Rhodopsins are photoreceptive proteins with seven-transmembrane alpha-helices and a covalently bound retinal. Based on their protein sequences, rhodopsins can be classified into microbial rhodopsins and metazoan rhodopsins. Because there is no clearly detectable sequence identity between these two groups, their evolutionary relationship was difficult to decide. Through ancestral state inference, we found that microbial rhodopsins and metazoan rhodopsins are divergently related in their seven-transmembrane domains. Our result proposes that they are homologous proteins and metazoan rhodopsins originated from microbial rhodopsins. Structure alignment shows that microbial rhodopsins and metazoan rhodopsins share a remarkable structural homology while the position of retinal-binding lysine is different between them. It suggests that the function of photoreception was once lost during the evolution of rhodopsin genes. This result explains why there is no clearly detectable sequence similarity between the two rhodopsin groups: after losing the photoreception function, rhodopsin gene was freed from the functional constraint and the process of divergence could quickly change its original sequence beyond recognition.

We previously showed that the chimeric proteins of microbial rhodopsins, such as light-driven proton pump bacteriorhodopsin (BR) and Gloeobacter rhodopsin (GR) that contain cytoplasmic loops of bovine rhodopsin, are able to activate Gt protein upon light absorption. These facts suggest similar protein structural changes in both the light-driven proton pump and animal rhodopsin. Here we report two trials to engineer chimeric rhodopsins, one for the inserted loop, and another for the microbial rhodopsin template. For the former, we successfully activated Gs protein by light through the incorporation of the cytoplasmic loop of β2-adrenergic receptor (β2AR). For the latter, we did not observe any G-protein activation for the light-driven sodium pump from Indibacter alkaliphilus (IndiR2) or a light-driven chloride pump halorhodopsin from Natronomonas pharaonis (NpHR), whereas the light-driven proton pump GR showed light-dependent G-protein activation. This fact suggests that a helix opening motion is common to G protein coupled receptor (GPCR) and GR, but not to IndiR2 and NpHR. Light-induced difference FTIR spectroscopy revealed similar structural changes between WT and the third loop chimera for each light-driven pump. A helical structural perturbation, which was largest for GR, was further enhanced in the chimera. We conclude that similar structural dynamics that occur on the cytoplasmic side of GPCR are needed to design chimeric microbial rhodopsins.

Recent accumulation of sequence and structural data, in conjunction with systematical classification into a set of families, has significantly advanced our understanding of diverse and specific protein functions. Analysis and interpretation of protein family data requires comprehensive sequence and structural alignments. Here, we present a simple scheme for analyzing a set of experimental structures of a given protein or family of proteins, using microbial rhodopsins as an example. For a data set comprised of around a dozen highly similar structures to each other (overall pairwise root-mean-squared deviation < 2.3 Å), intramolecular distance scoring analysis yielded valuable information with respect to structural properties, such as differences in the relative variability of transmembrane helices. Furthermore, a comparison with recent results for G protein-coupled receptors demonstrates how the results of the present analysis can be interpreted and effectively utilized for structural characterization of diverse protein families in general.

For billions of years, life has continuously adapted to dynamic physical conditions near the Earth's surface. Fossils and other preserved biosignatures in the paleontological record are the most direct evidence for reconstructing the broad historical contours of this adaptive interplay. However, biosignatures dating to Earth's earliest history are exceedingly rare. Here, we combine phylogenetic inference of primordial rhodopsin proteins with modeled spectral features of the Precambrian Earth environment to reconstruct the paleobiological history of this essential family of photoactive transmembrane proteins. Our results suggest that ancestral microbial rhodopsins likely acted as light-driven proton pumps and were spectrally tuned toward the absorption of green light, which would have enabled their hosts to occupy depths in a water column or biofilm where UV wavelengths were attenuated. Subsequent diversification of rhodopsin functions and peak absorption frequencies was enabled by the expansion of surface ecological niches induced by the accumulation of atmospheric oxygen. Inferred ancestors retain distinct associations between extant functions and peak absorption frequencies. Our findings suggest that novel information encoded by biomolecules can be used as "paleosensors" for conditions of ancient, inhabited niches of host organisms not represented elsewhere in the paleontological record. The coupling of functional diversification and spectral tuning of this taxonomically diverse protein family underscores the utility of rhodopsins as universal testbeds for inferring remotely detectable biosignatures on inhabited planetary bodies.

