­‐
Laue-­‐Str.  7,  60438  Frankfurt,  Germany.  
f)  Department  of  Chemistry,  University  of  Southampton,  Southampton  SO17  1BJ,  United  
Kingdom.  
g)  Bruker  Biospin  GmbH,  Silberstreifen  4,  76287  Rheinstetten,  Germany.  
 

(*)  Correspondence:     Institute  of  Biophysical  Chemistry  

Goethe  University  Frankfurt  
Max-­‐von-­‐Laue-­‐Str.  9  
60438  Frankfurt  
Germany  
Tel/Fax:   0049-­‐69-­‐798-­‐29927/29    
Email:     glaubitz@em.uni-­‐frankfurt.de  

           

   

  1  
(A)  Material  and  Methods  

Preparing  non-­‐labeled  and    15N-­‐labeled  ChR2  

Non-­‐labeled   ChR2   (amino   acids   1   to   315)   was   purified   as   described   before   from   membranes  
prepared  from  P.  pastoris  cultures  grown  in  complex  medium  without  retinal  (1).    

So   far,   isotope   enriched   ChR2   has   not   been   produced.   Therefore,   a   new   approach   had   to   be  
established   to   express   recombinant   15N-­‐labeled   ChR2   in   P.   pastoris.   Due   to   the   repeated   observation  
that   the   recombinant   production   of   this   protein   in   minimal   medium   with   stable   isotopes   led   to  
growth   arrest   of   P.   pastoris   we   developed   a   specific   expression   protocol   for   this   purpose.   While  
recombinant   ChRs   and   chimera   have   been   generated   in   cells   from   several   eukaryotic   species   like  
insect   cells   (2)   that   require   expensive   rich   media   for   isotopic   labeling   this   is   the   first   report   of   stable-­‐
15 15
isotope   N   labeled   ChR2   expression.   For   uniformly   N   labeled   ChR2   production,   cells   were  
precultured   in   buffered   minimal   glycerol   medium   BMGS   supplemented   with   the   Trace   metal   solution  
and   a   vitamin   solution   (10   µM   thiamine,   10   µM   NAD,   10   µM   vitamin   B12   (3)):     1.34%   yeast  
nitrogen  base   without  amino  acid,  0.00004%  biotin,  1%  glycerol,  0.1  M  phosphate   buffer  at  pH  6¸  20  
mL  culture  in  a  100  mL  baffled  flask)  containing  10  g/L  (14NH4)2SO4  as  the  sole  nitrogen  source  at  30  
°C,   200   rpm   (Infors   multitron   shaker,   shaking   hub   50   mm),   until   an  OD600   of   5-­‐10   was   reached.  
Adaptation  of  the  cells  from  14N  to  (15NH4)2SO4  (Eurisotop,  Saarbrücken,  Germany)  as  nitrogen  source  
was   performed   by   diluting   (1:20)   the   cells   to   OD600   of   0.3   by   addition   of   fresh   15N   BMG   medium.   This  
culture  was  grown  to  OD600  of  7-­‐12  at  30   oC  and  200  rpm  (50  ml  in  in  1-­‐L  baffled  flask).  Cells  were  
subsequently  diluted  to  OD600   of  0.3  by  addition  of  fresh   15N  BMG  medium  (final  volume  1L  in  a  5  L  
baffled   flask),   incubated   at   30   °C,   150   rpm   until   an  OD600   of   12-­‐14   was   reached   and   harvested   by  
centrifugation  at  3000  g  for  10  min  at  room  temperature.  Target  protein  expression  was  induced  by  
carefully  resuspending  the  pellet  in  12  L  of  induction  medium  BMMS  (BMGS  with  glycerol  replaced  by  
5  mL/L  methanol)  with  a  starting  OD600  of  2-­‐3  in  5-­‐L  baffled  flasks.  Cultures  were  incubated  at  30°C  
with  shaking  (140  RPM)  for  5  -­‐7  days  and  methanol  (5  ml  MeOH  plus  50  ml  water)  was  added  every  
24   hours   to   a   final   concentration   of   0.5%   provided   that   the   cultures   did   grow.   Finally,   cells   were  
pelleted  at  3000  g  for  10  min  and  subsequently  used  for  membrane  preparation  (Fig.  S6).  

ChR2  reconstituted  with  14-­‐15-­‐13C2-­‐all-­‐trans  retinal    

For   reconstitution   with   the   isotope  labeled   or   unlabeled   retinal   compounds,   we   incubated   the   crude  
membrane   preparation   with   5   µM   retinal   for   2   h   on   ice   before   starting   the   solubilization   with   1   %  
[m/v]   β-­‐decyl-­‐maltoside   (DM).   14,15-­‐13C2-­‐retinal   was   synthesized   as   described   before   (4).   The   final  
samples  were  concentrated  in  20  mM  Hepes,  pH  7.4,  100  mM  NaCl,  0.15%  DM.  

