Supplementary material
Characterization of the Ground State Dynamics of
Proteorhodopsin by NMR and Optical Spectroscopies
Jochen Stehle1,4Frank Scholz2, Frank Löhr3,4, Sina Reckel3,4, Christian Roos3,4, Michaela
Blum3,4, Markus Braun2, Clemens Glaubitz3,4, Volker Dötsch3,4, Josef Wachtveitl2,
Harald Schwalbe1,4,*
1
Institute for Organic Chemistry and Chemical Biology, Goethe University Frankfurt, 60438 Frankfurt
am Main, Germany
2
Institute of Physical and Theoretical Chemistry, Goethe University Frankfurt, 60438 Frankfurt am Main,
Germany
3
Institute of Biophysical Chemistry, Goethe University Frankfurt, 60438 Frankfurt am Main, Germany
4
Center of Biomolecular Magnetic Resonance, Goethe University Frankfurt, 60438 Frankfurt am Main,
Germany
Figure S1
Figure S1[15N, 1H] –TROSY spectrumof PR in DDMat different thresholds recorded at pH 5, at a
1
H Larmor frequency of 800 MHz. NS=32, 256 complex points in 1, 1024 complex points in 2,
relaxation delay =1s, total measurement time: 9h 7min
Figure S2
Figure S2Chemical shift behavior of selected residues at different pH values (from pH 5-9). Left:
expansions of TROSY spectra of PR at pH 5 (red), pH 6.5 (orange), pH 7.5 (green), pH 8 (purple), pH
9 (light blue). Right: the absolute value of a resonance shift at the various pH intervals
Flash Photolysis
Light absorption induces an isomerization of the retinal from the all-trans to the 13-cis conformation
(K-like intermediate). The absorption spectrum of this photointermediate is red-shifted compared to
the initial state (max. = 520 nm at pH 9). For PR-wt in DDM, at =590 nm a decay of absorption can
be observed on a µs time-scale after laser excitation (green area). This decay is attributed to the
depopulation of the K-intermediate. On the same time-scale an increase of absorption at =400 nm is
monitored, typically indicative for a deprotonated Schiff-base species (M1/M2-intermediate). Therefore
this process is attributed to the transfer of the Schiff-base proton to the primary proton acceptor D97
(green arrow in B). After roughly 300 µs this signal decays, again associated with an increase of
absorption (monitored at = 590 nm) attributed to the population of the N/O-intermediates (blue area).
This signature reflectsthe reprotonation of the Schiff-base through a proton transfer from the primary
proton donor (E108) to the deprotonated Schiff-base (blue arrow in B). Afterwards the signal decays
to zero simultaneously with the signal of the depleted initial state (monitored at = 510 nm), indicating
the completion of the photocycle within hundreds of ms (white area).Therefore, for PR-wt solubilized
in DDM a clear subsequentinterconversion of the different proton transfer steps is observed. This is
not the case for PR in diC7PC. At = 590 nm, the decay of the K-intermediate is observed on a µstimescale. This signal decay is accompanied by an absorption increase monitored at =400 nm,
indicating again the deprotonation of the Schiff base. But in contrast to PR solubilized in DDM this
signal decays to an offset with constant amplitude within 100 µs, which last until the end of the
photocycle. Moreover, an increase of absorption monitored at =590 nm on the ms timescale is absent
for PR in diC7PC. For all wavelengths a constant amplitude is present between 100 µs and 100
ms.Subsequently all signals decay on the same timescale, suggesting an equilibration between the
M2,N and O intermediates.
