UV-Spectroscopy
We apply stationary and transient UV/VIS spectroscopy to study photocycle intermediates of light activated proteins like Channelrhodopsins (ChR). The characteristic wavelength at which ChR absorbs light is regulated by interactions between protein (opsin) and retinylidene Schiff base chromophore. The all-trans retinal chromophore is linked to the opsin through a protonated Schiff base at position Lys-257. ChR-2 has a visible absorbance maximum at 470 nm. Illumination isomerizes the all-trans retinal to the 13-cis-form, and the protein proceeds through a series of transient photointermediates. The photocycle is a result of structural rearrangements in the protein that yield shifts in the absorption maximum. Recent spectroscopic studies have shown that the P520 ChR2 photocycle intermediate reflects the channel open state absorbing maximally at 520 nm. P520 is in a pH-dependent equilibrium with the blue shifted state P390 that contains a deprotonated Schiff base species. Residues that interact with the retinal-polyene system, including the counter ion, influence photo-isomerization, protonation, reisomerization and conformational changes. These critical residues can be substituted by site-directed mutagenesis. The resulting spectroscopic changes we observe in terms of absorbance behavior and photocycle kinetics give us clues about the gating mechanism of ChRs.