Article

Atomistic Study of Intramolecular Interactions in the

Closed-State Channelrhodopsin Chimera, C1C2
Monika R. VanGordon,1 Gaurav Gyawali,2 Steven W. Rick,1 and Susan B. Rempe3,*
1
Department of Chemistry and 2Department of Physics, University of New Orleans, New Orleans, Louisiana; and 3Sandia National
Laboratories, Albuquerque, New Mexico

ABSTRACT Channelrhodopsins (ChR1 and ChR2) are light-activated ion channels that enable photomobility of microalgae from
the genus Chlamydomonas. Despite common use of ChR2 in optogenetics for selective control and monitoring of individual neurons
in living tissue, the protein structures remain unresolved. Instead, a crystal structure of the ChR chimera (C1C2), an engineered com-
bination of helices I–V from ChR1, without its C-terminus, and helices VI–VII from ChR2, is used as a template for ChR2 structure
prediction. Surprisingly few studies have focused in detail on the chimera. Here, we present atomistic molecular dynamics studies of
the closed-state, non-conducting C1C2 structure and protonation states. A new and comprehensive characterization of interactions
in the vicinity of the gating region of the pore, namely between residues E90, E123, D253, N258, and the protonated Schiff base
(SBH), as well as nearby residues K93, T127, and C128, indicates that the equilibrated C1C2 structure with both E123 and D253
deprotonated closely resembles the available crystal structure. In agreement with experimental studies on C1C2, no direct or
water-mediated hydrogen bonding between an aspartate and a cysteine (D156-O.S-C128) that would define a direct-current
gate in C1C2 was observed in our simulations. Finally, we show that a single hydrogen bond between a glutamic acid (E90) and
an asparagine (N258) residue suffices to keep the gate of C1C2 closed and to disable free water and ion passage through the pu-
tative pore, in contrast to the double bond proposed earlier for ChR2. We anticipate that this work will provide context for studies of
both the gating process and water and ion transport in C1C2, and will spark interest in further experimental studies on the chimera.

INTRODUCTION
Channelrhodopsin-1 (ChR1) and channelrhodopsin-2 (ChR2)
are light-activated ion channels that enable photomobility of
microalgae from the genus Chlamydomonas toward or away
from the conditions optimal for photosynthetic growth (1).
Both ChRs accommodate retinal, a polyene chromophore
that links covalently to a lysine residue (K257 (ChR2
numbering)) on helix VII via a protonated Schiff base
(SBH) (Fig. 1 a) (2). Photo-activation of ChRs (lmax
(ChR1) ¼ 500 nm and lmax (ChR2) ¼ 470 nm) causes a
cascade of events that lead to opening of the channel and
non-selective cation permeation (Hþ, Naþ, Kþ, and Ca2þ).
Channelrhodopsins have become a common tool used in
optogenetics, a biological method in which membranes of
excitable cells (e.g., neurons, muscle cells) are enhanced
with light-sensitive ion channels and membrane depolariza-
tion is selectively controlled using light of specific wave-
lengths. Despite the diverse applications of ChR1 (3) and
Submitted September 20, 2016, and accepted for publication January 24,
2017.
*Correspondence: slrempe@sandia.gov
Editor: Carmen Domene.
http://dx.doi.org/10.1016/j.bpj.2017.01.023
Ó 2017 Biophysical Society.
ChR2 (4–6) in optogenetics and some information about
intermediates in the ChR2 photocycle (described briefly
below), little is known about the molecular mechanisms of
action of ChRs. This problem is partly attributable to chal-
lenges in determining the atomistic structures of ChR1 and
ChR2.
Conductance, point-mutation, and spectroscopy studies
(1,7–10) predict the presence of four photointermediates,
P1–P4, in the photocycle of ChR2 (Fig. 1 b). The sequence
of conformational changes within ChR2 is initiated by
light-induced all-trans to 13-cis retinal isomerization. The
resulting changes to the hydrogen-bond network cause
conformational changes in the transmembrane region of
the channel and yield formation of a non-conducting photo-
intermediate, P1. Deprotonation of the Schiff base (SB) and
protonation of aspartic acid D253 initializes Hþ transfer and
release to the extracellular side of the membrane, assisted by
formation of the P2 pre-open intermediate. It is speculated
that the protein at this point is still in a non-conducting state,
but there is little structural information about the two initial
intermediates.
Formation of the open-state ChR2, P3, is associated with
re-protonation of the SB from the cytoplasmic side of the

Biophysical Journal 112, 943–952, March 14, 2017 943

VanGordon et al.
FIGURE 1 (a) Schematic representation of the seven-helix transmem-
brane regions of ChR with all-trans retinal bound to the seventh helix
(HVII) via a SBH. In the C1C2 chimera, helices I–V (gray) derive from
ChR1, and helices VI–VII (blue) from ChR2. (b) Photocycle of ChR2. To
see this figure in color, go online.
protein, minor changes within the side chains of a few
amino acids in the vicinity of the glutamic acid residue
E90, and entry of bulk water into the previously dry trans-
membrane region. From the open, conducting P3 intermedi-
ate, there are two possible routes to pore closing (Fig. 1 b):
1) the fast route (10 ms), which leads directly to the ground/
closed state; or 2) desensitization, a long-lasting (20–25 s)
inactivation process that is observed under continuous illu-
mination conditions. The latter path originates from ChR2
temporarily occupying a late low-conducting state, P4, or
a mixture of P4 and conducting states. In either case, the re-
turn to the ChR2 ground state is a consequence of the 13-cis
to all-trans retinal isomerization and retrieval of the non-
conducting properties.
Recently, Kato et al. (11) reported an x-ray structure
resolved to 2.3 Å of an engineered combination of ChR1
and ChR2, called C1C2 chimera. The protein was crystal-
lized as a fully dark-adapted dimer, each subunit being
in a non-conductive state. The 342-residue region of
944 Biophysical Journal 112, 943–952, March 14, 2017
monomeric C1C2 consists of an engineered combination
of helices I–V from ChR1, without its C-terminus, and
helices VI–VII from ChR2 (Fig. 1 a). The putative pore is
localized among helices I–III and VII. Helix II is lined
with acidic glutamic acid residues E90, E97, E101, and
E123, thus suggesting that ion selectivity and conductance
depend on this helix. Despite the structural similarities be-
tween the well-studied proton-pumping bacteriorhodopsin
(1) and ChR, little is known about the underlying molecular
mechanism of light-induced channel gating that regulates
non-selective cation permeation (Hþ, Naþ, Kþ, and Ca2þ)
via the putative ion channel of C1C2 ChR.
Recent time-resolved Fourier transform infrared spectro-
scopic and molecular dynamics (MD) studies (12,13) of the
wild-type ChR2 in both dark-adapted (closed) and pre-open
(non-conducting) states suggest an E90-helix-II-tilt (EHT)
model to explain channel gating (Fig. 2, a and b). According
to the former study, the protonated glutamic acid E90 on he-
lix II (HII) and asparagine N258 on helix VII (HVII) form a
double hydrogen bond, an inner gate. After the all-trans to
13-cis isomerization of retinal, the E90-N258 interaction
supposedly weakens. Helices II and VII disconnect and hy-
drophilicity increases in the central section of the pore due
to deprotonation and rearrangement of E90 toward E83.
Experimental studies of the dark-state, wild-type ChR2
suggest that a hydrogen bond between the side chains of
aspartic acid D156 and cysteine C128 is crucial for on/off
kinetics of the conducting state (14,15). This so-called
aspartic acid - cysteine (DC) gate is located in the vicinity
of the retinal chromophore (Fig. 2, a and b). A single point
mutation, D156C, results in a ChR2 variant that exhibits an
extended lifetime of the conducting state, high expression in
host cells, improved subcellular localization, elevated
retinal affinity, and superior photocurrent amplitudes when
compared with all other published variants (16). In the
FIGURE 2 (a) Side and (b) top views of func-
tionally important residues of C1C2 (sticks and
spheres), including E90 and N258 (EHT gating
model), C128 and D156 (the DC gate), D253 (the
deprotonated acceptor of the SB proton, SBH),
and E123 (close neighbor of D253 and the SBH).
Water, lipids, and ions are not shown. Asterisks
(*) denote deprotonated residues. (c) Simulation
snapshot of the cross section of the equilibrated ter-
tiary structure of the wild-type ChR chimera C1C2
(tubes represent a-helices), with all-trans retinal
bound to K257 (sticks) surrounded by lipids (trans-
parent sticks), water (blue surface), Naþ (yellow
spheres), and Cl (cyan spheres). Water spontane-
ously enters from the extracellular side, but does
not permeate past the narrowest region of the
pore, which is found in the vicinity of E90 and
N258. To see this figure in color, go online.