The transient data set is fitted to a kinetic reaction model described in Materials and Methods (C and
D). The transition of M1 to M2 and O to PR are assumed to be irreversible. All other
interconversionslead to an equilibrium between forward reactions (black time constants) and back
reactions (red time constants). The experimental data are simulated by varying the wavelengthdependent extinction coefficient and the time dependent population of the different
photointermediates. Due to the fact that the absorption spectra of the photointermediates of PR
solubilized in DDM and diC7PC are equal (Figure 6), the different curvature of the transient
absorption changes in A are caused by the different time-dependent populations. The time constants
obtained for the forward and back reactions of the M2 to N and N to O states differ for PR in DDM by
a factor of 10 for the previous (167 µs vs. 1.3 ms) and by a factor of 5 (100 ms vs. 500 ms) for the
latter. In contrast to this thetime constants are nearly equal for the M2 to N (20 µs vs. 32 µs) and the N
to O transition (4 ms vs. 2 ms) for PR in diC7PC. Therefore, for PR in DDM a clear accumulation of
the late red-shifted intermediates (N/O) can be observed, while an equilibrium mixture of the M 2, N
and O state is found for PRin diC7PC.These photointermediatesshould accumulate under continuous
illumination conditions applied for the NMR studies presented here.
Figure S3
A
C
B
D
Figure S3Transient absorption changes for PR-wt solubilized in DDM or diC7PC (A) at three distinct
wavelengths, with main contributions from K, N and O intermediates (590 nm), the M 1/M2
intermediate (400 nm) and the PR ground state bleach (510 nm). In (B) the corresponding proton
transfer steps are indicated.The photocycle model of PR in DDM (C) and diC7PC (D) derived from
the simulation of the photocycle population dynamics described in Material and Methods.
Table S1Summary table with NMR spectroscopic data of PR (data used for Figures 2, 3, 4, and 5)
1H-15N
hetNOE
G 21
D 22
L 23
D 24
A 25
S 26
D 27
Y 28
T 29
G 30
V 31
S 32
F 33
W 34
L 35
V 36
T 37
A 38
A 39
L 40
L 41
A 42
S 43
T 44
V 45
F 46
F 47
F 48
V 49
E 50
R 51
D 52
R 53
V 54
S 55
A 56
K 57
W 58
K 59
T 60
S 61
L 62
T 63
V 64
S 65
G 66
L 67
V 68
T 69
G 70
I 71
A 72
F 73
W 74
H 75
Y 76
M 77
Y 78
M 79
R 80
G 81
V 82
W 83
I 84
E 85
T 86
G 87
D 88
S 89
P 90
T 91
V 92
F 93
-0.095
0.075
0.116
0.258
0.358
0.399
0.522
0.650
0.741
0.834
0.762
0.881
0.701
0.763
0.841
0.803
0.885
0.784
0.730
H/D
exchange
X
X
X
X
change of signal intensity due to different magnetic field
strengths
500 MHz
600
MHz
700
MHz
800
MHz
950
MHz
950 MHz500 MHz
pH 5-6.5
[ppm]
[ppm]
[ppm]
[ppm]
[ppm]
[ppm]
[ppm]
[ppm]
1
0.284
1
0.257
1
0.156
1
0.159
1
0.107
0
-0.176
0.013
0.017
0.012
0.036
0.032
0.044
-0.030
-0.030
-0.030
-0.030
0.024
0.014
-0.030
0.011
0.062
0.035
0.008
0.032
0.011
0.021
-0.030
0.032
0.011
-0.030
0.014
0.029
0.022
0.030
-0.030
0.006
-0.030
0.014
