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	FIGURE 1. P480 to D470 relaxation rates of ChR2 WT and E90Q. Amplitude spectra of the relaxation rate from P480 to D470 of WT ChR2 (black) and the mutant E90Q (red) are shown. The negative carbonyl band of Glu-90 at 1718 cm−1 and the corresponding positive carboxylate band, either at 1381 cm−1 or 1439 cm−1, are missing in the mutant. The insets show the subtraction results of the WT and E90Q spectra. The C=O (1718 cm−1, red stripes) of Glu-90 and its corresponding COO− band (1381 cm−1 or 1439 cm−1, red stripes) disappear in the mutant. Therefore, these bands can be unambiguously assigned to Glu-90 deprotonation. In Gln-90, which has a fixed protonation state, a difference band at 1688/1678 cm−1 (blue stripes) is observed, which indicates its H-bond or environmental change.
	FIGURE 2. Time-resolved absorbance changes in the WT protein induced by 20 ns laser excitation. UV-visible (A) and FTIR (B) spectra are shown. The amplitude spectra (orange and red) were generated by a global fit of the complete combined dataset. In A: a P520-D470 difference spectrum (black) averaged over the first 10 ms is shown. The red shift of the absorption maximum indicates the accumulation of the P520 intermediate. In the corresponding IR difference spectrum, now assigned to the P520 intermediate, the deprotonation of Glu-90, indicated by the negative band at 1718 cm−1, is observed. The first observed rate of 30 ms represents the P520 to P480 transition (orange). Here, the band shift of a carboxylic residue from 1728 to 1737 cm−1 is monitored. The slowest rate (red) of 45 s represents the P480 to D470 transition. The negative band at 1718 cm−1 indicates the reprotonation of Glu-90. Individual time-resolved absorbance changes at 520 nm in C and at 1718 cm−1 in D (Glu-90) are shown. The time-resolved data confirm that Glu-90 is already deprotonated in P520, being the putative open state. Whether deprotonation takes place in an earlier transition or simultaneously with the appearance of the P520 intermediate cannot be distinguished yet. Furthermore, the absorbance changes at 1662 cm−1 (E) show that the large structural changes (not time-resolved) of the protein backbone relax in two steps, in the P520 to P480 and the P480 to D470 transitions.
	FIGURE 3. Electrophysiological characterization of ChR2-WT and mutants of Glu-90. Photocurrents of ChR2-WT (A) at pHo 7.5 and pH 4 and E90H (B) at pHo 7.5 in the absence and presence of 100 mm Na+ at −100 mV (blue bar: 470 nm excitation). NMG-Cl was substituted by NaCl. C, averaged initial photocurrents (I0) at pHo 7.5 and pHo 4 at −100 mV. Significance: p < 0.05 (*), p < 0.005 (***). Current-voltage relation of the initial current (I0) at 100 mm Na+ for wild-type ChR2 (D) and the mutants E90Q, E90A, E90H, and E90K (E–H). Measurements at pHo 7.5 and −100 mV were used as reference currents (Iref); error bars: ±S.E.
	FIGURE 4. Water densities (43) reflecting the protein-internal volume in the ChR2 homology model occupied by water molecules during MD simulations depending on the protonation state of Glu-90. Water density distribution changes within ChR2 due to deprotonation of Glu-90. When Glu-90 is protonated (left panel), two distinct regions of intruding water molecules are observed, with Glu-90 being found in the middle of the barrier between them. When Glu-90 is deprotonated (right panel), the water influx is altered. The former unsolvated region around Glu-90 starts to fill up with water. The extracellular side is still more solvated than the intracellular side of the protein. The alteration of water densities results in an almost continuously spanning water-filled pore.
	FIGURE 5. Putative location of the conductive pore within the helical bundle of the BR-based ChR2 homology model (intracellular view). ChR2 is shown in gray, the chromophore retinal is highlighted in yellow, Glu-90 is in purple, and penetrating water molecules are shown as blue surfaces. MD simulation reveals that the location of the conductive pore within the helical bundle of ChR2 is between helices A, B, C, and G, in direct contact with the chromophore and the RSB, indicated by the red circle. MD simulations reveal Glu-90 to be flanking the conductive pore.

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