C1C2 chimera, however, the direct D156-OH.HS-C128
interaction was not observed either in crystal (11) or pre-
viously simulated (17) structures. Conversely, a bridging
water, or formation of an indirect DC gate, has been sug-
gested for ChR2 (11,18).
Computer simulations can provide detailed information
about networks of inter- and intramolecular interactions
crucial for molecular function. Here, we present an exten-
sive MD study of closed-state C1C2 with all-trans retinal
in a physiologically relevant environment (lipid bilayer,
water, and ions). We focus on analysis of the functionally
important residues in the non-conducting, closed-state
C1C2 that affect pore opening and water access to the trans-
membrane region, and initiate proton transport in ChRs.
Those residues include the pairs C128 and D156, E90
and N258, and the SB (retinal and K257 bond) and its pro-
posed proton acceptor, D253. Throughout this article, all
residues follow the ChR2 numbering scheme unless other-
wise stated. Our analysis is based on a total of 12 equili-
brated systems containing closed C1C2, lipid bilayer,
water, and ions (Naþ and Cl). First, we varied the compo-
nents of the lipid bilayer to determine which lipid composi-
tion affects the C1C2 protein’s secondary structure the least.
Next, we performed eight separate simulations of protein
with altered protonation states among these functionally
important residues: E123, D156, D253 (Fig. 2). The gluta-
mate E123 and aspartate D253 are proximal to the proton-
ated SB, SBH, and both have been proposed to be the
SBH proton acceptor in ChR2. We found that the protein
with deprotonated E123 and D253 closely matches the crys-
tal structure reported by Kato et al. (11). To help decipher
the network of interactions among functionally important
Channelrhodopsin Chimera, C1C2
residues in the non-conducting C1C2 chimera, we analyzed
the atomistic, time-dependent properties of the closed-state
protein equilibrated in the representative environment.
MATERIALS AND METHODS
We based our simulations on a recently resolved crystal structure of the
non-conducting ChR chimera in the electronic ground state (C1C2 with
all-trans retinal (Protein Data Bank (PDB): 3UG9)) (11). With the aid of
the I-TASSER web server (19–21), we generated the missing secondary
structure of C1C2 (see the Supporting Material for details). We chose to
work with a seven-transmembrane-monomer fold (~300 residues), which
has been shown experimentally to retain photocurrents unaffected by the
truncation (22). We initially assigned C1C2 protonation based on Kato
et al.’s (11) crystallographic study of C1C2 and predicted for the closed-
state ChR2 (18): SBH, protonated D253, and all glutamates except E90
on helix II deprotonated. In this protonation state, we noticed that the
side chains of the protein equilibrated in the representative environment
consisting of lipids, water, and ions did not align well with the crystal struc-
ture (Fig. 3). This motivated us to vary protonation states among key resi-
dues and study the differences among intramolecular interactions in
systems I–VIII (Table S1).
We used the CHARMM-GUI (23) software to create a simulation box
containing a reconstructed C1C2 immersed in lipid bilayers with ~200
(~100 per layer) molecules of 1,2-dipalmitoyl-sn-glycero-3-phosphatidyl-
choline (DPPC), 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC),
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), or 1,2-di-
oleoyl-sn-glycero-3-phosphatidylcholine (DOPC), TIP3P water, and ions
(Naþ and Cl) to neutralize the protein net charge and provide 0.15 M
salt concentration. The total number of atoms in each studied system was
~96,000. Periodic boundary conditions were used in all directions to mimic
bulk conditions. Electrostatic interactions were calculated using particle
mesh Ewald with a real-space cutoff distance of 15 Å and grid width of
1 Å. The switching distance for non-bonded electrostatics and van der
Waals interactions was 13 Å. The size of the simulation box (95  95 
125 Å) was large enough to prevent interactions between the protein and
its periodic images (see Fig. S3).
FIGURE 3 (a) Backbone overlay of the crystal
structure of truncated C1C2 (PDB: 3UG9 (blue))
and C1C2 predicted by I-TASSER (red). (b and c)
Steric overlay of the functionally important residues,
including the SB (SBH) and all-trans retinal, in
PDB: 3UG9 (sticks) and the modeled C1C2 after
equilibration for 120 ns (bonds and spheres), where
(b) D253 and E123 were deprotonated (system VII)
and (c) D253 was deprotonated and E123 was
neutral (system VI). The analysis of steric overlay
of side chains and median pKa values showed higher
agreement between equilibrated system VII (b) and
the C1C2 crystal structure. Thus, we chose to inves-
tigate equilibrated system VII (b) further. To see this
figure in color, go online.
Biophysical Journal 112, 943–952, March 14, 2017 945
VanGordon et al.
MD simulations of the ChR chimera in lipid environments were per-
formed in the constant-particle-number and isothermal-isobaric ensemble
(NpT) using the NAMD code (24). The time step was set to 1 fs. The Lan-
gevin piston method (25) was used to maintain a constant pressure of 1 atm.
The temperature, set to 300 K (C1C2 in DPPC, DSPD, and POPC) as well
as 324 K (C1C2 in DPPC) and 368 K (C1C2 in DSPC), was controlled us-
ing Langevin dynamics with a coupling coefficient of 1 ps1. Our choice of
environmental conditions derives from general interest in the protein’s
behavior at diverse temperatures. Structural studies have been carried out
in a range of temperatures from cryogenic (26,27) to physiological (e.g.,
neuron excitability studies (5,6)). Also, progression among the intermedi-
ates of the ChR photocycle is thermally driven. System equilibration was
divided into steps I–IV, each followed by 1000 energy-minimization steps.
Step I involves relaxation of the lipid tails with protein, lipid headgroups,
and ions fixed (0.5 ns). Step II consists of relaxation of the system with pro-
