The Photocycle of Channelrhodopsin‐2: Ultrafast Reaction Dynamics and Subsequent Reaction Steps

Abstract

The photocycle of channelrhodopsin-2 is investigated in a comprehensive study by ultrafast absorption and fluorescence spectroscopy as well as flash photolysis in the visible spectral range. The ultrafast techniques reveal an excited-state decay mechanism analogous to that of the archaeal bacteriorhodopsin and sensory rhodopsin II from Natronomonas pharaonis. After a fast vibrational relaxation of the excited-state population with 150 fs its decay with mainly 400 fs is observed. Hereby, both the initial all-trans retinal ground state and the 13-cis-retinal K photoproduct are populated. The reaction proceeds with a 2.7 ps component assigned to cooling processes. Small spectral shifts are observed on a 200 ps timescale. They are attributed to conformational rearrangements in the retinal binding pocket. The subsequent dynamics progresses with the formation of an M-like intermediate (7 and 120 μs), which decays into red-shifted states within 3 ms. Ground-state recovery including channel closing and reisomerization of the retinal chromophore occurs in a triexponential manner (6 ms, 33 ms, 3.4 s). To learn more about the energy barriers between the different photocycle intermediates, temperature-dependent flash photolysis measurements are performed between 10 and 30°C. The first five time constants decrease with increasing temperature. The calculated thermodynamic parameters indicate that the closing mechanism is controlled by large negative entropy changes. The last time constant is temperature independent, which demonstrates that the photocycle is most likely completed by a series of individual steps recovering the initial structure.

Enlightening the photoactive site of channelrhodopsin-2 by DNP-enhanced solid-state NMR spectroscopy

“…Our data, therefore, show unambiguously that ground-state ChR2 contains an all-trans,15-anti chromophore, which also explains the previously reported monoexponential decay of the photoexcited state (12) and the absence of a light adaption step as observed in bacteriorhodopsin, because the amount of this conformer is already close to 100% in the dark. Observing ChR2 Photointermediates by DNP-Enhanced MAS NMR.…”

“…In contrast to bacteriorhodopsin, no light adaption was observed using resonance Raman techniques (20) or visual spectroscopy (12). The occurrence of a conformer mixture in the ground state without light adaption would make ChR2 unique among the microbial retinal proteins, but additional data are needed to confirm these observations more directly at improved atomic resolution.…”

A Photochromic Histidine Kinase Rhodopsin (HKR1) That Is Bimodally Switched by Ultraviolet and Blue Light

“…We included relaxation of the excited state, with subsequent biexponential decay to an intermediate (I1), and a final, nondecaying species, which is identical to the P560 intermediate from flash photolysis. Similar to previous investigations on channelrhodopsin-2 from C. reinhardtii (25), we observe species-associated difference spectra (Fig. 4F) characteristic of vibrational and/or structural relaxation of the excited state in Ͻ300 fs, followed by a biexponential decay with 0.6 and 5 ps into I1.…”

Platymonas subcordiformis Channelrhodopsin-2 Function

“…Furthermore, the data acquisition, which entails repetitive flashing of samples to acquire transients with adequate signal-to-noise ratio, is complicated for slow cycling sensory rhodopsins such as ChRs because it is difficult to avoid data distortion from secondary photoexcitation of long-lived photointermediates. The single photocycle model, based on the unidirectional scheme, obtained in absorption studies (11,12) reproduces reasonably well the channel opening and closing kinetics in laser experiments. However, it falls short in predicting the transient channel current produced by long light pulses.…”