Infrared spectral marker bands characterizing a transient water wire
inside a hydrophobic membrane protein
Steffen Wolf1, Erik Freier2, Qiang Cui3, and Klaus Gerwert2,1,a)
1
Department of Biophysics, Chinese Academy of Sciences Max-Planck-Gesellschaft Partner Institute
for Computational Biology, 320 Yue Yang Road, 200031 Shanghai, China
2
Department of Biophysics, Ruhr-University Bochum, Universitätsstraße 150, 44780 Bochum, Germany
3
Department of Chemistry and Theoretical Chemistry Institute, University of Wisconsin, Madison, 1101
University Avenue, Madison, WI 53706, USA
SUPPLEMENTAL MATERIAL
1
FIG. S1. Overview of representative full QM vacuum model. Atoms kept at fixed position during optimization
highlighted as spheres.
2
FIG. S2. Initial data presented in Fig. 3 without scaling of calculated spectra. The calculated dangling water
feature in the WT SCC-DFTB spectrum is found at 3807 cm-1. The calculated double-hump shaped feature
found in the range of strong hydrogen bonds has its central minima at 2730 cm-1.
FIG. S3. Baseline correction of IR spectra in the regime between 2850 cm-1 and 2500 cm-1. All simulated
systems (3 H2O in black, 4 H2O in blue, and V49A in orange) exhibit a slope-like spectral shape between 2900
cm-1 and 2500 cm-1, which comes from underlying single molecule vibrations of the water molecules in the
polarized water wires. To enhance the visibility of the spectral features coming from the coupled O–H vibrations
between 2850 cm-1 and 2500 cm-1, we modelled these single molecule vibrations as exponential functions (red
for 3 H2O, yellow for 4 H2O and V49A; see Materials and Methods for further details). The baseline corrected
respective spectra displayed in Figs. 3, S2, and S6 between 2850 cm-1 and 2500 cm-1 were created by subtracting
these baselines from the actual calculated spectra.
3
Fig. S4. Convergence of calculated IR spectra. As can be seen for both dangling O–H and combined stretch
vibration range, the spectra after 0.55 ns and 1.1 ns are similar. Spectra taken after 1.1 ns of simulated time can
therefore be seen as converged.
4
FIG. S5. O–H stretch vibration range of water molecules of the water molecule water wire determined from FFT
of the respective O–H distances over time. The analysis was performed on a 300 ps trajectory prolongation of
the final system structure after 1.1 ns QM/MM simulation. The “middle” and “bottom” water molecules show
prominent dangling water molecule absorptions between 3900 cm-1 and 3750 cm-1, while the “top” water
molecule does not exhibit such vibrations. Like in NMA analysis (see Figs. 2, S8 and Table SI), the “middle”
water O–H stretch is found at higher wavenumbers than the “bottom” water O–H stretch vibrations.
5
FIG. S6. Evaluation of theoretical spectra for different water wire models. Comparison of SCC-DFTB
calculated and scaled spectra (see Materials and Methods for details) for the water wire models out of three
(black) and four (blue) water molecules and the V49 mutant (orange) with experimental spectra (red). In the
region of dangling water vibrations (left), three water molecules result in a single peak at 3665 cm-1, which fits
well to the experimental double peak at 3670 cm-1 and 3658 cm-1. In the same regime, the water wire out of four
water molecules only shows a broad unspecific absorption between 3750 cm-1 and 3600 cm-1. In the region of
strong hydrogen bonds (right), three water molecules produce a double-hump shaped signal (see Fig. 3), which is
in good agreement with the experimentally observed signal. The four water molecule system exhibits a slopelike shape above 2750 cm-1, which is not observed in experiments. The V49A mutant exhibits a broad unspecific
absorption in the range of dangling water molecules between 3750 cm-1 and 3575 cm-1, and a slope-like shape in
the spectral range of strongly hydrogen bonded O–H bonds, which is comparable to the one of the four molecule
water wire. The experimentally observed signals therefore need to come from a water wire made of three water
molecules.
6
FIG. S7. Water distribution in the N’ crystal structure of the V49A mutant 49. Val49 from our WT model in grey
spheres. The present alanine at position 49 in the mutant leaves more space for water molecules compared to WT
protein. Thus, five water molecules are found to form a water chain in the V49A mutant.
7
FIG. S8. Normal mode analysis of polarized water wire vacuum QM model (see Table SI) compared to
experimentally determined absorptions35 of the water wire out of four water molecules. The analysis reveals that
three out of the four water molecules (termed “top 2”, “top”, and “middle”) exhibit O–H stretch vibrations in
both the regime of dangling water molecules and bulk water, as well (“top 2”: 3740-3385 cm-1; “top”: 3722-3385
cm-1; “middle”: 3715-3464 cm-1). The water molecule termed “bottom” only exhibits dangling water vibrations
(3729-3718 cm-1). The hydrogen bonded O–H bonds of all water molecules an the N–H bond of the retinal
Schiff base show combined symmetrical vibrations between 2734 cm-1 and 2626 cm-1, and combined asymmetric
vibrations between 2878 cm-1 and 2773 cm-1.
8
TABLE SI. NMA analysis of water wire model QM systems. Calculations were performed with B3LYP/ 631++G**. For name assignments of water molecules in three water systems, see Fig. 2; for name assignments in
four water molecule systems, see Fig. S8.
3 water
system
A
B
C
4 water
system
A
B
C
Coupled stretch vibrations (<3000 cm-1)
Waveasym-metry Inten- description
number / corrected /
sity /
cm-1
cm-1
a. u.
Dangling O–H vibrations (>3450 cm-1)
WaveasymIntendescription
number /
metry
sity / a.
cm-1
corrected / u.
cm-1
2641
2544
142
sym. stretch
3479
3351
140
2839
2735
437
3864
3722
59
2959
2850
820
3885
3742
38
OH middle
2643
2546
5762
asym.
stretch
asym.
stretch II
sym. stretch
OH top (not
dangling!)
OH bottom
3686
3550
228
OH top (not
dangling!)
2784
2682
1125
3867
3725
45
2926
2818
1252
3872
3730
48
2850
2745
5647
asym.
stretch
asym.
stretch II
sym. stretch
3565
3434
641
2885
2779
238
3858
3716
42
2998
2888
79
asym.
Stretch
asym.
stretch
(+Thr46 CH)
OH middle
(not
dangling)
OH bottom
3872
3730
32
OH top
2838
2734
3981
3521
3391
555
OH top (not
dangling)
2977
2867
228
sym. stretch
(only 3
water
molecules)
asym.
stretch
(only 3
water
molecules)
3596
3464
617
OH middle
(not
dangling)
3691
3555
208
3860
3883
3698
3718
3740
3562
45
38
312
3819
3678
158
OH top (not
dangling)
OH bottom
OH top 2
OH top 2
(not
dangling)
OH middle
3864
3722
31
OH top
3870
3728
51
OH bottom
3514
3385
460
combination
OH top 2 /
top (sym.)
2726
2626
4118
sym. stretch
2879
2773
2066
asym.
stretch
2980
2870
365
2988
2878
524
2799
2696
5406
asym.
stretch II
(+Thr46)
asym.
stretch III
sym. stretch
(only 3
water
OH middle
OH bottom
9
molecules)
2948
2840
524
asym.
stretch
(only 3
water
molecules)
3564
3433
797
3743
3857
3871
3605
3715
3729
185
121
56
(not
dangling)
combination
OH top 2 /
top (asym.)
(not
dangling)
OH top
OH middle
OH bottom
10