tein heavy atoms harmonically restrained (1 kcal/mol/Å, duration 1 ns).
Step III involves equilibration with protein constraints released (100 ns).
Step IV comprises the production runs (additional 20 ns). System equilibra-
tion was evaluated by monitoring the root mean-square deviation (RMSD)
of transmembrane sections of the protein with respect to the initial struc-
ture. In equilibrated systems, the RMSD values plateau to a constant value
(Fig. S1). All systems simulated at temperatures above the lipid melting
point reached equilibrium energy values at ~5 ns. Equilibration of the sys-
tems with DPPC and DSPC lipids simulated at 300 K was not observed until
20 and 120 ns, respectively. Nevertheless, each system was simulated for a
total of 120 ns.
The CHARMM36 force field (28) was chosen as it reliably reproduces
protein NMR properties when calculated from MD simulations (29), and
it allows for the use of tensionless ensembles in MD simulations of lipid bi-
layers. Force fields used previously, CHARMM 27 and 27r, overestimated
the surface tension (30–40 dyn/cm) in fluid-phase bilayers at the experi-
mentally determined surface area per lipid, causing shrinking of lipid bila-
yers. CHARMM36 allows for simulations of stable bilayers with or without
proteins (30). The all-trans retinal parameters were taken from prior works
(31–35).
Protein structures and properties were calculated from statistical aver-
ages of molecular coordinates obtained from the last 20 ns of the MD
production runs. The results were analyzed using visual MD (VMD),
home-built Tcl/Tk, and Python-based programs that use the MDAnalysis
toolkit (36). Representative frames were chosen as the simulation frames
with the lowest RMSD value (protein backbone) with respect to the equil-
ibrated protein structure averaged over the last 20 ns of 120-ns-long simu-
lations. The pKa values of ionizable C1C2 chimera residues were calculated
for each frame of the production run with PROPKA software (37,38).
PROPKA is a fast, empirical method for calculating pKa values of ionizable
groups as a function of perturbations arising from desolvation effects and
intra-protein interactions such as hydrogen bonds and Coulombic interac-
tions. The latter is a long-distance (up to 10 Å), pairwise interaction be-
tween ionizable residues dependent on the chemical nature of the
residues (acidic or basic), their distance from each other, and their distance
from the surface of the protein (buried ratios). Unless stated otherwise, we
report the values of pKa, number of water molecules in the protein’s pore,
and distances and angles averaged over the last 20 ns of MD simulations.
See the Supporting Material for the time evolution of the properties listed
above.
RESULTS
Validation of the predicted C1C2 fold
The truncated C1C2 chimera structure deposited in the
PDB: 3UG9 consists of residues 1–342. Due to the high
disorder in the loop region, residues 24–48, 110–117, and
343–356 are invisible in the electron density map. To predict
946 Biophysical Journal 112, 943–952, March 14, 2017
the positions of the missing residues, we submitted the
sequence of 350 N-terminal amino acids of C1C2 to
I-TASSER (19–21), a protein structure prediction server.
The resulting overlay is shown in Fig. 3.
The confidence score (Cscore) for estimating the quality of
the C1C2 model predicted by I-TASSER was 0.49. The
Cscore value is in the higher range [5, 2], thus indicating
a higher confidence in the structural prediction. As sug-
gested by Roy et al. (20), most protein models with Cscore
values >1.5 have a correct fold. The template modeling
score (TMscore) is a measure of the similarity between two
tertiary protein structures. The value of the TMscore is inde-
pendent of the protein’s local errors (e.g., misalignment of
highly flexible tails, and loops) and varies between 0 and
1, where 1 indicates a perfect match between the two
investigated structures. The TMscore of C1C2 rebuilt via
I-TASSER was 0.834, indicating a model of highly homol-
ogous topology with the truncated C1C2 crystal structure.
Upon visual inspection, a nearly perfect alignment of the
transmembrane region of C1C2 with the crystal structure
was observed. The most misaligned regions are the protein’s
termini (Fig. 3 a, red helix and coil regions extending
beyond the blue structure of the crystal). Those regions
were discarded before immersing the protein in a lipid
bilayer and aqueous solvent and initiating the MD simula-
tions. We also tested the reproducibility of the I-TASSER
structure predictions. For details, see Fig. S2.
Choice of the lipid bilayer
We investigated the influence of the lipid tail length (16–18
carbon atoms (Table 1)) and number of unsaturated carbon-
carbon bonds (0–2) on the stability of the transmembrane
region of the closed ChR chimera. The C1C2 chimera
was immersed in homogeneous bilayers consisting of
various lipids with phosphatidylcholine headgroups:
DPPC, DSPC, POPC, or DOPC, 0.15 M sodium chloride
(NaCl), and water. DPPC and DSPC were chosen to verify
the correctness of the CHARMM36 force field and the
behavior of the systems at temperatures below and above
the lipid melting temperatures. We chose to test POPC
because it is one of the most common lipids used in MD
simulations. DOPC was selected because its hydrophobic
tail, which consists of 18 carbon atoms and one unsaturated
carbon-carbon bond per tail, is similar to monoolein, the
compound used to grow C1C2 crystals (11). Each protein
was equilibrated for 120 ns in a homogeneous lipid bilayer
and solvent using the NAMD code.
Simulations of the systems at 300 K, that is, below the
melting points of DPPC (TMELT ¼ 314 K) and DSPC
(TMELT ¼ 328 K), yielded formation of gel-like lipid
patches (Fig. S4). Such local ordering in DPPC and DSPC
lipid bilayers at 300 K confirmed the suitability of the
CHARMM36 force field and the correctness of the simula-
tion parameter choice. Local ordering of DPPC around
 Channelrhodopsin Chimera, C1C2