  2  
Reconstitution  in  proteoliposomes  

ChR2   was   reconstituted   into   proteoliposomes   prepared   from   a   mixture   of   POPC:POPG:cholesterol  

[8:1:1,   m/m/m].   The   lipids   were   dissolved   in   2%   cholate   before   adding   the   protein   to   a   final  
lipid:protein   ratio   of   3:1   [m/m].   The   detergent   was   extracted   by   several   addition   of   BioBeads   over  
the  time  course  of  42  h  at  4  °C.  

Samples  for  DNP-­‐enhanced  MAS  NMR  

14,15-­‐13C2-­‐retinal-­‐15N-­‐ChR2   proteoliposoms,   containing   1.8   mg   protein,   where   pelleted   by  

ultracentrifugation   for   an   hour.   88   µl   of   DNP   buffer   (20   mM   AMUPOL   (5),   Fig   1),   30   v/v%   d-­‐8-­‐
Glycerol,  60  v/v%  D2O,  10  v/v%  H2O)  was  carefully  layered  above  the  pellet  and  incubated  over  night.  
Then  the  supernatant  was  removed  and  the  sample  was  divided  into  two  parts.  The  larger  fraction  
was  packed  into  a  standard  3.2  mm  ZrO2  rotor  and  the  smaller  one  into  a  3.2  mm  sapphire  rotor.  The  
sample  in  the  sapphire  rotor  was  then  spun  in  an  NMR  probe  with  a  few  kHz  at  room  temperature  to  
distribute   the   sample   on   the  inner   surface   of   the   rotor   to   optimize   light   penetration   into   the   sample.  
From  the  relative  signal  intensities  it  was  judged  that  sample  in  the  sapphire  rotor  contained  20%  of  
the  sample  packed  in  the  ZrO2  rotor,  which  correspond  to  0.3  mg  and  1.5  mg  of  protein,  respectively.    

DNP  enhanced  MAS  NMR  and  illumination  

DNP   enhanced   MAS   NMR   spectra   were   recorded   on   a   Bruker   400   DNP   system   consisting   of   a   400  
MHz  WB  Avance  II  NMR  spectrometer,  a  263  GHz  Gyrotron  as  microwave  source  and  a  3.2mm  HCN  
Cryo  MAS  probe.  All  experiments  were  conducted  with  8  kHz  MAS  and  the  microwave  power  at  the  
probe   was   10.5   W.   During   DNP   experiments   the   temperature   was   kept   at   around   110   K.   Referencing  
for  13C  and  15N  was  done  indirectly  to  DSS  using  the  low  field  13C-­‐signal  of  adamantane  at  40.49  ppm.  
For  all  experiments  100  kHz  decoupling  using  SPINAL-­‐64  (6)  was  applied  during  acquisition.  

13
C  and   15N  CP  experiments  were  recorded  using  ramped  CP  from   1H  to   13C  during  0.8  ms.  The   15N-­‐
detected  double  CP  experiment  was  performed  using  a  6  ms  specific  CP  (7)  step.  13C-­‐double  quantum  
filter   experiments   (DQF-­‐experiments)   were   obtained   using   the   POST-­‐C7   (8)   sequence   for   double  
quantum  excitation  and  reconversion.  By  varying  the  number  of  excitation  and  reconversion  blocks  
the   signal   intensity   in   the   double   quantum   filtered   spectra   could   be   optimized   or   the   full   double  
quantum  build-­‐up  curve  was  recorded.  Using  the  isolated  C15  signal,  the  double  quantum  efficiency  
was   estimated   to   be   55%.   For   HCCH   torsional   angle   measurements   a   double   quantum   heteronuclear  
local   field   experiment   (9)   was   applied   (HCCH-­‐experiment).   In   the   experiment   POST-­‐C7   during   two  
rotor  periods  was  used  for  double  quantum  excitation  and  reconversion,  PMLG-­‐9  (10)  at  106.2  kHz  
was   used   for   homonuclear   1H   decoupling   and   1H   CW   irradiation   at   106.2   kHz   was   used   during   the  
constant   time   periods.   All   experiments   were   recorded   with   a   recycle   delay   of   3   s.   Spectra   in   (Fig.   2b)  

  3  
were  recorded  with  128  scans  except  for  CP  at  RT  for  which  106496  scans  were  required.  For  each  
data   point   in   (Fig.   2c)   and   (Fig.   2d),   spectra   with   768   and   2048   scans   were   recorded,   respectively.  
Spectra  in  Fig.  3  were  recorded  with  128  and  24576  scans,  for  the  CP  and  DCP  spectra,  respectively.  
Number   of   scans   in   Fig.   4   were   8192   (13C-­‐DQF,   dark   and   illuminated),   16385   (15N   DCP,   dark)   and  
24576   (15N   DCP,   illuminated).   All   spectra   in   Fig.   5   were   recorded   with   8192   scans.   Illumination   was  
done  with  a  cold  light  lamp  (Zeiss,  KL  1500)  using  a  blue  filter  (width  400-­‐480  nm,  maximal  intensity  
460   nm).   Two   methods   of   illumination   were   tested.   Illumination   outside   of   the   NMR   magnet   was  
carried   out   by   fixing   the   NMR   rotor   at   its   cap   and   subjecting   it   to   a   stream   of   cold   nitrogen   gas.  
Illumination  was  then  switched  on  for  10  min,  then  the  lamp  was  positioned  on  the  opposite  side  of  
the  rotor  and  another  10  min  illumination  was  applied.  Then  the  lamp  was  switched  off  and  the  rotor  
was  kept  close  to  liquid  nitrogen  temperatures  during  transport  to  the  magnet.  Then  the  cold  rotor  
was  inserted  into  the  precooled  NMR  probe  (100  K).  

