Abstract:Channelrhodopsins (ChRs) are microbial light-gated ion channels utilized in optogenetics to control neural activity with light . Light absorption causes retinal chromophore isomerization and subsequent protein conformational changes visualized as optically distinguished intermediates, coupled with channel opening and closing. However, the detailed molecular events underlying channel gating remain unknown. We performed time-resolved serial femtosecond crystallographic analyses of ChR by using an X-ray free elec… Show more
“…2C). This retinal movement resembles that seen in the TR-SFX study of channelrhodopsin (ChR) at Δ t =1 ms, by Oda et al (44). However, helix C of ChR was pushed outward by the retinal conformational change, while helix C of NM-R3 slightly moved within 0.5 Å.…”
Section: Discussionsupporting
confidence: 81%
“…However, helix C of ChR was pushed outward by the retinal conformational change, while helix C of NM-R3 slightly moved within 0.5 Å. The authors proposed that the sequential movements of ChR induced water influx from the cytoplasmic side (44). The structural changes of the retinal and helix C in NM-R3 may assist the anion pump and/or disrupt the pathway for the reverse anion transfer.…”
Light-driven chloride-pumping rhodopsins actively transport anions, including various halide ions, across cell membranes. Recent studies using time-resolved serial femtosecond crystallography (TR-SFX) have uncovered the structural changes and ion transfer mechanisms in light-driven cation-pumping rhodopsins. However, the mechanism by which the conformational changes pump an anion to achieve unidirectional ion transport, from the extracellular side to the cytoplasmic side, in anion-pumping rhodopsins remains enigmatic. We have collected TR-SFX data of Nonlabens marinus rhodopsin-3 (NM-R3), derived from a marine flavobacterium, at 10 µs and 1 ms time-points after photoexcitation. Our structural analysis reveals the conformational alterations during ion transfer and after ion release. Movements of the retinal chromophore initially displace a conserved tryptophan to the cytoplasmic side of NM-R3, accompanied with a slight shift of the halide ion bound to the retinal. After ion release, the inward movements of helix C and helix G and the lateral displacements of the retinal block access to the extracellular side of NM-R3. Anomalous signal data have also been obtained from NM-R3 crystals containing iodide ions. The anomalous density maps provide insight into the halide binding site for ion transfer in NM-R3.
“…2C). This retinal movement resembles that seen in the TR-SFX study of channelrhodopsin (ChR) at Δ t =1 ms, by Oda et al (44). However, helix C of ChR was pushed outward by the retinal conformational change, while helix C of NM-R3 slightly moved within 0.5 Å.…”
Section: Discussionsupporting
confidence: 81%
“…However, helix C of ChR was pushed outward by the retinal conformational change, while helix C of NM-R3 slightly moved within 0.5 Å. The authors proposed that the sequential movements of ChR induced water influx from the cytoplasmic side (44). The structural changes of the retinal and helix C in NM-R3 may assist the anion pump and/or disrupt the pathway for the reverse anion transfer.…”
Light-driven chloride-pumping rhodopsins actively transport anions, including various halide ions, across cell membranes. Recent studies using time-resolved serial femtosecond crystallography (TR-SFX) have uncovered the structural changes and ion transfer mechanisms in light-driven cation-pumping rhodopsins. However, the mechanism by which the conformational changes pump an anion to achieve unidirectional ion transport, from the extracellular side to the cytoplasmic side, in anion-pumping rhodopsins remains enigmatic. We have collected TR-SFX data of Nonlabens marinus rhodopsin-3 (NM-R3), derived from a marine flavobacterium, at 10 µs and 1 ms time-points after photoexcitation. Our structural analysis reveals the conformational alterations during ion transfer and after ion release. Movements of the retinal chromophore initially displace a conserved tryptophan to the cytoplasmic side of NM-R3, accompanied with a slight shift of the halide ion bound to the retinal. After ion release, the inward movements of helix C and helix G and the lateral displacements of the retinal block access to the extracellular side of NM-R3. Anomalous signal data have also been obtained from NM-R3 crystals containing iodide ions. The anomalous density maps provide insight into the halide binding site for ion transfer in NM-R3.