TABLE 1 Properties of C1C2 in Water, Ions, and Homogeneous Lipid Bilayers of Varying Melting Temperatures
Number of C Atoms in Tail Number of C¼C Bonds TMELT (K) TSIMULATION (K) RMSD (Å) RGYR (Å)
Initial structure 18.3
DPPC 16 0 314 300 1.9 19.1
324 2.2 19.3
DSPC 18 0 328 300 1.5 18.8
368 2.4 19.1
POPC 16 and 18 1 271 300 1.7 18.9
DOPC 18 2 290 300 1.4 18.9
Bilayers were equilibrated at temperatures, TSIMULATION, ranging from 300 to 368 K. A standard deviation of 1 K in temperature was noted for all systems.
RMSD and radius of gyration, RGYR, were calculated for helical portions of C1C2 from representative frames with respect to the initial (predicted) structure
of the protein. Representative frames were chosen as the simulation frames with the lowest RMSD value (protein backbone) with respect to the equilibrated
protein structure averaged over the production run (the last 20 ns of 120-ns-long simulations). Timelines of the protein’s RMSD values with respect to the
corresponding pre-equilibrated structures are shown in Fig. S1. TMELT, melting temperature.

C1C2 was accompanied by changes in the equilibrium introduced by varying the positions of hydrogen atoms,
conformation, measured as an increase in the protein’s however, RMSDs between the simulated protein and the
radius of gyration, RGYR (calculated for the helical trans- crystal structure may not be the most effective measure of
membrane region of C1C2 only), to 19.1 Å compared protein alignment. Therefore, we decided first to compare
with 18.3 Å for the initial structure (Table 1). Those changes pKa values among the key residues of the equilibrated sys-
correspond to shifts in the positions of helices and crowding tems with the corresponding ones from the crystal structure
in the transmembrane region. C1C2 simulated in DSPC at reported by Kato et al. (11). The results, statistical averages,
300 K reveals the smallest helix dislocation (RGYR ¼ and variations over the last 20 ns of MD simulations for sys-
18.8 Å) among all analyzed systems when compared with tems I–VIII are shown in Fig. S7, Table S1, and Table 2.
the initial structure (RGYR ¼ 18.3 Å). However, the protein’s In assessing the pKa results, it should be noted that the
geometry may be constricted by the local organization of the values reported by Kato et al. (11) for a single structure
nearby lipids simulated below TMELT(DSPC). The largest (PDB: 3UG9) used PROPKA software version 2.0. For com-
protein distortions were observed in the systems simulated parison, we used PROPKA3.1 to calculate pKa values of the
at temperatures >300 K; backbone RMSD values of same crystal structure. The differences between the values
C1C2 in DPPC (TSIMULATION ¼ 324 K) and DSPC
(TSIMULATION ¼ 368 K) were 2.25 and 2.44, respectively. TABLE 2 Protonation States and pKa Values of Functionally
C1C2 in POPC and DOPC simulated above the lipid melting Important Residues of C1C2
points were among the least affected by the lipid type and
E90 K257 K93 E123 D156 C128 D253
temperature choice. Hence, we chose DOPC for the simula-
A 4.50 10.50 4.50 3.80 9.00 3.80
tion studies of closed-state C1C2, pursued next.
B 9.31 5.83 3.21
C 9.25 14.39 12.97 5.62 7.75 13.53 2.96
VI 11.21 15.37 13.89 10.69 7.71 14.85 7.57
Characterization of the closed-state C1C2 8.62** 14.12** 12.21** 8.92** 6.79** 13.11** 2.06**
The protonation states of key amino acid residues can 8.51** 14.13** 12.24** 9.08** 6.83** 13.06** 2.02**
6.26 12.66 10.22 1.40 5.55 11.47 0.91
affect protein structure and function (39). In classical MD 0.77 0.43 0.47 0.98 0.34 0.62 0.50
simulations, the protonation states of residues do not change *
spontaneously. Nevertheless, protonation states of specific VII 10.03 15.04 13.69 5.98 8.12 14.83 5.84
residues can be modified manually and the consequent 8.79** 14.03** 11.59** 3.94** 7.28** 13.24** 2.78**
adjustment of the local environment tracked. Our goal 8.83** 14.08** 11.59** 4.46** 7.29** 13.26** 2.18**
7.13 11.61 10.79 1.33 6.12 11.90 0.75
here is to protonate or deprotonate residues of interest and
0.43 0.38 0.30 1.11 0.26 0.38 1.24
determine which of the studied systems (I–VIII) most * *
closely resembles the crystal structure reported by Kato
The asterisk (*) denotes deprotonated residues. For example, in system VI,
et al. (11). Therefore, our study of C1C2 included equilibra- D253 is deprotonated; in system VII, both E123 and D253 are deproto-
tion of eight structures (I–VIII) in representative environ- nated. Row A: pKa values of the isolated residues. Row B shows pKa values
ments, each differing in the protonation of the functionally reported by Kato et al. (11), calculated using PROPKA2.0. Row C shows
important residues E123, D156, and D253. pKa values calculated here for C1C2 in PDB: 3UG9 using PROPKA3.1.
The same software version (3.1) was used for the pKa calculations for sys-
To assess the results from varied protonation states,
tems I–VIII (see Table S1). Here, we report the maximum, mean and me-
RMSD values most often are calculated for backbone dian (indicated by double asterisks), minimum, and standard deviation of
atoms and indicate global dissimilarities among aligned pro- pKa values calculated for each frame of the 20 ns of equilibrated production
tein structures. In the case of fine structural differences run (the last 20 ns of 120-ns-long simulations) for systems VI and VII.

