The microbial opsin family of optogenetic tools

Abstract

The capture and utilization of light is an exquisitely evolved process. The single-component microbial opsins, although more limited than multicomponent cascades in processing, display unparalleled compactness and speed. Recent advances in understanding microbial opsins have been driven by molecular engineering for optogenetics and by comparative genomics. Here we provide a Primer on these light-activated ion channels and pumps, describe a group of opsins bridging prior categories, and explore the convergence of molecular engineering and genomic discovery for the utilization and understanding of these remarkable molecular machines.

Figure 1. Type I Microbial Rhodopsins

BR (and PRs) pump protons from the cytoplasm to the extracellular medium, and HRs pump chloride into the cytoplasm; all three hyperpolarize the cell. SRs lack TM ion transport in the presence of the His kinase transducer protein Htr; and algal ChRs conduct cations across the membrane in both directions but always along the electrochemical gradient of the transported ions. In SRs and ChRs, proton translocation within the protein is linked to efficient photocycle progression, but these protons are not necessarily exchanged between the intra- and extracellular spaces.

Figure 2. Photoreaction Mechanism

(A) Light-mediated isomerization of the retinal Schiff base (RSB). Top: retinal in the all-trans state, as found in the dark-adapted state of microbial rhodopsins and in the light-activated forms of type II rhodopsins of higher eukaryotes. The absorption of a photon converts the retinal from the all-trans to the 11-cis configuration. Bottom: 11-cis retinal is found only in type II rhodopsins, where it binds to the opsin in the dark state before isomerizing to the all-trans position after photon absorption. (B) The photocycle of BR is initiated from the dark state where photon absorption activates a sequence of photochemical reactions and structural changes represented by the indicated photointermediates. Also shown is the configuration of the RSB in each step (in red) and the wavelength at which each intermediate maximally absorbs light (in blue). (C) Summary of proton transport reactions during the BR photocycle. Photon absorption (1) initiates the conformational switch in the RSB, leading to transfer of a proton to Asp85 (2), release of a proton from the proton release complex (PRC, 3), reprotonation of the RSB by Asp96 (4), uptake of a proton from the cytoplasm to reprotonate Asp96 (5), and the reprotonation of the PRC from Asp85 (6), followed by a final proton transfer from D85 to R82 (7). (D) Light-induced switching of dipole orientation in response to photon absorption in BR, ChR, and HR. In BR and the ChRs, the configuration switch triggers the transfer of the RSB proton to Asp85/Glu123 (for BR/ChR2, respectively). In HR, dipole switching facilitates the transfer of a Cl⁻ ion from the cavity formed between the RSB and Thr143 to a Cl⁻ binding site cytoplasmic to the RSB, enabling the key transport steps of these transporters. Curved arrows indicate isomerization (top row) and ion movement (bottom row).

Figure 3. Structural and Functional Homology between BR and ChR

(A) Homology model-based structural alignment of ChR showing the 7-TM helices, next to the BR structural representation. For ChR2, the sequence of the illustrated residues may create a polar environment for water molecules and cation permeation. In BR, R82 functions as a connector between counterion and proton release complex, and D85 is the counterion to which the RSB proton is transferred. To emphasize the spatial discontinuity involved in pumping, only the proton transfer steps after photon absorption and proton transfer to D85 are shown. (B) Simplified model for the photocycle of ChRs. The D470 dark state is converted by a light-induced isomerization of retinal via the early intermediate P500 and the transient P390 intermediate to the conducting-state P520. The recovery of the D470 dark state proceeds either thermally via the nonconducting P480 intermediate or photochemically via possible short-lived intermediates (green arrow). The late or desensitized P480 state can also be activated (blue arrow) to yield the early intermediate P500. Additional parallel cycles may be present (Yizhar et al., 2011b). (C) Sample photocurrents show the key kinetic properties that govern function, including inactivation (inact), deactivation (deact), and recovery (rec). (D) Homology near the retinal-binding pocket between BR and ChR2. The BR retinal-binding pocket is shown based on structure 1KGB (Facciotti et al., 2001) with key amino acids that are involved in the proton transfer reaction. The ChR residues are shown based on sequence homology in the relevant positions.

Figure 4. Characterization of a ChR from Dunaliella salina

(A) The halophilic unicellular alga Dunaliella salina. (B) Sequence homology between the algal ChRs and BR within the third TM helix. The typically conserved E123 position has been replaced with an Ala in DChR1 (and is shown on a yellow background), conserved residues are shown on a blue background, and amino acids likely interacting with the chromophore are shown in red. (C) Lack of a proton acceptor in DChR1 (A178), compared with BR (D85) and Chlamydomonas ChR2 (CChR2; E123). ASR (Anabaena sensory rhodopsin) has been crystallized with a mixture of all-trans and 13-cis retinal seen as an overlay (Vogeley et al., 2004). (D) DChR1 photocurrents are unaffected by changes in the extracellular cation composition (sole cation present in each condition shown on category x axis). Cation exchange was performed in 5 mM Mops-NMG, 0.1 mM MgCl₂ with 100 mM LiCl, KCl, NaCl, guanidium chloride, or NMG chloride (pH 7.5). We used a human codonadapted DChR sequence (amino acid residues 1–339) as a template for capped RNA synthesis by T7 RNA polymerase (mMessage mMachine, Ambion). Oocyte preparation and injection of capped RNA were carried out as described previously (Berthold et al., 2008), and two-electrode voltage clamp was performed with a Turbo Tec-05 (NPI Electronic) or a GeneClamp 500 (Molecular Devices) amplifier on an oocyte after 3–7 days of the capped RNA injection. Continuous light was provided by a75-W Xenon lamp (Jena Instruments) and delivered to the oocytes via a 3 mm light guide. The light passed through a 500 ± 25 nm broadband filter (Balzers) with an intensity of 46 mW/cm². (E) In contrast, DChR1 photocurrent is highly sensitive to changes in the pH environment. Solutions contained 100 mM NMG chloride, 0.1 mM MgCl₂, 0.1 mM CaCl₂ with 5 mM glycine (pH 9.0), 5 mM Mops-NMG (pH 7.5), 5 mM citrate (pH 6, 5.5, 5.0, 4.6, 4.2). (F) Introduction or alteration of a proton acceptor (A178E or E309D) in the DChR1 retinal-binding pocket causes a pronounced red-shift in the absorption spectrum. We applied 10 ns laser flashes as described previously (Berthold et al., 2008); solutions for action spectra recording contained 100 mM NaCl, 0.1 mM MgCl₂, 0.1 mM CaCl₂, and 5 mM citrate (pH 4.2).

Figure 5. Phylogenetic Analysis of Microbial Opsins

Phylogenetic tree of the microbial opsins from algae, bacteria, and fungi. The tree was constructed by the neighbor-joining method based on amino acid sequences using MEGA5 (Tamura et l., 2011). The scale bar indicates the number of substitutions per site. H⁺ and Cl⁻ indicate proton and chloride pumping capability, respectively. Detailed opsin information is listed in Table 1. Sequences are provided in the Supplemental Information (“Sequences”).