Later   the   probe   was   equipped   with   a   light   guide   illuminating   the   spinning   rotor   from   the   walls  
through   the   openings   of   the   coil.   Illumination   was   done   for   10-­‐30   min.   Both   illumination   protocols  
give   indistinguishable   results   whereas   the   latter   method   is   easier   to   handle,   allows   better  
temperature   control   but   does   not   enable   for   rapid   freezing,   as   the   whole   probe   has   to   be   cooled  
down.  

For  thermal  relaxation  experiments,  as  described  by  Mak-­‐Jurkauskas  et  al.  (11),  the  temperature  of  
the   spinning   sample   was   changed   after   illumination   at   110   K   by   changing   the   temperature   of   the  
bearing,   drive   and   variable   temperature-­‐gas   flows.   Once   the   desired   temperature  had   been   reached,  
the   sample   was   kept   there   for   10   min   and   then   cooled   down   to   around   110   K   for   performing   the  
DNP-­‐enhanced  MAS  NMR  experiments.  

For   experiments   with   illumination   at   higher   temperatures   (thermal   trapping)   the   rotor   was   either  
illuminated  at  the  desired  temperature  and  then  quickly  frozen  and  inserted  into  the  cold  probe  or  
the  sample  was  spun  in  the  MAS  probe  at  around  8  kHz  and  illuminated  at  the  chosen  temperature.  
Then  the  spinning  sample  was  cooled  down  to  110  K  for  recording  of  the  DNP-­‐enhanced  MAS  NMR  
spectra.  

Ambient  temperature  experiments  

14,15-­‐13C2-­‐retinal-­‐15N-­‐ChR2  proteoliposomes  were  pelleted  by  ultracentrifugation  for  one  hour.  The  
obtained   wet   pellet   was   transferred   to   a   3.2   mm   MAS   rotor.   13C   CP   spectra   were   recorded   on   a  
Bruker  850MHz  WB  Avance  III  NMR  spectrometer  equipped  with  a  Bruker  3.2  mm  HCN  MAS  probe.  
The   nominal   temperature   set   was   270   K,   the   temperature   inside  the   rotor   is   estimated   to   be   10-­‐20   K  
higher.   13C   polarization   was   generated   by   a   ramped   CP   from   1H   to   13C   during   1   ms   and   100   kHz  

  4  
decoupling   using   SPINAL-­‐64   (6)   was   employed   during   acquisition.   The   recycle   delay   was   3   s.  
Referencing   was   done   indirectly   to   DSS   using   the   low-­‐field   13C-­‐signal   of   adamantane   at   40.49   ppm.  
The  spectra  at  an  MAS  rate  of  13  kHz  and  16.6  kHz  were  recorded  with  106496  transients  each.  

Data  Analysis  and  Simulations  

All  spectra  were  analyzed  and  if  appropriate  integrated  using  TOPSPIN  2.1  (Bruker).  If  appropriate  the  
signals   of   the   C14   and   C15   atoms   where   added   and   analyzed   together.   Deconvolution   was   applied  
prior  to  integration  for  overlapped  signals.  

Fitting   of   DQ   build-­‐up   data:   DQ   build-­‐up   data   were   simulated   with   SIMPSON   using   an   input   file  
adapted  from  an  example  by  Bak  2000  et  al.  (12).  CSA  parameters  where  taken  from  Smith  et  al.  (13).  
DQ  build-­‐up  curves  where  calculated  with  varying  13C-­‐13C  dipolar  couplings.  The  obtained  curves  were  
fitted  to  the  experimental  data  by  multiplying  them  with  a  mono  exponential  decay  function  and  the  
13
C-­‐13C  dipolar  couplings  was  then  obtained  from  the  best  fitting  curve.  

