Light-Dark Adaptation of Channelrhodopsin Involves Photoconversion between the all-trans and 13-cis Retinal Isomers.

Channelrhodopsins (ChR) are light-gated ion channels of green algae that are widely used to probe the function of neuronal cells with light. Most ChRs show a substantial reduction in photocurrents during illumination, a process named "light adaptation". The main objective of this spectroscopic study was to elucidate the molecular processes associated with light-dark adaptation. Here we show by liquid and solid-state nuclear magnetic resonance spectroscopy that the retinal chromophore of fully dark-adapted ChR is exclusively in an all-trans configuration. Resonance Raman (RR) spectroscopy, however, revealed that already low light intensities establish a photostationary equilibrium between all-trans,15-anti and 13-cis,15-syn configurations at a ratio of 3:1. The underlying photoreactions involve simultaneous isomerization of the C(13)═C(14) and C(15)═N bonds. Both isomers of this DAapp state may run through photoinduced reaction cycles initiated by photoisomerization of only the C(13)═C(14) bond. RR spectroscopic experiments further demonstrated that photoinduced conversion of the apparent dark-adapted (DAapp) state to the photocycle intermediates P500 and P390 is distinctly more efficient for the all-trans isomer than for the 13-cis isomer, possibly because of different chromophore-water interactions. Our data demonstrating two complementary photocycles of the DAapp isomers are fully consistent with the existence of two conducting states that vary in quantitative relation during light-dark adaptation, as suggested previously by electrical measurements.

A photochromic histidine kinase rhodopsin (HKR1) that is bimodally switched by ultraviolet and blue light.

Rhodopsins are light-activated chromoproteins that mediate signaling processes via transducer proteins or promote active or passive ion transport as ion pumps or directly light-activated channels. Here, we provide spectroscopic characterization of a rhodopsin from the Chlamydomonas eyespot. It belongs to a recently discovered but so far uncharacterized family of histidine kinase rhodopsins (HKRs). These are modular proteins consisting of rhodopsin, a histidine kinase, a response regulator, and in some cases an effector domain such as an adenylyl or guanylyl cyclase, all encoded in a single protein as a two-component system. The recombinant rhodopsin fragment, Rh, of HKR1 is a UVA receptor (λ(max) = 380 nm) that is photoconverted by UV light into a stable blue light-absorbing meta state Rh-Bl (λ(max) = 490 nm). Rh-Bl is converted back to Rh-UV by blue light. Raman spectroscopy revealed that the Rh-UV chromophore is in an unusual 13-cis,15-anti configuration, which explains why the chromophore is deprotonated. The excited state lifetime of Rh-UV is exceptionally stable, probably caused by a relatively unpolar retinal binding pocket, converting into the photoproduct within about 100 ps, whereas the blue form reacts 100 times faster. We propose that the photochromic HKR1 plays a role in the adaptation of behavioral responses in the presence of UVA light.

Photochemical chromophore isomerization in histidine kinase rhodopsin HKR1.

Histidine kinase rhodopsin 1 is a photoreceptor in green algae functioning as a UV-light sensor. It switches between a UV-absorbing state (Rh-UV) and a blue-absorbing state (Rh-Bl) with a protonated retinal Schiff base (RSB) cofactor in a mixture of 13-trans,15-anti and 13-cis,15-syn isomers. The present spectroscopic study now shows that cofactor-protein assembly stabilizes the protonated 13-trans,15-anti RSB isomer. Formation of the active photoswitch requires the photoinduced conversion to Rh-UV. The transitions between the Rh-Bl isomers and the deprotonated 13-cis,15-anti and 13-trans,15-syn isomers of Rh-UV proceed via multiple photoisomerizations of one or simultaneously two double bonds.

The chromophore structure of the long-lived intermediate of the C128T channelrhodopsin-2 variant.

The photocycle of the light-activated channel, channelrhodopsin-2 C128T, has been studied by resonance Raman (RR) spectroscopy focussing on the intermediates P380 and P353 that constitute a side pathway in the recovery of the parent state. The P353 species displays a UV-vis absorption spectrum with a fine-structure reminiscent of the reduced-retro form of bacteriorhodopsin, whereas the respective RR spectra differ substantially. Instead, the RR spectra of the P380/P353 intermediate couple are closely related to that of a free retinal in the all-trans configuration. These findings imply that the parent state recovery via P380/P353 involves the transient hydrolysis and re-formation of the retinal-protein linkage.