Time-resolved serial femtosecond crystallography reveals early structural changes in channelrhodopsin.

Channelrhodopsins (ChRs) are microbial light-gated ion channels utilized in optogenetics to control neural activity with light . Light absorption causes retinal chromophore isomerization and subsequent protein conformational changes visualized as optically distinguished intermediates, coupled with channel opening and closing. However, the detailed molecular events underlying channel gating remain unknown. We performed time-resolved serial femtosecond crystallographic analyses of ChR by using an X-ray free electron laser, which revealed conformational changes following photoactivation. The isomerized retinal adopts a twisted conformation and shifts toward the putative internal proton donor residues, consequently inducing an outward shift of TM3, as well as a local deformation in TM7. These early conformational changes in the pore-forming helices should be the triggers that lead to opening of the ion conducting pore.

Crystal structure of the red light-activated channelrhodopsin Chrimson.

Channelrhodopsins are light-activated ion channels that mediate cation permeation across cell membranes upon light absorption. Red-light-activated channelrhodopsins are of particular interest, because red light penetrates deeper into biological tissues and also enables dual-color experiments in combination with blue-light-activated optogenetic tools. Here we report the crystal structure of the most red-shifted channelrhodopsin from the algae Chlamydomonas noctigama, Chrimson, at 2.6 Å resolution. Chrimson resembles prokaryotic proton pumps in the retinal binding pocket, while sharing similarity with other channelrhodopsins in the ion-conducting pore. Concomitant mutation analysis identified the structural features that are responsible for Chrimson's red light sensitivity; namely, the protonation of the counterion for the retinal Schiff base, and the polar residue distribution and rigidity of the retinal binding pocket. Based on these mechanistic insights, we engineered ChrimsonSA, a mutant with a maximum activation wavelength red-shifted beyond 605 nm and accelerated closing kinetics.

Chimeras of channelrhodopsin-1 and -2 from Chlamydomonas reinhardtii exhibit distinctive light-induced structural changes from channelrhodopsin-2.

Channelrhodopsin-2 (ChR2) from the green alga Chlamydomonas reinhardtii functions as a light-gated cation channel that has been developed as an optogenetic tool to stimulate specific nerve cells in animals and control their behavior by illumination. The molecular mechanism of ChR2 has been extensively studied by a variety of spectroscopic methods, including light-induced difference Fourier transform infrared (FTIR) spectroscopy, which is sensitive to structural changes in the protein upon light activation. An atomic structure of channelrhodopsin was recently determined by x-ray crystallography using a chimera of channelrhodopsin-1 (ChR1) and ChR2. Electrophysiological studies have shown that ChR1/ChR2 chimeras are less desensitized upon continuous illumination than native ChR2, implying that there are some structural differences between ChR2 and chimeras. In this study, we applied light-induced difference FTIR spectroscopy to ChR2 and ChR1/ChR2 chimeras to determine the molecular basis underlying these functional differences. Upon continuous illumination, ChR1/ChR2 chimeras exhibited structural changes distinct from those in ChR2. In particular, the protonation state of a glutamate residue, Glu-129 (Glu-90 in ChR2 numbering), in the ChR chimeras is not changed as dramatically as in ChR2. Moreover, using mutants stabilizing particular photointermediates as well as time-resolved measurements, we identified some differences between the major photointermediates of ChR2 and ChR1/ChR2 chimeras. Taken together, our data indicate that the gating and desensitizing processes in ChR1/ChR2 chimeras are different from those in ChR2 and that these differences should be considered in the rational design of new optogenetic tools based on channelrhodopsins.

Exciton circular dichroism in channelrhodopsin.

Channelrhodopsins (ChRs) are of great interest currently because of their important applications in optogenetics, the photostimulation of neurons. The absorption and circular dichroism (CD) spectra of C1C2, a chimera of ChR1 and ChR2 of Chlamydomonas reinhardtii, have been studied experimentally and theoretically. The visible absorption spectrum of C1C2 shows vibronic fine structure in the 470 nm band, consistent with the relatively nonpolar binding site. The CD spectrum has a negative band at 492 nm (Δε(max) = -6.17 M(-1) cm(-1)) and a positive band at 434 nm (Δε(max) = +6.65 M(-1) cm(-1)), indicating exciton coupling within the C1C2 dimer. Time-dependent density functional theory (TDDFT) calculations are reported for three models of the C1C2 chromophore: (1) the isolated protonated retinal Schiff base (retPSB); (2) an ion pair, including the retPSB chromophore, two carboxylate side chains (Asp 292, Glu 162), modeled by acetate, and a water molecule; and (3) a hybrid quantum mechanical/molecular mechanical (QM/MM) model depicting the binding pocket, in which the QM part consists of the same ion pair as that in (2) and the MM part consists of the protein residues surrounding the ion pair within 10 Å. For each of these models, the CD of both the monomer and the dimer was calculated with TDDFT. For the dimer, DeVoe polarizability theory and exciton calculations were also performed. The exciton calculations were supplemented by calculations of the coupling of the retinal transition with aromatic and peptide group transitions. For the dimer, all three methods and three models give a long-wavelength C2-axis-polarized band, negative in CD, and a short-wavelength band polarized perpendicular to the C2 axis with positive CD, differing in wavelength by 1-5 nm. Only the retPSB model gives an exciton couplet that agrees qualitatively with experiment. The other two models give a predominantly or solely positive band. We further analyze an N-terminal truncated mutant because it was assumed that the N-terminal domain has a crucial role in the dimerization of ChRs. However, the CD spectrum of this mutant has an exciton couplet comparable to that of the wild-type, demonstrating that it is dimeric. Patch-clamp experiments suggest that the N-terminal domain is involved in protein stabilization and channel kinetics rather than dimerization or channel activity.