The   correct   performance   of   the   DQ   build-­‐up   experiments   was   validated   using   2,3-­‐13C2-­‐disodium  
fumarate  (Sigma  Aldrich).  Data  were  recorded  at  100  K  using  the  same  parameters  as  for  ChR2  but  
without   microwave   irradiation   and   with   a   recycle   delay   of   50   s.   The   spin   system   used   for   the  
SIMPSON  calculation  was  taken  from  Carravetta  et  al.,  who  used  2,3-­‐13C2-­‐diammonium  fumarate  and  
determined  a  bond  length  of  1.345  ±  0.013  Å  (14).    Experimental  data  and  simulations  are  shown  in  
Fig.  S7.  The  obtained  distance  of  1.37  Å  compares  well  within  the  experimental  error  (±0.025Å)  with  
the   previously   reported   NMR   and   X-­‐ray   data   (14,   15).   Small   deviations   are   caused   by   the  
temperature  difference  (100  K  vs.  RT),  the  usage  of  a  slightly  different  compound  (2,3-­‐13C2-­‐disodium  
fumarate  vs.  2,3-­‐13C2-­‐diammonium  fumarate),  which  could  result  in  altered  CSA  tensor  orientations  
and   different   DQ   excitation   schemes.   It   should   be   noted   that   the   exact   distance   determination  
depends   on   the   knowledge   of   the   Euler   angles   of   the   involved   CSA   and   dipolar   tensors.   As   these  
angles   are   unknown   for   our   retinal   compound   a   systematic   error   remains.   In   addition,   distances  
determined   by   this   method   using   solid   state   NMR   are   generally   found   to   be   slightly   larger   compared  
to   what   is   observed   by   X-­‐ray   crystallography   (14).   It   can   be   concluded   that   the   absolute   distances  
determined  contain  a  larger  uncertainty  than  when  comparing  distance  differences  using  the  same  
method  as  done  here.  However,  the  deviations  are  estimated  to  be  within  the  numerical  error.  

Fitting   of   HCCH   torsional   angle   data:   The   data   was   simulated   with   SIMPSON   and   the   input   file   was  
adapted  from  Mao  et  al  (16).  The  spin  system  was  calculated  based  on  a  1H-­‐13C  distance  of  1.13  Å  (17,  
18),  a   13C-­‐13C  distance  as  obtained  from  the  DQ  build-­‐up  data  of  1.51  Å  and  a  CHH  angle  of  115°  (17,  
19).  The  CH  dipolar  coupling  was  then  multiplied  with  the  PMLG  scaling  factor  of  0.57  as  the  PMLG  
part  was  not  explicitly  simulated.  The  obtained  curves  were  then  fitted  to  the  experimental  data  by  

  5  
multiplying  them  with  a  mono  exponential  decay  function  and  the   1H-­‐13C-­‐13C-­‐1H  torsional  angles  was  
then  obtained  from  the  best  fitting  curve.  

Validation  of  the  HCCH  experiment  was  done  using  2,3-­‐13C2-­‐disodium  fumarate  (Sigma  Aldrich).  The  
experiment  was  recorded  at  100  K  using  the  same  parameters  as  for  the  ChR2  samples  but  without  
microwave.   The   obtained   angle   of   180°   (Fig.   S8)   agrees   perfectly   with   the   dihedral   angle   in   the  
fumarate   anion   and   with   previous   experimental   data   (20-­‐22).     The   spin   system   for   the   calculations  
was  based  on  standard  values  as  used  by  Feng  et  al.  (23).    

UV/vis  Spectroscopy  under  cryogenic  conditions  

UV/vis-­‐difference  spectra  were  recorded  with  the  help  of  a  fiber-­‐optic  spectrometer  (Ocean  Optics,  
USB2000+).   Probe   light   was   provided   by   a   balanced   deuterium   halogen-­‐source   (Ocean   Optics,   DH-­‐
2000-­‐BAL).  The  sample  temperature  was  controlled  with  the  help  of  a  liquid  nitrogen  cooled  cryostat  
(Oxford   Instruments,   OptistatDN).   For   illumination   a   cold   light   lamp   (Zeiss,   KL   1500)   with   a   blue   filter  
(width   400-­‐480   nm,   maximal   intensity   460   nm)   was   coupled   into   the   cryostat   and   the   sample   was  
irradiated  for  10-­‐20  minutes  at  the  target  temperature.      

The   sample   was   prepared   in   a   shortened   1x10   mm   quartz   cuvette   (Hellma,   100-­‐QS).   For  
cryoprotection  and  reduction  of  light  scattering  60%vol  glycerol  (Sigma-­‐Aldrich,  spectrophotometric  
grade)  was  added  to  the  proteoliposomes.  

All  spectra  where  recorded  at  150  K  and  subtracted  from  a  dark  spectrum  recorded  before  one  of  the  
following   illumination   protocols:   a)   The   sample   was   illuminated   for   20   min   at   150   K   (Fig.     4).   b)   A  
thermal  relaxation  experiment  where  the  sample  illuminated  as  in  a)  and  then  heated  up  to  245  K.  
The   sample   was   kept   at   this   temperature   for   10   min.   Then   it   was   again   cooled   to   150   K   for   recording  
the  UV/vis  spectrum  (Fig.    S4).  c)  The  sample  was  heated  to  245  K  and  illuminated  for  10  min.  Finally;  
it  was  cooled  without  illumination  to  150  K  for  recording  the  UV/vis  spectrum  (Fig.    S4).  

The   spectra   were   measured   with   an   integration   time   of   100   ms   and   400   scans   were   averaged.   As  
reference   for   the   absorption   spectra   the   empty   cryostat   was   used.   To   correct   the   influence   of  
scattered  light,  all  spectra  were  baseline  corrected  (OriginLab,  OriginPro  9.0.0G).  After  subtraction  of  
the   dark   spectrum,   the   resulting   difference   spectra   were   smoothed   with   the   help   of   a   moving  
average  over  5  points.  

