Abstract
Channelrhodopsin (ChR) is a key protein of the optogenetic toolkit. C1C2, a functional chimeric protein of Chlamydomonas reinhardtii ChR1 and ChR2, is the only ChR whose crystal structure has been solved, and thus uniquely suitable for structure-based analysis. We report C1C2 photoreaction dynamics with ultrafast transient absorption and multi-pulse spectroscopy combined with target analysis and structure-based hybrid quantum mechanics/molecular mechanics calculations. Two relaxation pathways exist on the excited (S1) state through two conical intersections CI1 and CI2, that are reached via clockwise and counter-clockwise rotations: (i) the C13=C14 isomerization path with 450 fs via CI1 and (ii) a relaxation path to the initial ground state with 2.0 ps and 11 ps via CI2, depending on the hydrogen-bonding network, hence indicating active-site structural heterogeneity. The presence of the additional conical intersection CI2 rationalizes the relatively low quantum yield of photoisomerization (30 ± 3%), reported here. Furthermore, we show the photoreaction dynamics from picoseconds to seconds, characterizing the complete photocycle of C1C2.
Highlights
The discovery of channelrhodopsins (ChRs)[1, 2] lead to a breakthrough in optogenetics[3], which became a key technology in current neuroscience to optically control neural activity with high spatio-temporal precision[4,5,6]
The analysis program calculates both evolution-associated difference spectra (EADS) and decay-associated difference spectra (DADS) and the time constants that follow from the analysis apply to both[20]
We investigated the excited-state and photoproduct-state dynamics of a functional chimeric channelrhodopsin C1C2, with known X-ray structure and which has properties similar to Chlamydomonas reinhardtii ChR1 and ChR2 with respect to absorbance, ion selectivity and photocurrent kinetics
Summary
The discovery of channelrhodopsins (ChRs)[1, 2] lead to a breakthrough in optogenetics[3], which became a key technology in current neuroscience to optically control neural activity with high spatio-temporal precision[4,5,6]. Observation of the molecular dynamics from the femtosecond to millisecond time scales is essential for elucidation of the activation mechanisms of ChRs. A combination of time-resolved information on photoactivation with structure-based calculations is a powerful approach to obtain a molecular picture of the photoinduced initial structural dynamics of ChRs. C1C2, a chimeric protein of Chlamydomonas reinhardtii ChR1 and Chlamydomonas reinhardtii ChR2, is the only channelrhodopsin whose crystal structures were solved up to now[13, 14], providing the first direct molecular basis to understand the activation process of ChRs (Fig. 1). In combination with pump-dump-probe spectroscopy, target analysis and structure-based hybrid quantum mechanics/molecular mechanics calculations, we propose a multi-path excited-state dynamics model on C1C2 with two distinct conical intersections; a 450 fs isomerization path and a relaxation path with 2.0 ps and 11 ps. We further present a complete photocycle model of C1C2 from femtoseconds to seconds, which will be beneficial for further understanding of the activation mechanism of ChRs, and reveal striking kinetic differences on ground state dynamics between C1C2 and ChR2
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