Biophysical Journal 112, 943–952, March 14, 2017 947

VanGordon et al.
obtained using PROPKA2.0 and those obtained using
PROPKA3.1 ranged between 0.06 and 0.25 pKa units (Table
2), well below the typical standard deviation of pKa values
calculated from our MD production runs (the last 20 ns of
120-ns-long simulations). Due to the empirical nature of
the PROPKA code, the absolute pKa values should not be
compared, but rather the trends should be analyzed.
The variations in protonation of residues E123, D156, and
D253 had little effect on the pKa values of many residues.
We focus attention first on the putative ion channel of
ChRs, located along helix II. This helix contains negatively
charged residues E82, E83, E97, and E101, which point to-
ward the interior of the protein, allowing water to enter the
pore. The discontinuity of water in the protein’s closed state
is due to the presence of protonated E90 on helix II, which
forms a gate disabling unrestricted water and ion permeation
through the protein (Fig. 2 c). In our study, E90 was kept
protonated in all eight simulated systems to model the
closed channel. With the exception of system IV (described
in Table S2), the observed median pKa (E90) values ranged
between 8.03 and 9.62 and appeared not to vary remarkably
from 9.31, the pKa value reported by Kato et al. (11), or
9.25, the value calculated using PROPKA3.1, for the crystal
structure. The pKa values of 12.97 and 14.39 for the
neighboring residues, lysines K93 and K257, respectively,
resembled values calculated for the unequilibrated crystal
structure of C1C2. A similar trend appeared away from
helix II, where the median pKa values of the DC-gate resi-
dues (D156 and C128) remained close to 7.75 and 13.53
in each of the systems studied. These observations suggest
that the networks of interactions among residues E90,
K93, K257, D156, and C128 were not strongly affected by
changes in the E123, D156, and D253 protonation states.
In contrast to the residues relevant to the putative ion
channel and DC gate, the pKa values of residues E123 and
D253 varied strongly (by up to ~7 pKa units) depending
on the equilibrated system under study. The steric superim-
position of the functionally important residues of the closed-
state C1C2 crystal (PDB: 3UG9) and the C1C2 structure
predicted by I-TASSER is shown in Fig. 3 a. C1C2 with
both E123 and D253 deprotonated (system VII) and C1C2
with E123 protonated and D253 deprotonated (system VI),
both equilibrated in DOPC, water, and ions at 300 K for
120 ns, are shown in Fig. 3, b and c, respectively. All-trans
retinal and the side chains of functionally important residues
E90 and N258 of the EHT model, C128 and D156 of the DC
model, and E123 and D253 (both deprotonated) were found
to overlay accurately (Fig. 3 b). The backbone average
RMSD of the protein structure (without inclusion of the
flexible N-terminal tail) throughout the last 20 ns of the
120-ns-long simulations was 0.5 5 0.1 Å. For comparison,
we found an RMSD value of 1.7 Å between the protein ex-
tracted from a representative simulation frame and the pre-
equilibrated structure of system VII (Fig. 3 b). The RMSD
value of 1.7 Å accounts for the contribution from flexible
948 Biophysical Journal 112, 943–952, March 14, 2017
loops that link transmembrane helices and extend into the
solvent, but it also indicates that the equilibrated and crystal
structures are structurally similar. We chose to investigate
equilibrated system VII (with both E123 and D253 deproto-
nated) further because the pKa values for functionally
important residues and the side-chain positions in this sys-
tem aligned the best with the corresponding properties in
the crystal structure.
The extended open-state lifetime of a D156C ChR2 mutant
(16) suggests that the D156-C128 hydrogen bond (DC gate)
plays a key role in ChR kinetics. The predicted C1C2 chimera
structure presented here was based on the crystal structure of
wild-type C1C2 (11), where the distances between the sulfur
atom (S) of the C128 thiol group (SH) and carboxyl oxygen
atoms (O) of protonated D156 are 4.4 Å and 4.6 Å, respec-
tively, and the SH group of C128 was associated with the
p-electron system of the all-trans retinal chromophore
(Fig. 3 b). After equilibration, the mean separations between
the sulfur of C128 and the oxygen of D156 as a proton
acceptor (C128-SH.O-D156) or donor (C128-S.HO-
D156) remain large at 4.6 5 0.3 Å or 4.8 5 0.4 Å. Both
distances extended beyond the maximum length of a con-
ventional hydrogen bond (40). The mean values of the
D156-O-D156-H-C128-S and D156-O-C128-H-C128-S
angles were 157.4 5 11.6 and 61.7 5 14.0 , respectively.
Moreover, no water molecules were found to bridge C128
and D156. Those results exclude the existence of the direct
and indirect DC gate in our model. The high value of the
C128 median ionization constant, pKa (C128) ¼ 13.26 (Table
2), predicted by the PROPKA software (41), originates from
hydrogen bonding between side chains of C128 with D156
and T127, as well as Coulombic interactions with D156,
K273, K93, E123, E253, and E90, and weak backbone
hydrogen bonding of C128 with itself.
The EHT gating model (Fig. 4) suggests the presence of a
double hydrogen bond between E90 and N258 in the closed
ChR2 protein. Throughout the simulations of the dark-
adapted C1C2, the mean distances between the side chains
of protonated E90 and N258 were r(O...O) ¼ 2.7 5 0.1 Å
and r(O.N) ¼ 3.7 5 0.6 Å, respectively. The mean values
of the E90-O-E90-H.N258-O and E90-O.N258-H-
N258-N angles were 160.2 5 10.4 and 100.8 5 14.9 ,
respectively. The weak O.N hydrogen bond, indicated by
the large deviation from a linear donor-proton-acceptor
angle and typical hydrogen bond (donor-acceptor) distance
cutoff of 3.0 Å (40), dissociated frequently. Serine S63
competed for the hydrogen bonding with E90. Additionally,
significant electrostatic influences of D253, K93, E123, and
K257 on E90 were observed. Those interactions contributed
to a significant increase of the pKa (E90) value from 4.5 in
an isolated amino acid (Table 2) to the median value of 8.83
in equilibrated C1C2 (Fig. S7; Table 2). A similarly high
value, pKa (E90) ¼ 9.31, was reported for the crystal struc-
ture of C1C2 described by Kato et al. (11). That behavior
suggests that the O.O interaction between E90 and N258
 Channelrhodopsin Chimera, C1C2

deprotonated D253 and E123 were 2.18 and 4.46 (system

VII in Table 2), ~1 pKa unit lower compared with the values
calculated for the resolved C1C2 crystal structure. It should
be noted, however, that during the course of the production
run, the pKa values of the two residues fluctuated between
1.1 and 5.9 pKa units (Fig. 5), suggesting highly mobile side
chains for these residues. The median separation between car-
bon atoms of carboxylate ions of E123 and D253 during the
20 ns production run was 5.1 Å, and both side chains experi-
enced strong, mutual Coulombic interactions. Moreover,
E123 and D253 were affected by interactions with neigh-
boring K253, E97, K273, and R120. Additionally, E123
showed a propensity for forming hydrogen bonds with T127
FIGURE 4 (a) The EHT gating model in ChR2 predicts a double E90- and W124, which affected the E123-O.HN-SB interaction
O...N258-O and E90-O.N258-N bond. In the crystal structure of C1C2,
the corresponding distances are 2.86 Å and 3.01 Å. (b) In the equilibrated
by significantly decreasing the strength of the side-chain
C1C2, system VII, the mean distances between the side chains of proton- hydrogen bond. Despite the previous suggestion that D253
ated E90 and N258 were r(O...O) ¼ 2.7 5 0.1 Å and r(O.N) ¼ 3.7 5 is the SB proton acceptor during the ChR2 photocycle (7),
0.6 Å, respectively. This suggests that the O.O interaction between E90 our study suggests that in C1C2, the side chains of deproto-
and N258 alone can hold helices II and VII of C1C2 together, thus prevent- nated D253 and E123 may compete for the SB proton.
ing pore opening. To see this figure in color, go online.
Based on results from the PROPKA calculations and geo-
metric considerations (distances and angles among resi-
alone can hold helices II and VII together, thus preventing dues), we mapped selected key interactions branching out
pore opening. from the SB (denoted as K257) and its proton acceptor
During equilibration of the closed C1C2 protein in the (E123 or D253), the EHT gate (E90-N258), and the DC
lipid bilayer and electrolyte solution, we first allowed the gate (D156-C128). All those residues are found in the
water to relax and enter the constrained protein (see Mate- vicinity of the C1C2 ion channel and retinal molecule
rials and Methods for details of the simulation setup). An (Fig. 6); thus, retinal isomerization can be expected to
average of 32 water molecules (Fig. S5) entered the putative
transmembrane channel region from the extracellular side of
the protein, but they did not form a continuous pathway
(Fig. 2 c). The discontinuity of water in the vicinity of the
protonated E90 is consistent with previous modeling studies
(12,13). The average separation between the centers of mass
of helices HII and HVII was measured in three locations: the
vicinity of the extracellular, central, and intracellular parts
of the protein’s pore, yielding 14.7 5 0.3 Å, 9.9 5 0.2 Å,
and 9.4 5 0.2 Å, respectively. The separation of the helices
was commensurate with a wide, water-filled opening of the
pore toward the extracellular side of the protein and a nar-
row, dry region in the intracellular region of the pore
(Fig. 2 c). Narrowing of the pore in the vicinity of the gating
region (E90-N258), the close proximity of helices, and the
presence of hydrophobic residues such as bulky Y70 at
the intracellular side of the pore prevented water molecules
from continuously filling the pore of closed C1C2.
In the ChR2 photocycle (Fig. 1 b), deprotonated aspartic
acid D253 is believed to accept the SB proton. In our simula-
tions of C1C2 (Fig. 3 b), the mean separation between the
nitrogen atom of the all-trans retinal SB, SBH, and the depro-
tonated D253 centered around 3.3 5 0.3 Å and the SB- FIGURE 5 pKa values of functionally important residues of C1C2, (a)
NH..O-D253 angle took on a mean value of 92.5 5 16.5 E123 and (b) D253 (both deprotonated). Individual pKa values (black
line) and the running average of pKa values (red line) were calculated for
in the equilibrated closed C1C2 protein. Similarly, the mean
each frame of the 20 ns equilibrated production run (the last 20 ns of
distance between the SBH nitrogen atom and the oxygen of 120-ns-long simulations) using PROPKA3.1. pKa standard deviations of
deprotonated E123 was 2.8 5 0.2 Å and the SB-NH..O- 1.11 and 1.24 pKa units were found for deprotonated E123 and D253,
E123 angle was 158.9 5 15.9 . The median pKa values for respectively. To see this figure in color, go online.