  6  
  

(B)  C14  and  C15  Chemical  Shifts  of  ChR2,  Bacteriorhodopsin  and  Proteorhodopsin    

Table  S1:  Chemical  shifts  and  absorption  maxima  of  different  retinal  proteins.  All  chemical  shifts  
were  determined  at  around  100  K  under  DNP  conditions  if  not  otherwise  mentioned.  
15
  C14   C15   N  pSB   λ max   Conformation  
[ppm]   [ppm]   [ppm]   [nm]  
ChR2   126.3   166.5   196.5   470   all-­‐trans,  15-­‐anti  
Bacteriorhodopsin  light  adapted  (11,  24)     123.1   160.0   165.2   568   all-­‐trans,  15-­‐anti  
Bacteriorhodopsin,  dark  adapted  (11,  24)     123.1   160.0   165.2   568   all-­‐trans,  15-­‐anti  
111.0   163.2   173.5   555   13-­‐cis,  15-­‐syn  
Proteorhodopsin  (4)     120.2   161.1   182.0   520   all-­‐trans,  15-­‐anti  
Proteorhodopsin  A178R  (4)   122.1   160.7   182.1   533   all-­‐trans,  15-­‐anti  
110.7   165.6   -­‐   13-­‐cis,  15-­‐syn  
 

(C)  Comparison  to  previously  reported  data  from  vibrational  spectroscopy    

Resonance  Raman  experiments  are  non-­‐invasive  and  should  give  reliable  information  on  the  retinal  
chromophore.  However,  assignment  of  the  vibrational  bands  is   very  challenging  and  cannot  easily  be  
transferred   between   different   systems.   In   dark   adapted   Bacteriorhodopsin   a   C14H   out-­‐of-­‐plane  
wagging  vibration  at  800  cm-­‐1  is  detected,  which  vanishes  after  light  adaption  and  is  a  marker  band  
for  the  13-­‐cis  conformer  (25).  However,  this  marker  band  is  covered  in  resonance  Raman  spectra  of  
ChR2   by   lipid   or   detergent   signals.   Thus   the   ChR2   finger   print   region   of   the   resonance   Raman  
spectrum   was   analyzed   in   analogy   to   other   retinal   proteins   (26).   Assignment   of   the   vibrational   bands  
in  the  finger  print  regions  is  not  straightforward  and  usually  needs  a  rigorous  assignment  based  on  
differently   isotope   labeled   retinals   as   done   for   bacteriorhodopsin   (27).   To   our   knowledge,   such   an  
assignment   is   missing   for   ChR2   and   therefore   the   interpretation   of   the   resonance   Raman   data  
remains  ambiguous.  In  contrast  solid  state  MAS  NMR  can  easily  distinguish  between  the  numbers  of  
conformers  present  in  the  functional  protein  by  simply  counting  the  NMR  signals.    

The  data  in  Fig.  S4.  show  that  the  C14-­‐C15  bond  stretching  and  twisting  is  conserved  in  the  trapped  
states  within  the  experimental  error  limits.  At  first  glance,  some  changes  in  the  K-­‐  and  M-­‐like  states  
might   be   expected.     So   far,   no   comparable   K-­‐state   NMR   data   have   been   reported   for   other   retinal  
proteins   such   as   bacteriorhodopsin   or   proteorhodopsin.   Raman   data   however   suggested   for  
bacteriorhodopsin   a   non-­‐quantified   bond   twisting   based   on   the   occurrence   of   a   “hydrogen-­‐out-­‐of-­‐
plane  (HOOP)”-­‐band  assigned  to  the  proton  bound  to  C15  (28,  29).  This  band  disappears  already  in  
the   L-­‐state.   On   the   other   hand,   solid-­‐state   NMR   experiments   on   bacteriorhodopsin   have   shown,   that  
the  bond  is  already  twisted  in  the  ground  state  and  its  out-­‐of-­‐plane  orientation  increases  in  the  M-­‐
state   (22).   In   case   of   ChR2,   HOOP   bands   (986   cm-­‐1)   have   been   reported,   which   were   assigned   and  

  7  
interpreted   based   on   bacteriorhodopsin   data   (30).   It   is   currently   unresolved   how   these   vibrational  
bands   relate   to   the   H-­‐C14-­‐C15-­‐H   out-­‐of-­‐plane   twist   directly   observed   by   solid-­‐state   NMR.   Schiff   base  
deprotonation   in   the   M-­‐state   should   also   affect   the   C14-­‐C15   bond.   Indeed,   solid-­‐state   NMR   has  
shown  for  bacteriorhodopsin  that  the  H-­‐C14-­‐C15-­‐H  torsion  angle  changes  from  164°  to  150°  (22).  In  
our   case   however,   no   data   for   the   ChR2   M-­‐like   state   could   be   recorded,   as   this   state   could   not   be  
trapped.        

