## 3 Results
### Spectroscopic Properties of the Dark State of the T127 Variants of CrChR2
Inspection of the vibrational band at 1674 cm\({}^{-1}\) revealed a strong asymmetry in the shape, with a pronounced shoulder at lower wavenumbers (green trace in Figure 5B). Consequently, the band was fitted by two Gaussians with frequency maxima at 1674 cm\({}^{-1}\) and 1661 cm\({}^{-1}\). Such heterogeneity in the retinal binding pocket has been suggested from molecular dynamics simulations on WT [31] to be a result of two rotamers of the side chain of E123. In one rotamer configuration, E123 was in direct hydrogen-bonding interaction with the protonated Schiff base, competing with a water molecule and D253. The other rotamer configuration positioned the carboxylic group of E123 in a remote position from the Schiff base. A water molecule or the carboxylic group of D253 can then form a hydrogen bond with the Schiff base proton. The resulting effect on the strength of the N-H bond of the Schiff base was acutely determined by Raman spectroscopy. The vibrational band of the Schiff base in WT has so far been treated as a single vibration [32]. In light of our results for the T127A variant, we revisited the Raman spectrum of the WT (Figure 5, black trace). The slight asymmetry in band shape was again considered by fitting two Gaussians, resulting in maxima at 1665 cm\({}^{-1}\) and 1661 cm\({}^{-1}\). From these results, we assigned the lower frequency of the C=N-H vibration at 1661 cm\({}^{-1}\) to the Schiff base being hydrogen-bonded to a water molecule and/or directly to D253. In this scenario, the substitution of T127 by A did not affect the frequency of the Schiff base vibration, as this residue did not interact directly with D253. This explained the low-frequency shoulder of the C=N-H band at 1661 cm\({}^{-1}\) which did not shift after amino acid exchange. The high-frequency band at 1674 cm\({}^{-1}\) in T127A corresponded to the population in which E123 was hydrogen bonded to the Schiff base and, with respect to the WT, lacked the hydrogen bond to the threonine (see Figure 2, right panel). We tested this hypothesis by recording resonance Raman spectra for two other CrChR2 variants: T127S, where the hydroxyl group of the threonine side chain is conserved; and E123D, where the carboxylate side chain is shorter. In agreement with the WT and the T127A variant, all spectral features of the retinal chromophore were retained in the spectra of the T127S (blue traces in Figure 5) and E123D variants (red traces in Figure 5) with only minor differences. Analysis of the Schiff base vibration (Figure 5b) showed that the C=N-H band in E123D could be fitted sufficiently well with one Gaussian, whereas for T127S, two Gaussians were needed to fit the band. The band with a maximum at 1661 cm\({}^{-1}\) was present in both variants, supporting the hypothesis that this Schiff base vibration did not involve interaction with E123. Figure 5: (**A**) Resonance Raman spectra of CrChR2-WT and the variants E123D, T127S, and T127A in H\({}_{2}\)O. (**B**) Zoom-in of the frequency range of the Schiff base vibration after a three-point moving average smooth. Blue circles are the data points. Dashed grey lines are Gaussians fitted to the data points and continuous lines are sums of the fitted Gaussians. In T127S, where the exchange retained the hydroxyl group that is hydrogen-bonded to E123 (see Figure 2), the high-frequency shoulder of the peak was only slightly blue-shifted by 5 cm\({}^{-1}\), showing strong similarities to the C=N-H vibrational band of the WT. In E123D, however, the high frequency band was not present at all as a consequence of the shorter amino acid side chain length that prevented formation of a hydrogen bond with the Schiff base. We focused our QM/MM calculations on the structure with the rotamer configuration of E123 in the upward orientation to be able to accept a hydrogen bond from the Schiff base proton. This configuration was in fact the one in which the C=N-H vibration exhibited the strongest effect in our T127 variants.