Abstract
Channelrhodopsins (ChRs) belong to the unique class of light-gated ion channels. The structure of channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2) has been resolved, but the mechanistic link between light-induced isomerization of the chromophore retinal and channel gating remains elusive. Replacements of residues C128 and D156 (DC gate) resulted in drastic effects in channel closure. T127 is localized close to the retinal Schiff base and links the DC gate to the Schiff base. The homologous residue in bacteriorhodopsin (T89) has been shown to be crucial for the visible absorption maximum and dark–light adaptation, suggesting an interaction with the retinylidene chromophore, but the replacement had little effect on photocycle kinetics and proton pumping activity. Here, we show that the T127A and T127S variants of CrChR2 leave the visible absorption maximum unaffected. We inferred from hybrid quantum mechanics/molecular mechanics (QM/MM) calculations and resonance Raman spectroscopy that the hydroxylic side chain of T127 is hydrogen-bonded to E123 and the latter is hydrogen-bonded to the retinal Schiff base. The C=N–H vibration of the Schiff base in the T127A variant was 1674 cm−1, the highest among all rhodopsins reported to date. We also found heterogeneity in the Schiff base ground state vibrational properties due to different rotamer conformations of E123. The photoreaction of T127A is characterized by a long-lived P2380 state during which the Schiff base is deprotonated. The conservative replacement of T127S hardly affected the photocycle kinetics. Thus, we inferred that the hydroxyl group at position 127 is part of the proton transfer pathway from D156 to the Schiff base during rise of the P3530 intermediate. This finding provides molecular reasons for the evolutionary conservation of the chemically homologous residues threonine, serine, and cysteine at this position in all channelrhodopsins known so far.
Highlights
Channelrhodopsins (ChRs) are members of the group of microbial rhodopsins that are light-gated cation channels
We report here the exceptionally high frequency of the Schiff base vibration in the T127A variant, indicating strong interaction between the protonated Schiff base and the carboxylic side chain of E123 in this variant
Due to insertion of the corresponding coding region with sequences for a C-terminal 10xHis tag and a linker behind the alpha factor signal sequence of the vector pPIC 9K into the EcoRI/NotI sites, the expressed protein led to a N-terminal extension of aa YVEFH and a C-terminal extension of aa ASHHHHHHHHHH. Based on this construct, which is referred in the following as can be triggered. Channelrhodopsin-2 (CrChR2)-WT or WT, T127 was substituted by serine (T127S) or alanine residues (T127A)
Summary
Channelrhodopsins (ChRs) are members of the group of microbial rhodopsins that are light-gated cation channels. They were originally found in the eyespot of the algae Chlamydomonas reinhardtii (Cr), Appl. Within the new research field of optogenetics, the coding sequences for light-responsive proteins together with regulating promoters can be introduced into complex organisms and expressed in a tissue-specific way, where cell processes can be triggered. Channelrhodopsin-2 (CrChR2) in neurons can depolarize the nerve cell when illuminated by light of the corresponding wavelength. This protein is already frequently used as optogenetic tool, aimed at medical approaches like restoring vision and hearing, the mechanism of action is still not completely understood. Comparison to related microbial rhodopsins where individual positions in the protein sequences are highly conserved could guide our approach towards mechanistically important amino acid residues which have demonstrated effects on photocycle kinetics, activity, and/or structural stability upon mutation
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
