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 P2 380 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 P3 530 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.
Bibliographical noteFunding Information:
The publication of this article was funded by Freie Universität Berlin.The research was funded by the German Research Foundation via the SFB-1078 projects B3 (to J.H.) and B4 (to R.S.). I.S. thanks the SFB 1078 for support within the Mercator program. R.K.K. acknowledges support from the Lady Davis Trust for Shunbrun postdoctoral fellowship. I.S. gratefully acknowledges funding by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (Grant No. 678169 "PhotoMutant").
© 2019 by the authors.
- Flash photolysis
- Hybrid QM/MM simulation
- Resonance Raman