(D)  Supporting  Data  

 
 

Fig.     S1:   Room   temperature   control   spectra   of   14,15-­‐13C2-­‐retinal-­‐ChR2   recorded   on   a   850   MHz  
spectrometer   using   13.0   kHz   and   16.6   kHz   MAS,   respectively.   All   small   signals   around   110   ppm  
originate   from   spinning   side   bands   and   no   13-­‐cis,15-­‐syn   chromophore   conformation   is   detectable.  
The   retinal   resonances   C14   (125.2   ppm)   and   C15   (166.2   ppm)   are   very   similar   to   those   recorded  
under   DNP   conditions.   The   small   differences   stem   from   temperature   effects.   The   experiment  
confirms   that   the   retinal   within   ChR2   exists   in   purely   all-­‐trans,15-­‐anti   conformation.   Some   of   the  
signals   detected   in   the   DNP-­‐spectra   in   Fig.   2   between   130   and   150   ppm   are   not   observed   here   at  
room   temperature   due   the   higher   lipid   dynamics   resulting   in   much   reduced   spinning   side   band  
signals   and   due   to   aromatic   side   chain   dynamics   which   either   lead   to   line   broadening   due   to  
intermediate  motion  and  reduced  cross  polarization  efficiencies.    

  8  
  
 

Fig.   S2:   DNP   enhanced   13C   DQF   spectra   of   ChR2   recorded   immediately   after   preparation   and   again  
after  storage  at  4°C  for  24  h.  Identical  signals  are  observed  in  both  spectra  showing  no  dark  adaption.  

  9  
  
Fig.     S3:   N-­‐DCP   filtered   spectra   of   ChR2   in   different   states.   (a)   Ground   state   ChR2470   with   a   pSB  
15

chemical   shift   at   196.5   ppm.   (b)   Spectra   obtained   using   the   thermal   relaxation   protocol   (see   Fig.   5a).  
A   resonance   similar   to   ChR2470   is   detected.   In   order   to   probe   whether   deprotonated   Schiff   base  
species   would   occur   around   300   ppm,   a   second   spectrum   with   shifted   spectrometer   offset   was  
recorded.   No   indication   for   a   deprotonated   species   was   found.   (c)   Spectra   obtained   using   the  
thermal  trapping  protocol  (see  Fig.  5b).  The  ground  state  signal  at  196.5  ppm  is  depleted  and  a  new  
signal   occurs   at   185   ppm,   which   is   not   visible   under   thermal   relaxation   conditions   and   which   is  
therefore  assigned  to  the  Px  state.  No  peak  could  be  identified  for  the  P4480  state,  which  might  be  due  
to  severe  line  broadening.  This  could  be  caused  by  an  ensemble  of  many  different  interactions  the  
Schiff  base  might  be  involved  in  during  this  long-­‐lived  state.    
 
 
 

  10  
  
Fig.     S4:   Optical   difference   absorption   spectra   of   ChR2   samples   similar   to   those   used   for   DNP-­‐
enhanced  MAS-­‐NMR  acquired  after  subjecting  the  samples  to  the  thermal  relaxation  and  the  thermal  
trapping  protocols  at  245  K.  The  difference  spectrum  of  ground  state  and  the  245  K  thermally  relaxed  
state  shows  some  ground  state  bleaching  and  increase  of  a  red  shifted  intermediate.  This  is  in  good  
agreement  with  the  corresponding  NMR  spectra,  which  contain  a  large  ground  state  signal  for   13C14  
and  a  smaller  signal  for  P4480.  In  contrast,  the  optical  difference  spectrum  of  ground  state  and  the  245  
K   thermally   trapped   state   shows   significant   differences.   The   large   ground   state   bleaching   agrees   well  
with   the   ground   state   depopulation   observed   by   solid   state   NMR   spectroscopy.   In   addition,   a  
pronounced  signal  maximum  is  observed  at  500  nm.  Thus,  the  optical  data  confirms  that  the  thermal  
relaxation   and   the   thermal   trapping   protocols   at   245   K   result   in   different   population   of   photo  
intermediates.   These   data   confirm   that   the   thermal   relaxation   and   the   thermal   trapping   protocols   at  
245  K  result  in  different  population  of  photo  intermediates.  

   

  11  
  
 

 
 

Fig.    S5:  a-­‐c)   13C14-­‐13C15  double  quantum  build-­‐up  curves  and  d-­‐f)  H13C14-­‐H13C15  dipolar  evolution  
curves  recorded  for  differently  trapped  ChR2  states:  a+d)  P1500,  K-­‐like  state,  from  the  deconvoluted  
13
C14   signal   as   seen   in   Fig.   4b)     b+e)   P4480,   from  the  deconvoluted   13C14   signal   as   seen   at   245   K   in   Fig.  
5a)  c+f)  Px,  from  the  deconvoluted   13C14  signal  as  seen  at  245  K  with  blue  light  in  Fig.  5b).  Our  data  
show   that   the   observed   stretching   and   twisting   of   the   C14-­‐C15   bond   remains   unchanged   in   our  
trapped   states   within   the   experimental   error.   Further   discussions   are   found   in   the   main   text   in   in  
section  (C)  of  the  supporting  information.    

