Structural Factors Determining the Absorption Spectrum of Channelrhodopsins: A Case Study of the Chimera C1C2

Abstract

Channelrhodopsins are photosensitive proteins that trigger flagella motion in single cell algae and have been successfully utilized in optogenetic applications. In optogenetics light is used to activate neural cells in living organisms, which can be achieved by exploiting the ion channel signaling of channelrhodopsins. Tailoring channelrhodopsins for such applications includes the tuning of the absorption maximum. In order to establish rational design and to obtain a desired spectral shift, a basic understanding of the absorption spectrum is required. We have studied the chimera C1C2 as a representative of this protein family and the first member with an available crystal structure. For this purpose, we sampled the conformations of C1C2 using QM/MM molecular dynamics, and subjected the resulting snapshots of the trajectory to excitation energy calculations using ADC(2) and simplified TD-DFT. In contrast to previous reports, we found that different hydrogen-bonding networks-involving the retinal protonated Schiff base, the putative counterions E162 and D292 as well as water molecules-had only a small impact on the absorption spectrum. However, in case of deprotonated E162 increasing the distance to the Schiff base hydrogen-bonding partner led to a systematic blue shift. The β-ionone ring rotation was identified as another important contributor. Yet the most important factor was found to be the bond length alternation and bond order alternation that were linearly correlated to the absorption maximum by up to 62 % and 82 %, respectively. We ascribe this novel insight into the structural basis of the absorption spectrum to our enhanced protein setup that includes membrane embedding as well as long and extensive sampling. File list (2) download file view on ChemRxiv manuscript_v1.30.pdf (1.81 MiB) download file view on ChemRxiv supplementary_v1.10.pdf (472.85 KiB)

The Impact of Retinal Configuration on the Protein–Chromophore Interactions in Bistable Jumping Spider Rhodopsin-1

“…The MM partition included the remaining protein, lipids, ions and waters that were treated with the classical ff14SB Amber force field using a 12-Å nonbonding cutoff [ 21 ]. DFTB was chosen to treat the QM region, because this method has been shown to yield structures in close agreement with those obtained using the B3LYP functional with medium-sized basis sets while remaining computationally feasible for trajectories in the nanosecond timescale [ 23 , 24 , 25 ]. These QM/MM trajectories were propagated for 1 ns with a timestep of 1 fs at 300 K using the Langevin thermostat and keeping the pressure at 1 atm.…”

Deciphering the Spectral Tuning Mechanism in Proteorhodopsin: The Dominant Role of Electrostatics Instead of Chromophore Geometry

“…This includes the BOA, BLA and C 14 À C 15 bond length which have been reported to correlate with the absorption maxima. [21,22,30] To gain further insight we analyzed structural factors from retinal geometries used for the spectrum generation (Figure 4A,C,E and Table S3 in Supporting Information).…”

“…Such correlations between the retinal structural factors and the predicted absorption maxima were reported by Choongkeun et al for BPR and Adam et al for Channelrhodopsin C1C2. [22,30] This high correlation raised the question of whether the change of the bond length, which is induced by the protein environment, is sufficient to explain the spectral shift as discussed by Mao et al [21] Hence, we recomputed the absorption maxima by omitting the protein environment from the simulation, while keeping the retinal geometry unchanged.…”

“…[21,22] In general, the bond length alternation (BLA) or bond order alternation (BOA), correlate strongly with the absorption maxima of rhodopsins. [30][31][32] The BOA is defined as the difference between the average bond order (BO) of the single bonds subtracted from the average BO of the double bonds. [32] Likewise, the BLA is the difference in the average length of single bonds from those of double bonds.…”

Insight into the isomerization mechanism of retinal proteins from hybrid quantum mechanics/molecular mechanics simulations

“…This is the first contribution to the reaction coordinate which is referred to as an inversion of the bond length alternation or the bond order alternation. 9 The second component of the reaction coordinate is the rotation of a double bond, which sets in as the bond is elongated. As a twist reaches 70 or larger, the ground and excited state are close such that a radiationless decay between these two states becomes feasible.…”