All-optical electrophysiology-spatially resolved simultaneous optical perturbation and measurement of membrane voltage-would open new vistas in neuroscience research. We evolved two archaerhodopsin-based voltage indicators, QuasAr1 and QuasAr2, which show improved brightness and voltage sensitivity, have microsecond response times and produce no photocurrent. We engineered a channelrhodopsin actuator, CheRiff, which shows high light sensitivity and rapid kinetics and is spectrally orthogonal to the QuasArs. A coexpression vector, Optopatch, enabled cross-talk-free genetically targeted all-optical electrophysiology. In cultured rat neurons, we combined Optopatch with patterned optical excitation to probe back-propagating action potentials (APs) in dendritic spines, synaptic transmission, subcellular microsecond-timescale details of AP propagation, and simultaneous firing of many neurons in a network. Optopatch measurements revealed homeostatic tuning of intrinsic excitability in human stem cell-derived neurons. In rat brain slices, Optopatch induced and reported APs and subthreshold events with high signal-to-noise ratios. The Optopatch platform enables high-throughput, spatially resolved electrophysiology without the use of conventional electrodes.

Oligomeric assembly is a common feature of membrane proteins and often relevant to their physiological functions. Determining the stoichiometry and the oligomeric state of membrane proteins in a lipid bilayer is generally challenging because of their large size, complexity, and structural alterations under experimental conditions. Here, we use high-speed atomic force microscopy (HS-AFM) to directly observe the oligomeric states in the lipid membrane of various microbial rhodopsins found within eubacteria to archaea. HS-AFM images show that eubacterial rhodopsins predominantly exist as pentamer forms, while archaeal rhodopsins are trimers in the lipid membrane. In addition, circular dichroism (CD) spectroscopy reveals that pentameric rhodopsins display inverted CD couplets compared to those of trimeric rhodopsins, indicating different types of exciton coupling of the retinal chromophore in each oligomer. The results clearly demonstrate that the stoichiometry of the fundamental oligomer of microbial rhodopsins strongly correlate with the phylogenetic tree, providing a new insight into the relationship between the oligomeric structure and function-structural evolution of microbial rhodopsins.

Even though microbial photosensitive proteins have been used for optogenetics, their use should be optimized to precisely control cell and tissue functions in vivo. We exploited GtCCR4 and KnChR, cation channelrhodopsins from algae, BeGC1, a guanylyl cyclase rhodopsin from a fungus, and photoactivated adenylyl cyclases (PACs) from cyanobacteria (OaPAC) or bacteria (bPAC), to control cell functions in zebrafish. Optical activation of GtCCR4 and KnChR in the hindbrain reticulospinal V2a neurons, which are involved in locomotion, induced swimming behavior at relatively short latencies, whereas activation of BeGC1 or PACs achieved it at long latencies. Activation of GtCCR4 and KnChR in cardiomyocytes induced cardiac arrest, whereas activation of bPAC gradually induced bradycardia. KnChR activation led to an increase in intracellular Ca2+ in the heart, suggesting that depolarization caused cardiac arrest. These data suggest that these optogenetic tools can be used to reveal the function and regulation of zebrafish neurons and cardiomyocytes.