“… Figure 5—figure supplement 1. Retinal Schiff base, Glu129 (Glu68 in Gt ACR1), and Asp292 (Asp234 in Gt ACR1) in the structures of the ground state (green sticks, PDB code 7C86; Oda et al, 2021 ) and 4 ms after light irradiation (cyan sticks, PDB code 7E6X; Oda et al, 2021 ) of cation channelrhodopsin C1C2. Values indicate the distances between the N atom of the Schiff base and the C α atom of Glu129 (Å).…”
Anion channelrhodopsin from Guillardia theta (GtACR1) has Asp234 (3.2 Å) and Glu68 (5.3 Å) near the protonated Schiff base. Here, we investigate mutant GtACR1s (e.g., E68Q/D234N) expressed in HEK293 cells. The influence of the acidic residues on the absorption wavelengths was also analyzed using a quantum mechanical/molecular mechanical approach. The calculated protonation pattern indicates that Asp234 is deprotonated and Glu68 is protonated in the original crystal structures. The D234E mutation and the E68Q/D234N mutation shorten and lengthen the measured and calculated absorption wavelengths, respectively, which suggests that Asp234 is deprotonated in the wild-type GtACR1. Molecular dynamics simulations show that upon mutation of deprotonated Asp234 to asparagine, deprotonated Glu68 reorients toward the Schiff base and the calculated absorption wavelength remains unchanged. The formation of the proton transfer pathway via Asp234 toward Glu68 and the disconnection of the anion conducting channel are likely a basis of the gating mechanism.
“…These channels, channelrhodopsins (737 aas), evolved from bacterio-rhodopsin consisting of seven transmembrane segments covalently linked to a retinal chromophore [ 276 ]. Light absorption induces isomerisation of the retinal, which, in turn, causes a set of conformational changes leading to the opening of a pore that allows ions to pass through [ 277 , 278 , 279 ]. In cryptophytes, a distinct group of green algae, there are also cation-conducting channel rhodopsins that specifically conduct anions [ 280 , 281 ] and another group of channels that are more structurally related to haloarchaeal rhodopsins and have different functional properties [ 282 ].…”
Section: Cell Signalling and Sensory Motricitymentioning
How can single cells without nervous systems perform complex behaviours such as habituation, associative learning and decision making, which are considered the hallmark of animals with a brain? Are there molecular systems that underlie cognitive properties equivalent to those of the brain? This review follows the development of the idea of molecular brains from Darwin’s “root brain hypothesis”, through bacterial chemotaxis, to the recent discovery of neuron-like r-protein networks in the ribosome. By combining a structural biology view with a Bayesian brain approach, this review explores the evolutionary labyrinth of information processing systems across scales. Ribosomal protein networks open a window into what were probably the earliest signalling systems to emerge before the radiation of the three kingdoms. While ribosomal networks are characterised by long-lasting interactions between their protein nodes, cell signalling networks are essentially based on transient interactions. As a corollary, while signals propagated in persistent networks may be ephemeral, networks whose interactions are transient constrain signals diffusing into the cytoplasm to be durable in time, such as post-translational modifications of proteins or second messenger synthesis. The duration and nature of the signals, in turn, implies different mechanisms for the integration of multiple signals and decision making. Evolution then reinvented networks with persistent interactions with the development of nervous systems in metazoans. Ribosomal protein networks and simple nervous systems display architectural and functional analogies whose comparison could suggest scale invariance in information processing. At the molecular level, the significant complexification of eukaryotic ribosomal protein networks is associated with a burst in the acquisition of new conserved aromatic amino acids. Knowing that aromatic residues play a critical role in allosteric receptors and channels, this observation suggests a general role of π systems and their interactions with charged amino acids in multiple signal integration and information processing. We think that these findings may provide the molecular basis for designing future computers with organic processors.
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