  12  
  
 

Fig.   S6:   Purification   of   15N-­‐labeled   ChR2.   A)   Elution   profile   of   IMAC   (Immobilized   metal   ion   affinity  
chromatography).  B)  SDS-­‐PAGE  of  IMAC  elution  fractions  with  a  molecular  weight  reference  marker  
(kDa)  on  the  left.  

  13  
  

 
13
Fig.  S7:  DQ  build-­‐up  experiment  and  SIMPSON  simulations  for  2,3-­‐ C2-­‐disodium  fumarate.  Data  were  
recorded   under   conditions   identical   to   those   used   for   the   ChR2   experiments   except   for   microwave  
irradiation.   Due   to   a   very   long   1H-­‐T1,   the   recycle   delay   time   had   to   be   set   to   50   s.   The   best   fit   is  
obtained   2950   ±   150   Hz,   which   is   in   good   agreement   with   the   expected   bond   length.   An   empirical  
mono-­‐exponential   damping   function   and   baseline   correction   was   used   to   describe   the   relaxation  
decay.  

Fig.   S8:   HCCH   dephasing   curve   for   2,3-­‐13C2-­‐disodium   fumarate   recorded   under   conditions   identical   to  
those  used  for  the  ChR2  experiments  except  for  microwave  irradiation.  The  data  agree  perfectly  well  
with  simulations  assuming  the  expected  180°  H-­‐C-­‐C-­‐H  torsion  angle  of  this  compound.  

  14  
References  

1.   Bamann  C,  Gueta  R,  Kleinlogel  S,  Nagel  G,  &  Bamberg  E  (2010)  Structural  guidance  of  the  
photocycle  of  channelrhodopsin-­‐2  by  an  interhelical  hydrogen  bond.  Biochemistry  49(2):267-­‐
278.  
2.   Kato  HE,  et  al.  (2012)  Crystal  structure  of  the  channelrhodopsin  light-­‐gated  cation  channel.  
Nature  482(7385):369-­‐374.  
3.   Studier  FW  (2005)  Protein  production  by  auto-­‐induction  in  high  density  shaking  cultures.  
Protein  Expr  Purif  41(1):207-­‐234.  
4.   Mehler  M,  et  al.  (2013)  The  EF  loop  in  green  proteorhodopsin  affects  conformation  and  
photocycle  dynamics.  Biophys.  J.  105(2):385-­‐397.  
5.   Sauvee  C,  et  al.  (2013)  Highly  efficient,  water-­‐soluble  polarizing  agents  for  dynamic  nuclear  
polarization  at  high  frequency.  Angew.  Chem.  Int.  Ed.  Engl.  52(41):10858-­‐10861.  
6.   Fung  BM,  Khitrin  AK,  &  Ermolaev  K  (2000)  An  improved  broadband  decoupling  sequence  for  
liquid  crystals  and  solids.  J.  Magn.  Reson.  142(1):97-­‐101.  
7.   Baldus  M,  Petkova  AT,  Herzfeld  J,  &  Griffin  RG  (1998)  Cross  polarization  in  the  tilted  frame:  
assignment  and  spectral  simplification  in  heteronuclear  spin  systems.  Mol.  Phys.  95(6):1197-­‐
1207.  
8.   Hohwy  M,  Jakobsen  HJ,  Edén  M,  Levitt  MH,  &  Nielsen  NC  (1998)  Broadband  dipolar  
recoupling  in  the  nuclear  magnetic  resonance  of  rotating  solids:  A  compensated  C7  pulse  
sequence.  J.  Chem.  Phys.  108(7):2686.  
9.   Concistre  M,  et  al.  (2012)  A  large  geometric  distortion  in  the  first  photointermediate  of  
rhodopsin,  determined  by  double-­‐quantum  solid-­‐state  NMR.  J.  Biomol.  NMR  53(3):247-­‐256.  
10.   Vinogradov  E,  Madhu  PK,  &  Vega  S  (1999)  High-­‐resolution  proton  solid-­‐state  NMR  
spectroscopy  by  phase-­‐modulated  Lee-­‐Goldburg  experiment.  Chem.  Phys.  Lett.  314(5-­‐6):443-­‐
450.  
11.   Mak-­‐Jurkauskas  ML,  et  al.  (2008)  Energy  transformations  early  in  the  bacteriorhodopsin  
photocycle  revealed  by  DNP-­‐enhanced  solid-­‐state  NMR.  Proc.  Natl.  Acad.  Sci.  USA  
105(3):883-­‐888.  
12.   Bak  M,  Rasmussen  JT,  &  Nielsen  NC  (2000)  SIMPSON:  A  general  simulation  program  for  solid-­‐
state  NMR  spectroscopy.  J.  Magn.  Reson.  147(2):296-­‐330.  
13.   Smith  SO,  et  al.  (1989)  Structure  and  protein  environment  of  the  retinal  chromophore  in  
light-­‐  and  dark-­‐adapted  bacteriorhodopsin  studied  by  solid-­‐state  NMR.  Biochemistry  
28(22):8897-­‐8904.  
14.   Carravetta  M,  et  al.  (2001)  Estimation  of  carbon-­‐carbon  bond  lengths  and  medium-­‐range  
internuclear:  Distances  by  solid-­‐state  nuclear  magnetic  resonance.  J.  Am.  Chem.  Soc.  
123(43):10628-­‐10638.  
15.   Gupta  MP  &  Sahu  RG  (1970)  The  Crystal  Structure  of  Sodium  Hydrogen  Fumarate.  Acta.  
Crystallogr.  B  B26:1964-­‐1968.  
16.   Mao  J,  et  al.  (2014)  Structural  Basis  of  the  Green–Blue  Color  Switching  in  Proteorhodopsin  as  
Determined  by  NMR  Spectroscopy.  J.  Am.  Chem.  Soc.  136(50):17578-­‐17590.  
17.   Feng  X,  et  al.  (2000)  Determination  of  a  molecular  torsional  angle  in  the  metarhodopsin-­‐I  
photointermediate  of  rhodopsin  by  double-­‐quantum  solid-­‐state  NMR.  J.  Biomol.  NMR  
16(1):1-­‐8.  
18.   Nakai  T,  Ashida  J,  &  Terao  T  (1989)  Influence  of  small-­‐amplitude  motions  on  two-­‐dimensional  
N.M.R.  powder  patterns.  Mol.  Phys.  67(4):839-­‐847.  
19.   Hamanaka  T,  Mitsui  T,  Ashida  T,  &  Kakudo  M  (1972)  The  crystal  structure  of  all-­‐trans  retinal1.  
Acta  Crystallogr.  Sect.  B:  Struct.  Sci.  28(1):214-­‐222.  
20.   Feng  X,  et  al.  (2000)  Determination  of  a  molecular  torsional  angle  in  the  metarhodopsin-­‐I  
photointermediate  of  rhodopsin  by  double-­‐quantum  solid-­‐state  NMR.  J.  Biomol.  NMR  
16(1):1-­‐8.  
21.   Hosomi  H,  Ito  Y,  &  Ohba  S  (1998)  Ammonium  and  Isopropylammonium  Salts  of  the  Fumaric  
Acid  Dianion.  Acta  Crystallogr.  Sect.  C-­‐Cryst.  Struct.  Commun.  54(1):142-­‐145.  