Rhodopsins are widely distributed across all domains of life where they perform a plethora of functions through the conversion of electromagnetic radiation into physicochemical signals. As a result of an extensive survey of available genomic and metagenomic sequencing data, we reported the existence of novel clades and exotic sequence motifs scattered throughout the evolutionary radiations of both Type-1 and Type-3 rhodopsins that will likely enlarge the optogenetics toolbox. We expanded the typical rhodopsin blueprint by showing that a highly conserved and functionally important arginine residue (i.e., Arg82) was substituted multiple times during evolution by an extensive amino acid spectrum. We proposed the umbrella term Alt-rhodopsins (AltRs) for all such proteins that departed Arg82 orthodoxy. Some AltRs formed novel clades in the rhodopsin phylogeny and were found in giant viruses. Some newly uncovered AltRs were phylogenetically close to heliorhodopsins, which allowed a closer examination of the phylogenetic border between Type-1 rhodopsins and heliorhodopsins. Comprehensive phylogenetic trees and ancestral sequence reconstructions allowed us to advance the hypothesis that proto-heliorhodopsins were a eukaryotic innovation before their subsequent diversification into the extant Type-3 rhodopsins. IMPORTANCE The rhodopsin scaffold is remarkably versatile and widespread, coupling light availability to energy production and other light-dependent cellular responses with minor alterations to critical residues. We described an unprecedented spectrum of substitutions at one of the most conserved amino acids in the rhodopsin fold, Arg82. We denoted such phylogenetically diverse rhodopsins with the umbrella name Alt-rhodopsins (AltR) and described a distinct branch of AltRs in giant viruses. Intriguingly, some AltRs were the closest phylogenetic neighbors to Heliorhodopsins (HeRs) whose origins have remained enigmatic. Our analyses of HeR origins in the light of AltRs led us to posit a most unusual evolutionary trajectory that suggested a eukaryotic origin for HeRs before their diversification in prokaryotes.

Since the discovery of proteorhodopsins, the ubiquitous marine light-driven proton pumps of eubacteria, a large number of other eubacterial rhodopsins with diverse structures and functions have been characterized. Here, we review the body of knowledge accumulated on the four major groups of eubacterial rhodopsins, with the focus on their biophysical characterization. We discuss advances and controversies on the unique eubacterial sensory rhodopsins (as represented by Anabaena sensory rhodopsin), proton-pumping proteorhodopsins and xanthorhodopsins, as well as novel non-proton ion pumps. This article is part of a Special Issue entitled: Retinal Proteins - You can teach an old dog new tricks.

Microbial rhodopsins (M-Rho) are found in Archaea, Bacteria and some species of Eukarya and serve as light-driven ion pumps or mediate phototaxis responses in various biological systems. We previously reported an expression system using a highly expressible mutant, D94N-HmBRI (HEBR) from Haloarcula marismortui, as a leading tag to assist in the expression of membrane proteins that were otherwise difficult to express in E. coli. In this study, we show a universal strategy for the expression of two M-Rho proteins, either the same or different types, as one fusion protein with the HEBR system. One extra transmembrane domain was engineered to the C-terminal of HEBR to express another target M-Rho. The average expression yield in this new system reached a minimum of 2 mg/L culture, and the maximum absorbance of the target M-Rho remained unaltered in the fusion forms. The fusion protein showed a combined absorbance spectrum of a lone HEBR and target M-Rho. The function of the target M-Rho was not affected after examination with functional tests, including the photocycle and proton pumping activity of fusion proteins. In addition, an otherwise unstable sensory rhodopsin, HmSRM, showed the same or even improved stability under various temperatures, salt concentrations, and a wide range of pH conditions. This HEBR platform provides the possibility to construct multi-functional, stoichiometric and color-tuning fusion proteins using M-Rho from haloarchaea.

Targeted manipulation of activity in specific populations of neurons is important for investigating the neural circuit basis of behavior. Optogenetic approaches using light-sensitive microbial rhodopsins have permitted manipulations to reach a level of temporal precision that is enabling functional circuit dissection. As demand for more precise perturbations to serve specific experimental goals increases, a palette of opsins with diverse selectivity, kinetics, and spectral properties will be needed. Here, we introduce a novel approach of "topological engineering"-inversion of opsins in the plasma membrane-and demonstrate that it can produce variants with unique functional properties of interest for circuit neuroscience. In one striking example, inversion of a Channelrhodopsin variant converted it from a potent activator into a fast-acting inhibitor that operates as a cation pump. Our findings argue that membrane topology provides a useful orthogonal dimension of protein engineering that immediately permits as much as a doubling of the available toolkit.