Biophysical Journal 112, 943–952, March 14, 2017 949

VanGordon et al.
FIGURE 6 Schematic diagram of the side-chain hydrogen bonds (red
lines), Coulombic interactions (blue lines), and backbone interaction with
itself (green) most commonly observed among functionally important
residues of C1C2 equilibrated in a lipid bilayer and electrolyte solution.
The putative ion channel in ChRs has been located along HII. In C1C2,
the majority of interactions spanned helices II and III (originally from
ChR1), as well as helix VII (from ChR2). HVII is depicted as the longest
of the helices, in agreement with the crystal structure of C1C2, where
HVII extends toward the intracellular side of the protein. The asterisks
(*) denote deprotonated residues. To see this figure in color, go online.
influence the network of interactions leading to pore open-
ing. The least involved in the network of interactions were
helices HI, HV (originally from ChR1), and HVI (ChR2).
In addition to the interactions listed above between E90-
N258, D156-C128, and the SB and its proton acceptors,
two more residue pairs link HII, HIII, and HVII, the three
helices shown experimentally to form the key network
among functionally important residues. Those residues are
D253-O.N-K93 and E123-O.HN-K93, which form
hydrogen bonds at 2.7 5 0.1 Å, 112.4 5 13.8 , and 2.6
5 0.1 Å, 160.1 5 8.8 , respectively.
DISCUSSION
We equilibrated multiple wild-type, closed-state C1C2
chimera structures in a physiologically representative envi-
ronment of lipid bilayer (DOPC) and electrolyte solution.
All proteins consisted of deprotonated residues E82, E83,
E97, and E101 along helix II and protonated E90—a compo-
nent of the EHT gate in the central region of the pore. The
structures differed in protonation of the functionally impor-
tant residues E123, D156, and D253. We compared the
mean separations among side-chain atoms, median pKa
values of functionally important residues, and alignment of
the representative structures extracted from each of the sim-
ulations. We determined that the equilibrated C1C2 with de-
protonated E123 and D253 (system VII in Table 2) resembles
950 Biophysical Journal 112, 943–952, March 14, 2017
the crystal structure of C1C2 reported by Kato et al. (11) most
closely. That motivated further study of the atomistic details
of system VII, as discussed below.
The double hydrogen bond between protonated E90 on HII
and N258 on HVII in the dark-adapted ChR2 was recently
indicated to be an inner gate (12,13). Upon all-trans to
13-cis isomerization of retinal, the E90 deprotonates and
the E90-N258 interaction supposedly weakens, causing
disconnection of helices II and VII as well as an increase in
hydrophilicity in the central section of the pore. This EHT
model (13) predicts a reaction path for formation of the
continuous water pore through the transmembrane section
of the protein; a prerequisite for ion transport. However, the
EHT model is not yet well understood. We tested the EHT
mechanistic model in wild-type C1C2 via all-atom MD
studies. Our simulations showed that the average distance be-
tween oxygen and nitrogen atoms of side chains of E90 and
N258 fluctuated around r(O...O) ¼ 2.7 5 0.1 Å and
r(O.N) ¼ 3.7 5 0.6 Å. This difference suggests that the
presence of a single OH.O hydrogen bond between E90
and N258 sufficed to keep the gate in C1C2 closed.
The presence of the DC gate has been demonstrated in
Fourier transform infrared spectroscopy studies by Nack
et al. (14). In the dark state of ChR2, the side chains of
D156 and C128 form a hydrogen bond that links helices IV
and III, respectively. A single point mutation, D156C, results
in a ChR2 variant of high expression and long open state due
to the slow channel closure (16). Although the existence and
functional relevance of the DC gate in ChR2 is well sup-
ported, the nature of the hydrogen bond between C128 and
D156 is still an open issue. Nevertheless, the presence of a
direct or water-mediated C128-S.O-D156 interaction has
not been observed in the crystal structure of C1C2. In
C1C2 analyzed here, a bridging water between residues
128 and 156 was not found. The thiol group of C128 most
commonly formed a hydrogen bond with the side chain of
the neighboring T127, rarely with D156. According to
PROPKA simulations, C128 was also affected by the electro-
static potential originating from residues found in the vicinity
of the gating region of the pore, namely E90, K93, E123,
D156, and D253. Both C128 and T127 reside in the immedi-
ate vicinity of the all-trans retinal. Interestingly, the back-
bone oxygen of the deprotonated E123 was found forming
an intra-helical hydrogen bond with T127 at 2.8 5 0.2 Å
and 158.2 5 18.8 . It can be expected that upon the all-trans
to 13-cis isomerization of the chromophore, the polyene
chain displacement could affect the C128-T127 hydrogen
bonding and, consequently, influence the network of interac-
tions among the SB proton acceptor (E123 or D253) and
other residues involved in the channel gating.
In this study, both deprotonated residues E123 and D253
were found to form hydrogen bonds with the SB nitrogen.
Moreover, the two residues were incorporated into the largest
number of contacts, both Coulombic and hydrogen bonds,
within the network of functionally important interactions