  15  
22.   Lansing  JC,  et  al.  (2002)  Chromophore  distortions  in  the  bacteriorhodopsin  photocycle:  
evolution  of  the  H-­‐C14-­‐C15-­‐H  dihedral  angle  measured  by  solid-­‐state  NMR.  Biochemistry  
41(2):431-­‐438.  
23.   Feng  X,  et  al.  (1996)  Direct  determination  of  a  molecular  torsional  angle  by  solid-­‐state  NMR.  
Chem.  Phys.  Lett.  257(3-­‐4):314-­‐320.  
24.   Bajaj  VS,  Mak-­‐Jurkauskas  ML,  Belenky  M,  Herzfeld  J,  &  Griffin  RG  (2009)  Functional  and  shunt  
states  of  bacteriorhodopsin  resolved  by  250  GHz  dynamic  nuclear  polarization-­‐enhanced  
solid-­‐state  NMR.  Proc.  Natl.  Acad.  Sci.  USA  106(23):9244-­‐9249.  
25.   Smith  SO,  Pardoen  JA,  Lugtenburg  J,  &  Mathies  RA  (1987)  Vibrational  analysis  of  the  13-­‐cis-­‐
retinal  chromophore  in  dark-­‐adapted  bacteriorhodopsin.  J.  Phys.  Chem.  91(4):804-­‐819.  
26.   Nack  M,  Radu  I,  Bamann  C,  Bamberg  E,  &  Heberle  J  (2009)  The  retinal  structure  of  
channelrhodopsin-­‐2  assessed  by  resonance  Raman  spectroscopy.  FEBS  Lett.  583(22):3676-­‐
3680.  
27.   Smith  SO,  Lugtenburg  J,  &  Mathies  RA  (1985)  Determination  of  retinal  chromophore  
structure  in  bacteriorhodopsin  with  resonance  Raman  spectroscopy.  J.  Membr.  Biol.  
85(2):95-­‐109.  
28.   Ames  JB,  et  al.  (1989)  Bacteriorhodopsin's  M412  intermediate  contains  a  13-­‐cis,14-­‐s-­‐
trans,15-­‐anti-­‐retinal  Schiff  base  chromophore.  Biochemistry  28(9):3681-­‐3687.  
29.   Rodig  C,  Chizhov  I,  Weidlich  O,  &  Siebert  F  (1999)  Time-­‐resolved  step-­‐scan  Fourier  transform  
infrared  spectroscopy  reveals  differences  between  early  and  late  M  intermediates  of  
bacteriorhodopsin.  Biophys.  J.  76(5):2687-­‐2701.  
30.   Lorenz-­‐Fonfria  VA,  et  al.  (2013)  Transient  protonation  changes  in  channelrhodopsin-­‐2  and  
their  relevance  to  channel  gating.  Proc.  Natl.  Acad.  Sci.  USA  110(14):E1273-­‐1281.  
 

  16