Sodium pumping rhodopsins (NaRs) are a unique member of the microbial-type I rhodopsin family which actively transport Na+ and H+ depending on ionic condition. In this study, we surveyed 12 different NaRs from various sources of eubacteria for their electrophysiological as well as spectroscopic properties. In mammalian cells several of these NaRs exhibited a Na+ based pump photocurrent and four interesting candidates were chosen for further characterization. Voltage dependent photocurrent amplitudes revealed a membrane potential-sensitive turnover rate, indicating the presence of an electrically-charged intermediate(s) in the photocycle reaction. The NaR from Salinarimonas rosea DSM21201 exhibited a red-shifted absorption spectrum, and slower kinetics compared to the first described sodium pump, KR2. Although the ratio of Na+ to H+ ion transport varied among the NaRs we tested, the NaRs from Flagellimonas sp_DIK and Nonlabens sp_YIK_SED-11 showed significantly higher Na+ selectivity when compared to KR2. All four further investigated NaRs showed a functional expression in dissociated hippocampal neuron culture and hyperpolarizing activity upon light-stimulation. Additionally, all four NaRs allowed optical inhibition of electrically-evoked neuronal spiking. Although efficiency of silencing was 3-5 times lower than silencing with the enhanced version of the proton pump AR3 from Halorubrum sodomense, our data outlines a new approach for hyperpolarization of excitable cells without affecting the intracellular and extracellular proton environment.

G-protein-coupled receptors (GPCRs) transmit stimuli to intracellular signaling systems. Rhodopsin (Rh), which is a prototypical GPCR, possesses an 11-cis retinal. Photoisomerization of 11-cis to all-trans leads to structural changes in the protein of cytoplasmic loops, activating G-protein. Microbial rhodopsins are similar heptahelical membrane proteins that function as bacterial sensors, light-driven ion-pumps, or light-gated channels. They possess an all-trans retinal, and photoisomerization to 13-cis triggers structural changes in protein. Despite these similarities, there is no sequence homology between visual and microbial rhodopsins, and microbial rhodopsins do not activate G-proteins. In this study, new chimeric proton-pumping rhodopsins, proteorhodopsin (PR) and Gloeobacter rhodopsin (GR) were designed by replacing cytoplasmic loops with bovine Rh loops. Although G-protein was not activated by the PR chimeras, all 12 GR chimeras activated G-protein. The GR chimera containing the second cytoplasmic loop of bovine Rh did not activate G-protein. However, the chimera with a second and third double-loop further enhanced G-protein activation. Introduction of an E132Q mutation slowed the photocycle 30-fold and enhanced activation. The highest catalytic activity of the GR chimera was still 3,200 times lower than bovine Rh but only 64 times lower than amphioxus Go-rhodopsin. This GR chimera showed a strong absorption change of the amide-I band on a light-minus-dark difference FTIR spectrum which could represent a larger helical opening, important for G-protein activation. The light-dependent catalytic activity of this GR chimera makes it a potential optogenetic tool for enzymatic activation by light.