0.018
-0.030
-0.030
0.012
0.012
0.030
0.029
0.036
-0.030
-0.030
0.008
0.019
-0.030
0.007
0.009
0.030
0.056
-0.030
0.017
0.038
0.015
0.010
0.001
0.017
0.036
0.024
0.035
0.029
0.053
0.002
0.013
0.004
0.002
0.038
0.042
0.012
0.022
0.018
0.001
0.012
0.207
X
X
X
X
X
X
X
X
0.178
0.155
0.140
0.121
-0.086
0.071
0.161
0.101
0.034
0.090
0.081
0.016
0.061
0.057
0.013
0.048
0.049
0.007
0.033
0.035
-0.064
-0.128
-0.065
0.190
0.183
0.118
0.146
0.118
0.133
0.115
0.099
0.086
0.102
0.073
0.088
0.086
0.063
0.054
-0.103
-0.120
-0.064
0.015
0.048
0.034
0.044
0.020
0.043
0.019
0.036
0.012
0.037
-0.003
-0.011
0.123
0.078
0.060
0.046
0.034
-0.089
0.226
0.052
0.183
0.109
0.129
0.062
0.109
0.077
0.079
0.045
-0.146
-0.006
X
X
X
0.859
0.925
0.850
0.731
0.768
0.862
X
X
X
0.751
0.706
0.743
0.609
0.718
0.647
0.810
0.754
0.742
0.767
0.676
0.849
0.880
0.875
0.823
0.966
0.810
0.836
0.728
0.810
0.626
0.843
0.934
0.566
0.745
0.722
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
0.225
0.217
0.334
0.473
0.205
0.191
0.273
0.384
0.162
0.152
0.231
0.322
0.153
0.153
0.221
0.293
0.131
0.131
0.185
0.233
-0.094
-0.086
-0.150
-0.240
0.132
0.144
0.101
0.099
0.072
-0.060
0.191
0.155
0.127
0.120
0.095
-0.095
0.136
0.089
0.058
0.055
0.027
-0.110
0.534
0.209
0.105
0.017
0.512
0.176
0.179
0.083
0.563
0.152
0.131
0.044
0.524
0.129
0.139
0.051
0.568
0.111
0.094
0.028
0.034
-0.098
-0.012
0.011
0.065
0.040
0.118
0.067
0.080
0.032
0.076
0.038
0.048
0.017
-0.017
-0.023
0.005
0.062
0.031
0.028
0.016
0.010
0.148
0.090
0.059
0.060
0.039
-0.109
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
change of signal intensity due to pH
shift
0.108
0.212
0.207
0.077
0.199
0.164
0.046
0.142
0.114
0.030
0.126
0.105
0.021
0.084
0.072
-0.088
-0.129
-0.136
0.024
0.033
0.040
-0.030
0.020
0.016
0.054
0.043
0.026
0.039
0.040
-0.030
0.014
-0.030
0.020
0.028
0.104
0.019
0.074
0.011
0.058
0.008
0.044
0.007
0.031
-0.021
-0.073
0.463
0.351
0.271
0.233
0.182
-0.281
0.309
0.257
0.181
0.152
0.096
-0.213
0.034
0.024
0.027
-0.030
-0.030
-0.030
0.047
-0.030
0.013
0.150
0.119
0.092
0.077
0.061
-0.089
-0.030
pH 6.57.5
pH 7.58
pH 89
[ppm]
[ppm
]
0.008
0.003
0.015
0.068
0.047
0.029
0.077
0.040
0.052
0.005
0.005
0.065
0.024
0.006
-0.030
0.034
-0.030
0.035
0.001
0.014
0.015
0.034
0.002
0.036
0.009
0.017
0.020
0.018
0.012
0.015
0.022
0.003
0.003
0.001
0.003
0.007
0.021
0.013
0.014
0.010
0.010
0.002
0.017
0.004
0.014
0.025
0.052
0.036
0.009
0.010
0.004
0.000
0.002
-0.030
0.009
0.023
0.015
0.016
0.009
0.025
-0.030
-0.030
0.007
0.013
-0.030
0.047
-0.030
0.008
0.008
0.057
0.006
-0.030
0.054
0.012
0.017
R 94
Y 95
I 96
D 97
W 98
L 99
L 100
T 101
V 102
P 103
L 104
L 105
I 106
C 107
E 108
F 109
Y 110
L 111
I 112
L 113
A 114
A 115
A116
T 117
N 118
V 119
A 120
G 121
S 122
L 123
F 124
K 125
K 126
L 127
L 128
V 129
G 130
S 131
L 132
V 133
M
134
L 135
V 136
F 137
G 138
Y 139
M
140
G 141
E 142
A 143
G 144
I 145
M
146
A 147
A 148
W
149
P 150
A 151
F 152
I 153
I 154
G 155
C 156
L 157
A 158
W
