Assessing the Role of R120 in the Gating of CrChR2 by Time-Resolved Spectroscopy from Femtoseconds to Seconds.

The light-gated ion channel channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2) is the most frequently used optogenetic tool in neurosciences. However, the precise molecular mechanism of the channel opening and the correlation among retinal isomerization, the photocycle, and the channel activity of the protein are missing. Here, we present electrophysiological and spectroscopic investigations on the R120H variant of CrChR2. R120 is a key residue in an extended network linking the retinal chromophore to several gates of the ion channel. We show that despite the deficient channel activity, the photocycle of the variant is intact. In a comparative study for R120H and the wild type, we resolve the vibrational changes in the spectral range of the retinal and amide I bands across the time range from femtoseconds to seconds. Analysis of the amide I mode reveals a significant impairment of the ultrafast protein response after retinal excitation. We conclude that channel opening in CrChR2 is prepared immediately after retinal excitation. Additionally, chromophore isomerization is essential for both photocycle and channel activities, although both processes can occur independently.

Ultrafast Protein Response in Channelrhodopsin-2 Studied by Time-Resolved Infrared Spectroscopy.

Ultrafast infrared transient absorption in the carbonyl vibrational region of protonated aspartate and glutamate residues in channelrhodopsin-2 from Chlamydomonas reinhardtii shows immediate protein response to retinal excitation. The observed difference bands are formed directly after the excitation on the subpicosecond time scale and were assigned to side chains in the retinal vicinity, such as D156 and E90. This finding implies an ultrafast and effective energy transfer from the retinal to its environment via hydrogen-bonded networks and reveals extraordinarily strong chromophore-protein coupling and intense interaction within the protein. Relevance to the protein function as an optically gated ion channel is discussed.

Fluorescence and excited state dynamics of the deprotonated Schiff base retinal in proteorhodopsin.

The UV light absorbing species of proteorhodopsin with deprotonated Schiff base retinal was investigated using steady-state fluorescence and femtosecond pump-probe spectroscopy. Compared to the all-trans retinal with protonated Schiff base, the deprotonated chromophore absorbs at 365 nm and exhibits a blue-shifted fluorescence spectrum. The unusually long-lived excited state decays bi-exponentially with time constants of 8 ps and 130 ps to form a deprotonated 13-cis retinal as the primary photo-product.