Microbial rhodopsins, comprising a protein moiety (rhodopsin apoprotein) bound to the light-absorbing chromophore retinal, function as ion pumps, ion channels, or light sensors. However, recent genomic and metagenomic surveys showed that some rhodopsin-possessing prokaryotes lack the known genes for retinal biosynthesis. Since rhodopsin apoproteins cannot absorb light energy, rhodopsins produced by prokaryotic strains lacking genes for retinal biosynthesis are hypothesized to be non-functional in cells. In the present study, we investigated whether Aurantimicrobium minutum KNCT, which is widely distributed in terrestrial environments and lacks any previously identified retinal biosynthesis genes, possesses functional rhodopsin. We initially measured ion transport activity in cultured cells. A light-induced pH change in a cell suspension of rhodopsin-possessing bacteria was detected in the absence of exogenous retinal. Furthermore, spectroscopic analyses of the cell lysate and HPLC-MS/MS analyses revealed that this strain contained an endogenous retinal. These results confirmed that A. minutum KNCT possesses functional rhodopsin and, hence, produces retinal via an unknown biosynthetic pathway. These results suggest that rhodopsin-possessing prokaryotes lacking known retinal biosynthesis genes also have functional rhodopsins.

Microbial rhodopsins and G-protein coupled receptors (GPCRs, which include animal rhodopsins) are two distinct (super) families of heptahelical (7TM) membrane proteins that share obvious structural similarities but no significant sequence similarity. Comparison of the recently solved high-resolution structures of the sodium-translocating bacterial rhodopsin and various Na(+)-binding GPCRs revealed striking similarity of their sodium-binding sites. This similarity allowed us to construct a structure-guided sequence alignment for the two (super)families, which highlighted their evolutionary relatedness. Our analysis supports a common underlying molecular mechanism for both families that involves a highly conserved aromatic residue playing a pivotal role in rotation of the 6th transmembrane helix.

Microbial rhodopsin is a photoreceptor protein found in various bacteria and archaea, and it is considered to be a light-utilization device unique to heterotrophs. Recent studies have shown that several cyanobacterial genomes also include genes that encode rhodopsins, indicating that these auxiliary light-utilizing proteins may have evolved within photoautotroph lineages. To explore this possibility, we performed a large-scale genomic survey to clarify the distribution of rhodopsin and its phylogeny. Our surveys revealed a novel rhodopsin clade, cyanorhodopsin (CyR), that is unique to cyanobacteria. Genomic analysis revealed that rhodopsin genes show a habitat-biased distribution in cyanobacterial taxa, and that the CyR clade is composed exclusively of non-marine cyanobacterial strains. Functional analysis using a heterologous expression system revealed that CyRs function as light-driven outward H+ pumps. Examination of the photochemical properties and crystal structure (2.65 Å resolution) of a representative CyR protein, N2098R from Calothrix sp. NIES-2098, revealed that the structure of the protein is very similar to that of other rhodopsins such as bacteriorhodopsin, but that its retinal configuration and spectroscopic characteristics (absorption maximum and photocycle) are distinct from those of bacteriorhodopsin. These results suggest that the CyR clade proteins evolved together with chlorophyll-based photosynthesis systems and may have been optimized for the cyanobacterial environment.

Schizorhodopsins (SzRs), a rhodopsin family first identified in Asgard archaea, the archaeal group closest to eukaryotes, are present at a phylogenetically intermediate position between typical microbial rhodopsins and heliorhodopsins. However, the biological function and molecular properties of SzRs have not been reported. Here, SzRs from Asgardarchaeota and from a yet unknown microorganism are expressed in Escherichia coli and mammalian cells, and ion transport assays and patch clamp analyses are used to demonstrate SzR as a novel type of light-driven inward H+ pump. The mutation of a cytoplasmic glutamate inhibited inward H+ transport, suggesting that it functions as a cytoplasmic H+ acceptor. The function, trimeric structure, and H+ transport mechanism of SzR are similar to that of xenorhodopsin (XeR), a light-driven inward H+ pumping microbial rhodopsins, implying that they evolved convergently. The inward H+ pump function of SzR provides new insight into the photobiological life cycle of the Asgardarchaeota.

