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The operation of bacteriorhodopsin (BR) from the archaeon Halobacterium salinarum is based on the photochromic reaction of isomerization of the chromophore group (the retinal protonated Schiff base, RPSB) from the all-trans to the 13-cis form. The ultrafast dynamics of the reverse 13-cis → all-trans photoreaction was studied using femtosecond transient absorption spectroscopy in comparison with the forward photoreaction. The forward photoreaction was initiated by photoexcitation of BR by pulse I (540 nm). The reverse photoreaction was initiated by photoexcitation of the product K590 at an early stage of its formation (5 ps) by pulse II (660 nm). The conversion of the excited K590 to the ground state proceeds at times of 0.19, 1.1, and 16 ps with the relative contributions of ~20/60/20, respectively. All these decay channels lead to the formation of the initial state of BR as a product with a quantum yield of ~1. This state is preceded by vibrationally excited intermediates, the relaxation of which occurs in the 16 ps time range. Likely, the heterogeneity of the excited state of K590 is determined by the heterogeneity of its chromophore center. The forward photoreaction includes two components—0.52 and 3.5 ps, with the relative contributions of 91/9, respectively. The reverse photoreaction initiated from K590 proceeds more efficiently in the conical intersection (CI) region but on the whole at a lower rate compared to the forward photoreaction, due to significant heterogeneity of the potential energy surface. [ABSTRACT FROM AUTHOR]

Objective: Rhodopsin from the eubacterium Exiguobacterium sibiricum (ESR) performs the lightdependent proton pumping function. The operation of ESR is based on the ultrafast photochemical reaction of isomerization of the retinal chromophore, which triggers dark processes closed in a photocycle. Many parameters of the photocycle are determined by the presence of a hydrogen bond between the primary counterion Asp85 and the chromophore. ESR in detergent micelles pumps protons most efficiently at pH > 9, when such a bond is most probable. Methods: In the present study, the photochemical reaction of ESR at pH 9.5 was investigated by femtosecond laser absorption spectroscopy. Results and Discussion: It was shown that photoisomerization of the chromophore group occurs in 0.51 ps, with the contribution from the reactive excited state being ca. 80%. A comparison with the earlier obtained data for pH 7.4 showed that, at pH 9.5, the reaction proceeds much faster and more efficiently. Conclusions: The present results confirm the important role of the chromophore group counterion in the photoactivated processes of rhodopsins. [ABSTRACT FROM AUTHOR]

Genetically encoded voltage indicators, particularly those based on microbial rhodopsins, are gaining traction in neuroscience as fluorescent sensors for imaging voltage dynamics with high-spatiotemporal precision. Here we establish a novel genetically encoded voltage indicator candidate based on the recently discovered subfamily of the microbial rhodopsin clade, termed heliorhodopsins. We discovered that upon excitation at 530 to 560 nm, wildtype heliorhodopsin exhibits near-infrared fluorescence, which is sensitive to membrane voltage. We characterized the fluorescence brightness, photostability, voltage sensitivity, and kinetics of wildtype heliorhodopsin in HEK293T cells and further examined the impact of mutating key residues near the retinal chromophore. The S237A mutation significantly improved the fluorescence response of heliorhodopsin by 76% providing a highly promising starting point for further protein evolution.

The functional properties of proteorhodopsin (PR) have been found to be strongly modulated by oligomeric distributions and lipid membrane mimetics. This study aims to distinguish and explain their effects by investigating how oligomer formation impacts PRs function of proton transport in lipid-based membrane mimetic environments. We find that PR forms stable hexamers and pentamers in both E. coli membranes and synthetic liposomes. Compared with the monomers, the photocycle kinetics of PR oligomers is ∼2 and ∼4.5 times slower for transitions between the K and M and the M and N photointermediates, respectively, indicating that oligomerization significantly slows PRs rate of proton transport in liposomes. In contrast, the apparent pKa of the key proton acceptor residue D97 (pKaD97) of liposome-embedded PR persists at 6.2-6.6, regardless of cross-protomer modulation of D97, suggesting that the liposome environment helps maintain PRs functional activity at neutral pH. By comparison, when extracted directly from E. coli membranes into styrene-maleic acid lipid particles, the pKaD97 of monomer-enriched E50Q PR drastically increases to 8.9, implying that there is a very low active PR population at neutral pH to engage in PRs photocycle. These findings demonstrate that oligomerization impacts PRs photocycle kinetics, while lipid-based membrane mimetics strongly affect PRs active population via different mechanisms.