159
V 160
Y 161
M
162
I 163
Y 164
E 165
L 166
W
167
A 168
G 169
X
0.859
0.108
0.102
0.077
0.060
0.037
-0.071
-0.030
0.060
X
X
X
0.014
0.017
-0.030
0.731
0.999
0.000
0.064
0.034
0.037
0.019
0.019
-0.030
-0.030
-0.030
0.849
0.010
0.051
0.029
0.034
0.022
0.011
0.030
0.035
0.079
0.079
0.037
0.014
-0.030
0.043
0.046
0.098
0.055
0.065
0.035
-0.011
0.033
0.038
0.015
0.053
0.036
0.029
0.030
0.026
0.014
0.018
-0.030
-0.030
-0.030
0.004
0.009
0.188
0.032
0.339
0.145
0.102
0.060
0.268
0.079
0.072
0.031
0.206
0.050
0.058
0.032
0.187
0.037
0.039
0.015
0.146
0.024
-0.150
-0.017
-0.193
-0.121
0.066
0.012
0.022
-0.030
0.005
0.004
0.003
0.028
0.009
0.016
0.345
0.267
0.206
0.099
0.178
0.140
0.054
0.052
0.115
0.166
0.077
0.040
0.077
0.141
0.064
0.023
0.041
0.089
0.007
0.013
-0.304
-0.178
-0.199
-0.086
0.025
-0.030
0.036
-0.030
0.001
0.005
0.025
-0.030
0.143
0.140
0.080
0.097
0.026
-0.117
0.033
0.227
0.036
0.055
0.172
0.072
0.024
0.144
0.040
0.035
0.121
0.044
0.011
0.104
0.027
-0.022
-0.123
-0.009
0.044
0.042
0.007
0.005
0.043
0.007
X
0.753
0.718
0.732
0.890
0.537
0.804
0.587
0.542
0.626
0.482
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
-0.030
-0.030
0.039
0.043
0.014
0.027
-0.030
0.004
0.018
0.038
0.009
-0.030
-0.030
-0.030
0.839
0.780
X
-0.030
-0.030
-0.030
0.004
0.002
0.701
X
0.039
0.041
0.004
0.044
0.038
0.007
-0.030
0.041
0.032
0.028
0.043
0.016
0.003
0.018
0.019
0.017
0.043
0.012
0.002
0.033
0.011
0.038
0.056
0.005
0.055
0.030
0.030
0.009
0.002
0.053
0.000
0.791
X
0.616
X
0.900
0.783
0.841
0.053
0.032
0.034
0.013
0.838
0.116
0.078
0.081
0.044
0.850
0.816
0.769
0.814
0.171
0.149
0.109
0.105
0.084
-0.087
X
X
X
X
0.125
0.204
0.147
0.178
0.105
0.143
0.105
0.133
0.075
0.112
-0.050
-0.092
0.769
X
0.145
0.129
0.100
0.091
0.077
-0.068
0.027
0.045
0.003
0.013
0.625
0.833
X
X
0.159
0.416
0.117
0.403
0.093
0.313
0.074
0.294
0.060
0.246
-0.099
-0.171
-0.030
0.018
0.048
0.004
0.017
0.826
X
0.367
0.286
0.247
0.210
0.187
-0.180
0.024
0.007
0.003
0.017
0.017
0.000
0.017
0.002
0.003
0.001
0.015
0.001
0.015
0.014
-0.030
0.007
0.009
-0.030
-0.030
0.039
0.001
0.019
0.017
0.039
0.005
0.006
0.015
0.017
0.016
-0.030
0.012
-0.030
0.007
0.003
0.009
X
0.034
0.051
0.019
X
0.012
0.021
0.006
0.002
0.036
0.004
0.056
0.001
0.013
0.067
0.039
0.003
0.005
0.026
0.014
X
0.856
0.816
X
X
X
0.665
0.802
X
0.076
0.085
0.020
0.052
0.141
0.142
0.147
0.019
0.054
0.492
0.090
0.096
0.011
0.029
0.232
0.106
0.107
0.012
0.035
0.403
0.067
0.076
0.007
0.018
0.165
-0.009
-0.009
-0.013
-0.034
0.024
0.884
X
0.854
0.186
0.113
0.128
0.113
0.132
-0.055
-0.030
0.037
0.045
-0.030
0.042
0.833
X
X
0.014
0.022
-0.030
0.027
0.707
X
-0.030
0.775
0.708
X
X
0.969
0.017
0.011
0.011
0.008
0.021
0.110
0.068
0.042
0.028
0.015
-0.095
0.031
0.008
E 170
G 171
K 172
S 173
A 174
C 175
N 176
T 177
A 178
S 179
P 180
A 181
V 182
Q 183
0.729
0.648
S 184
0.745
A 185
Y 186
N 187
T 188
M
189
M
190
Y 191
I 192
I 193
I 194
F 195
G 196
W
197
A 198
I 199
Y 200
P 201
V 202
G 203
Y 204
