Channelrhodopsin-2 (ChR2) is a microbial type rhodopsin and a light-gated cation channel that controls phototaxis in Chlamydomonas. We expressed ChR2 in COS-cells, purified it, and subsequently investigated this unusual photoreceptor by flash photolysis and UV-visible and Fourier transform infrared difference spectroscopy. Several transient photoproducts of the wild type ChR2 were identified, and their kinetics and molecular properties were compared with those of the ChR2 mutant E90Q. Based on the spectroscopic data we developed a model of the photocycle comprising six distinguishable intermediates. This photocycle shows similarities to the photocycle of the ChR2-related Channelrhodopsin of Volvox but also displays significant differences. We show that molecular changes include retinal isomerization, changes in hydrogen bonding of carboxylic acids, and large alterations of the protein backbone structure. These alterations are stronger than those observed in the photocycle of other microbial rhodopsins like bacteriorhodopsin and are related to those occurring in animal rhodopsins. UV-visible and Fourier transform infrared difference spectroscopy revealed two late intermediates with different time constants of ‫؍‬ 6 and 40 s that exist during the recovery of the dark state. The carboxylic side chain of Glu 90 is involved in the slow transition. The molecular changes during the ChR2 photocycle are discussed with respect to other members of the rhodopsin family. Channelrhodopsins (ChRs)3 are light-gated cation channels (1, 2) that share homology with other microbial rhodopsins such as bacteriorhodopsin (BR), halorhodopsin (HR), and sensory rhodopsin (SR). In nature they serve as sensory photoreceptors for phobic responses and phototaxis in green algae (3-5). The light-induced ion conductance leads to depolarization of the cell membrane within milliseconds. Because of this property, in recent years ChRs have been widely used in the neuroscience field as a tool for depolarization of selected cell types or cell ensembles (6, 7). Furthermore, channelrhodopsins were used to control neuronal activity in Caenorhabditis elegans, Drosophila, zebrafish, chicken embryos, and mice (8 -13).As is typical for rhodopsins, light absorption induces isomerization of the ChR-chromophore with subsequent conformational changes in the protein (photocycle). Based on UV-visible spectroscopic and electrophysiological measurements, several schemes for this photocycle have been presented. A recent model for recombinant Volvox channelrhodopsin (VChR), purified from green monkey COS cells, comprises the two dark states D470 and D480, characterized by a fine structured UVvisible absorption spectrum with maxima at 470 and 480 nm, respectively (14). These two states, which exist in a pH-dependent equilibrium, are both converted by light via retinal isomerization and transient Schiff base deprotonation to the conducting state P510 or, under acidic conditions, to P530. These intermediates thermally relax back to the dark state equilibrium in a biphas...

Channelrhodopsins (ChRs) are light-gated ion channels that are widely used in optogenetics. They allow precise control of neuronal activity with light, but a detailed understanding of how the channel is gated and the ions are conducted is still lacking. The recent determination of the X-ray structural model in the closed state marks an important milestone. Herein the open state structure is presented and the early formation of the ion conducting pore is elucidated in atomic detail using time-resolved FTIR spectroscopy. Photo-isomerization of the retinal-chromophore causes a downward movement of the highly conserved E90, which opens the pore. Molecular dynamic (MD) simulations show that water molecules invade through this opened pore, Helix 2 tilts and the channel fully opens within ms. Since E90 is a highly conserved residue, the proposed E90-Helix2-tilt (EHT) model might describe a general activation mechanism and provides a new avenue for further mechanistic studies and engineering.

Background: Channelrhodopsin-2 is a light-gated ion channel extensively used in optogenetics.Results: Glu-90 is deprotonated in the open state and is crucial for ion selectivity.Conclusion: Protonation change of Glu-90 is part of the opening/closing of the conductive pore, and the functional protein unit is assumed to be the monomer.Significance: Understanding the gating mechanism is necessary for optimizing this optogenetic tool.

Nanodiamonds exhibit exceptional colloidal properties in aqueous media that lead to a wide range of applications in nanomedicine and other fields. Nevertheless, the role of surface chemistry on the hydration of nanodiamonds remains poorly understood. Here, we probed the water hydrogen bond network in aqueous dispersions of nanodiamonds by infrared, Raman, and X-ray absorption spectroscopies applied in situ in aqueous environment. Aqueous dispersions of nanodiamonds with hydrogenated, carboxylated, hydroxylated, and polyfunctional surface terminations were compared. A different hydrogen bond network was found in hydrogenated nanodiamonds dispersions compared to dispersions of nanodiamonds with other surface terminations. Although no hydrogen bonds are formed between water and hydrogenated surface groups, a long-range disruption of the water hydrogen bond network is evidenced in hydrogenated nanodiamonds dispersion. We propose that this unusual hydration structure results from electron accumulation at the diamond–water interface.

In the retinal binding pocket of rhodopsin, a Schiff base links the retinal ligand covalently to the Lys296 side chain. Light transforms the inverse agonist 11-cis-retinal into the agonist all-trans-retinal, leading to the active Meta II state. Crystal structures of Meta II and the active conformation of the opsin apoprotein revealed two openings of the 7-transmembrane (TM) bundle towards the hydrophobic core of the membrane, one between TM1/TM7 and one between TM5/TM6, respectively. Computational analysis revealed a putative ligand channel connecting the openings and traversing the binding pocket. Identified constrictions within the channel motivated this study of 35 rhodopsin mutants in which single amino acids lining the channel were replaced. 11-cis-retinal uptake and all-trans-retinal release were measured using UV/visible and fluorescence spectroscopy. Most mutations slow or accelerate both uptake and release, often with opposite effects. Mutations closer to the Lys296 active site show larger effects. The nucleophile hydroxylamine accelerates retinal release 80 times but the action profile of the mutants remains very similar. The data show that the mutations do not probe local channel permeability but rather affect global protein dynamics, with the focal point in the ligand pocket. We propose a model for retinal/receptor interaction in which the active receptor conformation sets the open state of the channel for 11-cis-retinal and all-trans-retinal, with positioning of the ligand at the active site as the kinetic bottleneck. Although other G protein-coupled receptors lack the covalent link to the protein, the access of ligands to their binding pocket may follow similar schemes.G protein-coupled-receptor | regeneration | signal transduction T he photoreceptor rhodopsin is a prototypical member of the superfamily of seven transmembrane (7 TM) helix or G protein-coupled receptors (GPCRs). Rhodopsin consists of the apoprotein opsin and the covalently bound chromophoric ligand 11-cis-retinal, which acts as a powerful inverse agonist and holds the receptor in its inactive conformation. Absorption of a photon isomerizes the chromophore to the agonist all-trans-retinal which in turn triggers conformational changes of the protein leading to the active, G protein-binding form metarhodopsin II (Meta II). In rod cells Meta II decays within minutes by hydrolysis of the Schiff base and release of all-trans-retinal. The regeneration of the rhodopsin dark state by uptake of new 11-cis-retinal effectively suppresses the basal activity of opsin and primes it at the same time for photoactivation (1, 2).In the rhodopsin dark state, 11-cis-retinal is buried in its binding pocket in the core of the 7 TM bundle. The side chain of Lys296 in TM7 protrudes into the pocket and provides the active site for the protonated Schiff base linkage between ligand and protein. The protonated Schiff base is stabilized by a salt-bridge with its counterion, Glu113, and by residues in the second extracellular loop, which is folded deeply into the...