(Fig. 6). Those contacts also translated into large fluctuations
in pKa values for both residues (Fig. 5), which, as stated in
Materials and Methods, were calculated as neighbor-induced
perturbations. E123 of ChRs corresponds to D85, a SB proton
acceptor in H. salinarum bacteriorhodopsin (22,42). How-
ever, point-mutation studies on E123 (22,43) in ChR2 show
that the residue is not compulsory for the protein’s function,
suggesting that D253 acts as the proton acceptor from the SB
also in C1C2. Although in bacteriorhodopsin D96 was sug-
gested as a SB proton donor (44), the corresponding residue
in C1C2 is still unknown. To better understand the most prob-
able proton transfer pathway and to gain additional insight
into the treatment of the C1C2 protein’s protonation and its
effect on intramolecular interactions, future studies will
include constant pH simulations (45,46).
Based on the existing knowledge of the ChR2 photocycle
and our study of pairwise interactions in the closed-state
non-conducting ChR chimera, C1C2, we have deciphered
an extended network of potentially correlated interactions
among key residues in the latter (Fig. 6). We expect the
network of hydrogen bonds and Coulombic interactions
among E90, E123, D253, N258, and the SBH, as well as
contributions from K93, C128, and T127, to play a key
role in the C1C2 photocycle. In the early stage of C1C2 pho-
toactivation, the C128...T127 pair, which resides near the
polyene retinal moiety, would be affected by the isomeriza-
tion of retinal from all-trans to 13-cis. Changes in the vicin-
ity of T127, which forms a hydrogen bond with E123, could
affect Coluombic interaction between E123 and D253.
Those two residues reside in the vicinity of the SBH, and
both have been suggested as SBH proton acceptors (7).
Re-protonation of the SB in the pre-gating stage of the
ChR2 photocycle is expected via residues not yet specified.
Finally, we found that K93 interacts with E123 and D253.
Studies on the possible involvement of K93 in the proton
transfer pathway are underway.
CONCLUSIONS
With steadily growing interest in the application of ChRs
in optogenetics, there have been a surprising number of
unanswered questions regarding the atomistic details of
the action of these proteins. Although most research on the
medical applications of ChRs has focused on ChR2, the
structure of the protein has not been resolved. Most of the cur-
rent theories regarding ChR2 function have been derived
from ChR2 homology models built based on the crystal
structure of C1C2 chimera (PDB: 3UG9). Surprisingly
few detailed studies address C1C2 itself. To gain a better
understanding of the C1C2 structure-versus-function rela-
tionships, we conducted atomistic MD simulations of the
closed state, wild-type C1C2 in a physiologically representa-
tive environment of lipid bilayer and electrolyte solution.
Out of the eight equilibrated proteins with varied proton-
ation states of key residues (E123, D156, and D253), we found
Channelrhodopsin Chimera, C1C2
that the structure with E123 and D253 deprotonated closely
matches the crystal structure of C1C2 chimera reported by
Kato et al. (11). We discovered that the gating in C1C2 could
be achieved by a single E90-OH.N258-O hydrogen bond,
not a double E90-OH.N258-O and E90-O...N258-NH
bond, as previously suggested for ChR2. We suspect that
D253 is the probable SB proton acceptor, but E123 should
not be excluded from consideration. In agreement with previ-
ous studies on C1C2, our simulations show no proof of the ex-
istence of a direct or water-bridged interaction between the
all-trans retinal-neighboring DC-gate components, D156
and C128. Instead, the side chain of C128 formed hydrogen
bonds with T127, a residue in close vicinity to E123.
We have deciphered key interactions among the function-
ally important residues in the gating region and in the vicinity
of the all-trans retinal chromophore. We anticipate that this
work will provide context for studies of the gating process
as well as water and ion transport in C1C2, and spark interest
in further experimental studies on the chimera. In future
work, we will study pore opening and further investigate
the contributions of the key residues to structural rearrange-
ments leading to solvent transport through the C1C2 pore.
SUPPORTING MATERIAL
Seven figures and one table are available at http://www.biophysj.org/
biophysj/supplemental/S0006-3495(17)30144-3.
AUTHOR CONTRIBUTIONS
M.R.V. and S.B.R. conceived the study. S.B.R and S.W.R. supervised the
project. M.R.V. performed research and, together with G.G., analyzed
data. G.G. contributed analytic tools. M.R.V. drafted the manuscript.
S.B.R. and S.W.R. assisted in interpretation of data and writing the
manuscript.
ACKNOWLEDGMENTS
We gratefully acknowledge support by the Louisiana Optical Network Insti-
tute (LONI), the National Science Foundation award no. CHE-1301072, the
Defense Threat Reduction Agency (DTRA), and the Sandia National Lab-
oratories’ LDRD program. We also thank Andriy Anishkin for fruitful dis-
cussions and for providing the scripts to calculate pKa values. This work
was performed, in part, at the Center for Integrated Nanotechnologies, a
U.S. Department of Energy, Office of Basic Energy Sciences user facility
at Los Alamos National Laboratory (contract DE-AC52-06NA25396) and
Sandia National Laboratories (Contract DE-Ac04-94AL85000). Sandia Na-
tional Laboratories is a multi-mission laboratory managed and operated by
Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corpo-
ration, for the U.S. Department of Energy’s National Nuclear Security
Administration under contract DE-AC04-94AL85000.
REFERENCES
1. Lórenz-Fonfrı́a, V. A., and J. Heberle. 2014. Channelrhodopsin un-
chained: structure and mechanism of a light-gated cation channel. Bio-
chim. Biophys. Acta. 1837:626–642.
Biophysical Journal 112, 943–952, March 14, 2017 951
VanGordon et al.
2. Spudich, J. L., C. S. Yang, ., E. N. Spudich. 2000. Retinylidene pro-
teins: structures and functions from archaea to humans. Annu. Rev. Cell
Dev. Biol. 16:365–392.
3. Berthold, P., S. P. Tsunoda, ., P. Hegemann. 2008. Channelrhodopsin-1
initiates phototaxis and photophobic responses in Chlamydomonas by
immediate light-induced depolarization. Plant Cell. 20:1665–1677.
4. Yizhar, O., L. E. Fenno, ., K. Deisseroth. 2011. Optogenetics in
neural systems. Neuron. 71:9–34.
5. Mac
e, E., R. Caplette, ., D. Dalkara. 2015. Targeting channelrhodop-
sin-2 to ON-bipolar cells with vitreally administered AAV Restores ON
and OFF visual responses in blind mice. Mol. Ther. 23:7–16.
6. Bruegmann, T., T. van Bremen, ., P. Sasse. 2015. Optogenetic control
of contractile function in skeletal muscle. Nat. Commun. 6:7153.
7. Lórenz-Fonfrı́a, V. A., T. Resler, ., J. Heberle. 2013. Transient proton-