F 205
T 206
G 207
Y 208
L 209
M
210
G 211
D 212
G 213
0.815
0.795
0.793
X
X
X
X
0.647
X
0.517
0.674
0.750
0.401
0.487
0.456
0.716
0.716
0.896
X
X
X
X
X
X
X
X
X
X
X
X
X
X
0.129
0.065
0.034
0.023
0.009
-0.120
0.328
0.214
0.239
0.136
0.184
0.079
0.150
0.059
0.114
0.027
-0.214
-0.187
X
0.921
0.900
0.739
0.858
0.783
0.820
0.665
0.845
0.027
0.093
0.033
0.010
0.004
0.008
-0.030
-0.030
-0.030
0.045
0.047
0.041
0.030
0.007
0.003
-0.030
-0.030
0.080
0.189
0.321
0.049
0.142
0.274
-0.163
-0.237
-0.355
0.176
0.279
11313.00
0
0.189
0.172
0.280
0.141
0.221
0.117
0.155
0.104
0.106
0.086
0.076
-0.090
-0.204
0.820
0.770
0.698
0.667
-0.464
0.045
0.042
0.008
0.128
0.142
0.213
0.101
0.109
0.166
0.085
0.090
0.150
0.066
0.060
0.117
-0.123
-0.112
-0.164
-0.030
0.041
0.024
0.081
0.022
0.093
0.006
0.003
0.007
0.006
-0.030
0.016
0.018
0.029
0.009
0.003
0.007
0.039
0.002
0.010
0.033
-0.030
0.040
-0.030
0.017
0.004
0.034
0.034
0.006
0.018
-0.030
-0.030
0.114
0.184
0.140
0.122
0.111
-0.004
0.019
0.008
0.011
0.003
-0.004
-0.023
0.048
0.074
0.032
0.031
0.014
-0.035
0.080
-0.003
-0.030
0.011
-0.030
0.023
0.015
0.021
X
X
X
X
0.027
0.098
0.211
0.373
X
0.901
0.004
0.145
0.274
0.460
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0.123
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0.476
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A 216
L 217
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I 222
Y 223
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A 226
D 227
F 228
V 229
N 230
L 231
I 232
L 233
F 234
G 235
L 236
I 237
I 238
W
239
N 240
V 241
A 242
V 243
K 244
E 245
0.492
X
X
X
X
X
X
X
X
X
X
X
X
0.745
10698.00
0
0.803
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0.016
0.009
0.019
0.509
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0.013
0.406
0.006
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0.158
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0.053
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0.906
0.574
0.854
0.895
0.867
0.844
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X
X
0.750
X
0.845
0.554
0.682
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0.038
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0.151
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0.826
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X
X
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X
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0.752
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0.763
0.731
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0.213
0.146
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S 247
N 248
A 249
0.528
0.416
0.284
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X
X
X
X
0.250
0.415
0.188
0.352
0.156
0.297
0.140
0.278
0.114
0.232
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0.064
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0.005
0.011
0.075
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