Ultrafast Infrared Spectroscopy on Channelrhodopsin-2 Reveals Efficient Energy Transfer from the Retinal Chromophore to the Protein

Abstract

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.

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

“…For the retinal 13 C14 signal, one additional peak at 124.2 ppm is observed in addition to the groundstate signal, whereas the 13 C15 resonance broadens slightly. Similarly, a new 15 N signal upfield of the pSB ground-state resonance is detected at 181 ppm. It can be concluded that illumination at low temperatures leads to a mixture of two states, one of which corresponds to the ground state.…”

“…The authors in refs. 45 and 46 reported upfield shifts for both 13 C14-retinal and 15 N pSB signals with respect to the ground state by 4.9 and 8.7 ppm, respectively. The same trend is observed here, and the newly generated state is, therefore, assigned to the K-like intermediate P 500 1 containing a 13-cis,15-anti chromophore.…”

“…A chimera of ChR1 and ChR2 has been crystallized to yield a structure at 2.3-Å resolution (9). However, little is known on how this coupling functions on a molecular level, and a large number of studies based on visible (10-13), IR (11,[14][15][16][17][18][19], resonance Raman spectroscopy (20, 21), and EPR spectroscopy (22, 23) has been performed to address this question.The photocycles of microbial rhodopsins are usually compared with bacteriorhodopsin, the first discovered and most studied lightdriven proton pump (24). Without any illumination, microbial retinal proteins thermally equilibrate into a dark state (25).…”

“…A chimera of ChR1 and ChR2 has been crystallized to yield a structure at 2.3-Å resolution (9). However, little is known on how this coupling functions on a molecular level, and a large number of studies based on visible (10-13), IR (11,(14)(15)(16)(17)(18)(19), resonance Raman spectroscopy (20,21), and EPR spectroscopy (22,23) has been performed to address this question.…”

“…Here, DNP-enhanced solid-state NMR spectroscopy has been applied to 15 N-labeled ChR2 carrying 14,15-13 C 2 retinal reconstituted into lipid bilayers and incubated with the DNP polarizing agent AMUPOL (52) in a glycerol-water mixture. The labeling scheme adopted here is shown in Fig.…”