Rhodopsin-containing marine microbes such as those in the class Flavobacteriia play a pivotal role in the biogeochemical cycle of the euphotic zone (Fuhrman JA, Schwalbach MS, Stingl U. 2008. Proteorhodopsins: an array of physiological roles? Nat Rev Microbiol. 6:488-494). Deciphering the genome information of flavobacteria and accessing the diversity and ecological impact of microbial rhodopsins are important in understanding and preserving the global ecosystems. The genome sequence of the orange-pigmented marine flavobacterium Nonlabens dokdonensis (basonym: Donghaeana dokdonensis) DSW-6 was determined. As a marine photoheterotroph, DSW-6 has written in its genome physiological features that allow survival in the oligotrophic environments. The sequence analysis also uncovered a gene encoding an unexpected type of microbial rhodopsin containing a unique motif in addition to a proteorhodopsin gene and a number of photolyase or cryptochrome genes. Homologs of the novel rhodopsin gene were found in other flavobacteria, alphaproteobacteria, a species of Cytophagia, a deinococcus, and even a eukaryote diatom. They all contain the characteristic NQ motif and form a phylogenetically distinct group. Expression analysis of this rhodopsin gene in DSW-6 indicated that it is induced at high NaCl concentrations, as well as in the presence of light and the absence of nutrients. Genomic and metagenomic surveys demonstrate the diversity of the NQ rhodopsins in nature and the prevalent occurrence of the encoding genes among microbial communities inhabiting hypersaline niches, suggesting its involvement in sodium metabolism and the sodium-adapted lifestyle.

The isomerization of a covalently bound retinal is an integral part of both microbial and animal rhodopsin function. As such, detailed structure and conformational changes in the retinal binding pocket are of significant interest and are studied in various NMR, FTIR, and Raman spectroscopy experiments, which commonly require isotopic labeling of retinal. Unfortunately, the de novo organic synthesis of an isotopically-labeled retinal is complex and often cost-prohibitive, especially for large scale expression required for solid-state NMR. We present the novel protocol for biosynthetic production of an isotopically labeled retinal ligand concurrently with an apoprotein in E. coli as a cost-effective alternative to the de novo organic synthesis. Previously, the biosynthesis of a retinal precursor, β-carotene, has been introduced into many different organisms. We extended this system to the prototrophic E. coli expression strain BL21 in conjunction with the inducible expression of a β-dioxygenase and proteo-opsin. To demonstrate the applicability of this system, we were able to assign several new carbon resonances for proteorhodopsin-bound retinal by using fully 13C-labeled glucose as the sole carbon source. Furthermore, we demonstrated that this biosynthetically produced retinal can be extracted from E. coli cells by applying a hydrophobic solvent layer to the growth medium and reconstituted into an externally produced opsin of any desired labeling pattern.

Proton-pump rhodopsin (PPR) in marine microbes can convert solar energy to bioavailable chemical energy. Whereas bacterial PPR has been extensively studied, counterparts in microeukaryotes are less explored, and the relative importance of the two groups is poorly understood. Here, we sequenced whole-assemblage metatranscriptomes and investigated the diversity and expression dynamics of PPR in microbial eukaryotes and prokaryotes at a continental shelf and a slope site in the northern South China Sea. Data showed the whole PPRs transcript pool was dominated by Proteorhodopsins and Xanthorhodopsins, followed by Bacteriorhodopsin-like proteins, dominantly contributed by prokaryotes both in the number and expression levels of PPR unigenes, although at the continental slope station, microeukaryotes and prokaryotes contributed similarly in transcript abundance. Furthermore, eukaryotic PPRs are mainly contributed by dinoflagellates and showed significant correlation with nutrient concentrations. Green light-absorbing PPRs were mainly distributed in >3 μm organisms (including microeukaryotes and their associated bacteria), especially at surface layer at the shelf station, whereas blue light-absorbing PPRs dominated the <3 μm (mainly bacterial) communities at both study sites, especially at deeper layers at the slope station. Our study portrays a comparative PPR genotype and expression landscape for prokaryotes and eukaryotes in a subtropical marginal sea, suggesting PPR's role in niche differentiation and adaptation among marine microbes.