Channelrhodopsin-2 (ChR2) is a member of the new class of light-gated ion channels which serve as phototaxis receptors in the green alga Chlamydomonas reinhardtii. The protein is employed in optogenetics where neural circuits are optically stimulated under high spatiotemporal control. Despite its rapidly growing use in physiological experiments, the reaction mechanism of ChR2 is poorly understood. Here, we applied vibrational spectroscopy to trace structural changes of ChR2 after light-excitation of the retinal chromophore. FT-IR difference spectra of the various photocycle intermediates revealed that stages of the photoreaction preceding (P(1) state) and succeeding (P(4)) the conductive state of the channel (P(3)) are associated with large conformational changes of the protein backbone as indicate by strong differences in the amide I bands. Critical hydrogen-bonding changes of protonated carboxylic amino acid side chains (D156, E90) were detected and discussed with regard to the functional mechanism. We used the C128T mutant where the lifetime of P(3) is prolonged and applied FT-IR and resonance Raman spectroscopy to study the conductive P(3) state of ChR2. Finally, a mechanistic model is proposed that links the observed structural changes of ChR2 to the changes in the channel's conductance.
Channelrhodopsin-2 mediates phototaxis in green algae by acting as a light-gated cation channel. As a result of this property, it is used as a novel optogenetic tool in neurophysiological applications. Structural information is still scant and we present here the first resonance Raman spectra of channelrhodopsin-2. Spectra of detergent solubilized and lipid-reconstituted protein were recorded under pre-resonant conditions to exclusively probe retinal in its electronic ground state. All-trans retinal was identified to be the favoured configuration of the chromophore but significant contributions of 13-cis were detected. Pre-illumination hardly changed the isomeric composition but small amounts of presumably 9-cis retinal were found in the light-adapted state. Spectral analysis suggested that the Schiff base proton is strongly hydrogen-bonded to a nearby water molecule.
The light-gated cation channel Channelrhodopsin-2 (ChR2), a retinylidene protein found in the eye-spot of Chlamydomonas reinhardtii, became an optogenetic tool to trigger neurophysiological responses by light and, thus, revolutionized spatio-temporal studies of such processes. The reaction mechanism still remains elusive but recent vibrational spectroscopic experiments started to resolve details of the associated structural changes during the photocycle. Large alterations in the polypeptide backbone were observed by FT-IR spectroscopy that precede and succeed the opening and closing of the channel, respectively. However, the molecular switch that controls gating is still unknown. Here, we present difference spectra of the D156E mutant of ChR2 and assign the observed vibrational bands to crucial hydrogen bonding changes of this residue in various intermediate states of the photoreaction. By comparison with spectra of wild-type ChR2 and the C128T mutant and correlation to electrophysiological studies, we propose the DC gate as a crucial hydrogen-bonding interaction between D156 and C128 which may represent the valve of the channel.
The primary reaction dynamics of channelrhodopsin-2 was investigated using femtosecond vis-pump/mid-IR probe spectroscopy. Due to the fast deactivation of the excited state in channelrhodopsin-2, it is possible to observe the direct impact of retinal isomerization on the protein surrounding. We show that the dominant negative band at 1665 cm(-1) tentatively assigned to an amide I vibration is developed with a time constant of 0.5 ps. Also a variety of side-chain vibrations are formed or intensified on this time scale. The comparison of the light-induced FT-IR spectra of channelrhodopsin-2 in H2O and D2O at 80 K enabled us to tentatively identify the contribution of Arg side chain(s). The subsequently observed decay of nearly the whole difference pattern has a particularly high impact on the C═C and C═N stretching vibrations of the retinal. This suggests that the underlying mechanism describes a cooling process in which the excess energy is redirected toward the retinal surrounding, e.g., the protein and functional water molecules. The pronounced protein contributions in comparison to other rhodopsins point to a very efficient energy redistribution in channelrhodopsin-2.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.