ation changes in channelrhodopsin-2 and their relevance to channel
gating. Proc. Natl. Acad. Sci. USA. 110:E1273–E1281.
8. Radu, I., C. Bamann, ., J. Heberle. 2009. Conformational changes of
channelrhodopsin-2. J. Am. Chem. Soc. 131:7313–7319.
9. Ritter, E., K. Stehfest, ., F. J. Bartl. 2008. Monitoring light-induced
structural changes of Channelrhodopsin-2 by UV-visible and Fourier
transform infrared spectroscopy. J. Biol. Chem. 283:35033–35041.
10. Bamann, C., T. Kirsch, ., E. Bamberg. 2008. Spectral characteristics
of the photocycle of channelrhodopsin-2 and its implication for channel
function. J. Mol. Biol. 375:686–694.
11. Kato, H. E., F. Zhang, ., O. Nureki. 2012. Crystal structure of the
channelrhodopsin light-gated cation channel. Nature. 482:369–374.
12. Eisenhauer, K., J. Kuhne, ., K. Gerwert. 2012. In channelrhodopsin-2
Glu-90 is crucial for ion selectivity and is deprotonated during the
photocycle. J. Biol. Chem. 287:6904–6911.
13. Kuhne, J., K. Eisenhauer, ., F. Bartl. 2015. Early formation of the ion-
conducting pore in channelrhodopsin-2. Angew. Chem. Int. Ed. Engl.
54:4953–4957.
14. Nack, M., I. Radu, ., J. Heberle. 2010. The DC gate in Channelrho-
dopsin-2: crucial hydrogen bonding interaction between C128 and
D156. Photochem. Photobiol. Sci. 9:194–198.
15. Bamann, C., R. Gueta, ., E. Bamberg. 2010. Structural guidance of
the photocycle of channelrhodopsin-2 by an interhelical hydrogen
bond. Biochemistry. 49:267–278.
16. Dawydow, A., R. Gueta, ., R. J. Kittel. 2014. Channelrhodopsin-2-
XXL, a powerful optogenetic tool for low-light applications. Proc.
Natl. Acad. Sci. USA. 111:13972–13977.
17. Kamiya, M., H. E. Kato, ., S. Hayashi. 2013. Structural and spectral
characterizations of C1C2 channelrhodopsin and its mutants by molec-
ular simulations. Chem. Phys. Lett. 556:266–271.
18. Watanabe, H. C., K. Welke, ., M. Elstner. 2013. Towards an under-
standing of channelrhodopsin function: simulations lead to novel in-
sights of the channel mechanism. J. Mol. Biol. 425:1795–1814.
19. Yang, J., R. Yan, ., Y. Zhang. 2015. The I-TASSER Suite: protein
structure and function prediction. Nat. Methods. 12:7–8.
20. Roy, A., A. Kucukural, and Y. Zhang. 2010. I-TASSER: a unified
platform for automated protein structure and function prediction.
Nat. Protoc. 5:725–738.
21. Zhang, Y. 2008. I-TASSER server for protein 3D structure prediction.
BMC Bioinformatics. 9:40.
22. Nagel, G., T. Szellas, ., E. Bamberg. 2003. Channelrhodopsin-2, a
directly light-gated cation-selective membrane channel. Proc. Natl.
Acad. Sci. USA. 100:13940–13945.
23. Jo, S., T. Kim, ., W. Im. 2008. CHARMM-GUI: a web-based graph-
ical user interface for CHARMM. J. Comput. Chem. 29:1859–1865.
24. Phillips, J. C., R. Braun, ., K. Schulten. 2005. Scalable molecular
dynamics with NAMD. J. Comput. Chem. 26:1781–1802.
25. Feller, S. E., Y. Zhang, ., B. R. Brooks. 1995. Constant pressure mo-
lecular dynamics simulation: the Langevin piston method. J. Chem.
Phys. 103:4613–4621.
952 Biophysical Journal 112, 943–952, March 14, 2017
26. M€uller, M., C. Bamann, ., W. K€uhlbrandt. 2015. Light-induced helix
movements in channelrhodopsin-2. J. Mol. Biol. 427:341–349.
27. Ito, S., H. E. Kato, ., H. Kandori. 2014. Water-containing hydrogen-
bonding network in the active center of channelrhodopsin. J. Am.
Chem. Soc. 136:3475–3482.
28. Best, R. B., X. Zhu, ., A. D. Mackerell, Jr. 2012. Optimization of the
additive CHARMM all-atom protein force field targeting improved
sampling of the backbone 4, j and side-chain c(1) and c(2) dihedral
angles. J. Chem. Theory Comput. 8:3257–3273.
29. Huang, J., and A. D. MacKerell, Jr. 2013. CHARMM36 all-atom addi-
tive protein force field: validation based on comparison to NMR data.
J. Comput. Chem. 34:2135–2145.
30. Klauda, J. B., R. M. Venable, ., R. W. Pastor. 2010. Update of the
CHARMM all-atom additive force field for lipids: validation on six
lipid types. J. Phys. Chem. B. 114:7830–7843.
31. Tajkhorshid, E., B. Paizs, and S. Suhai. 1997. Conformational effects
on the proton affinity of the Schiff base in bacteriorhodopsin: a density
functional study. J. Phys. Chem. B. 101:8021–8028.
32. Tajkhorshid, E., and S. Suhai. 1999. Influence of the methyl groups on
the structure, charge distribution, and proton affinity of the retinal
Schiff base. J. Phys. Chem. B. 103:5581–5590.
33. Tajkhorshid, E., J. Baudry, ., S. Suhai. 2000. Molecular dynamics
study of the nature and origin of retinal’s twisted structure in bacterio-
rhodopsin. Biophys. J. 78:683–693.
34. Nina, M., B. Roux, and J. C. Smith. 1995. Functional interactions in
bacteriorhodopsin: a theoretical analysis of retinal hydrogen bonding
with water. Biophys. J. 68:25–39.
35. Baudry, J., S. Crouzy, ., J. C. Smith. 1997. Quantum chemical and
free energy simulation analysis of retinal conformational energetics.
J. Chem. Inf. Comput. Sci. 37:1018–1024.
36. Michaud-Agrawal, N., E. J. Denning, ., O. Beckstein. 2011. MDA-
nalysis: a toolkit for the analysis of molecular dynamics simulations.
J. Comput. Chem. 32:2319–2327.
37. Søndergaard, C. R., M. H. M. Olsson, ., J. H. Jensen. 2011. Improved
treatment of ligands and coupling effects in empirical calculation and
rationalization of pKa values. J. Chem. Theory Comput. 7:2284–2295.
38. Olsson, M. H. M., C. R. Søndergaard, ., J. H. Jensen. 2011.
PROPKA3: consistent treatment of internal and surface residues in
empirical pKa predictions. J. Chem. Theory Comput. 7:525–537.
39. Jiao, D., and S. B. Rempe. 2012. Combined density functional theory
(DFT) and continuum calculations of pKa in carbonic anhydrase.
Biochemistry. 51:5979–5989.
40. Arunan, E., G. R. Desiraju, ., D. J. Nesbitt. 2011. Defining the
hydrogen bond: an acount (IUPAC technical report). Pure Appl.
Chem. 83:1619–1636.
41. Rostkowski, M., M. H. Olsson, ., J. H. Jensen. 2011. Graphical anal-
ysis of pH-dependent properties of proteins predicted using PROPKA.
BMC Struct. Biol. 11:6.
42. Braiman, M. S., T. Mogi, ., K. J. Rothschild. 1988. Vibrational spec-
troscopy of bacteriorhodopsin mutants: light-driven proton transport
involves protonation changes of aspartic acid residues 85, 96, and
212. Biochemistry. 27:8516–8520.
43. Gunaydin, L. A., O. Yizhar, ., P. Hegemann. 2010. Ultrafast optoge-
netic control. Nat. Neurosci. 13:387–392.
44. Wolf, S., E. Freier, ., K. Gerwert. 2014. Infrared spectral marker
bands characterizing a transient water wire inside a hydrophobic mem-
brane protein. J. Chem. Phys. 141:22D524.
45. Chen, W., and J. K. Shen. 2014. Effects of system net charge and elec-
trostatic truncation on all-atom constant pH molecular dynamics.
J. Comput. Chem. 35:1986–1996.
46. Huang, Y., W. Chen, ., J. Shen. 2016. Mechanism of pH-dependent
activation of the sodiumproton antiporter NhaA. Nat. Commun.
7:12940.