Microbial rhodopsins are a family of photoreceptive membrane proteins with a wide distribution across the Tree of Life. Within the candidate phyla radiation (CPR), a diverse group of putatively episymbiotic bacteria, the genetic potential to produce rhodopsins appears to be confined to a small clade of organisms from sunlit environments. Here, we characterize the metabolic context and biophysical features of Saccharibacteria Type-1 rhodopsin sequences derived from metagenomic surveys and show that these proteins function as outward proton pumps. This provides one of the only known mechanisms by which CPR can generate a proton gradient for ATP synthesis. These Saccharibacteria do not encode the genetic machinery to produce all-trans-retinal, the chromophore essential for rhodopsin function, but their rhodopsins are able to rapidly uptake this cofactor when provided in experimental assays. We found consistent evidence for the capacity to produce retinal from β-carotene in microorganisms co-occurring with Saccharibacteria, and this genetic potential was dominated by members of the Actinobacteria, which are known hosts of Saccharibacteria in other habitats. If Actinobacteria serve as hosts for Saccharibacteria in freshwater environments, exchange of retinal for use by rhodopsin may be a feature of their associations.

The operation of bacteriorhodopsin (BR) from the archaeon Halobacterium salinarum is based on the photochromic reaction of isomerization of the chromophore group (the retinal protonated Schiff base, RPSB) from the all-trans to the 13-cis form. The ultrafast dynamics of the reverse 13-cis → all-trans photoreaction was studied using femtosecond transient absorption spectroscopy in comparison with the forward photoreaction. The forward photoreaction was initiated by photoexcitation of BR by pulse I (540 nm). The reverse photoreaction was initiated by photoexcitation of the product K590 at an early stage of its formation (5 ps) by pulse II (660 nm). The conversion of the excited K590 to the ground state proceeds at times of 0.19, 1.1, and 16 ps with the relative contributions of ~20/60/20, respectively. All these decay channels lead to the formation of the initial state of BR as a product with a quantum yield of ~1. This state is preceded by vibrationally excited intermediates, the relaxation of which occurs in the 16 ps time range. Likely, the heterogeneity of the excited state of K590 is determined by the heterogeneity of its chromophore center. The forward photoreaction includes two components—0.52 and 3.5 ps, with the relative contributions of 91/9, respectively. The reverse photoreaction initiated from K590 proceeds more efficiently in the conical intersection (CI) region but on the whole at a lower rate compared to the forward photoreaction, due to significant heterogeneity of the potential energy surface.

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.

This article discusses the role of microbial rhodopsins in capturing solar energy in the ocean. While most photosynthetic organisms use chlorophyll to convert sunlight into energy, microbial rhodopsins use the pigment retinal. These proteins are found in both eukaryotes and prokaryotes, including bacteria and viruses. The discovery of microbial rhodopsins has expanded our understanding of energy capture and productivity in marine ecosystems. They are particularly abundant in low-nutrient environments and provide enough energy to sustain basal metabolism and promote survival. Additionally, microbial rhodopsins have potential applications in optogenetics and the engineering of non-phototrophic organisms to harvest energy from light. [Extracted from the article]

Fluorescence of the vast majority of natural opsin-based photoactive proteins is extremely low, in accordance with their functions that depend on efficient transduction of absorbed light energy. However, several recently proposed classes of engineered rhodopsins with enhanced fluorescence, along with the discovery of a new natural highly fluorescent rhodopsin, NeoR, opened a way to exploit these transmembrane proteins as fluorescent sensors and draw more attention to studies on this untypical rhodopsin property. Here, we review the available data on the fluorescence of the retinal chromophore in microbial and animal rhodopsins and their photocycle intermediates, as well as different isomers of the protonated retinal Schiff base in various solvents and the gas phase. [ABSTRACT FROM AUTHOR]

Channelrhodopsins stand out among other retinal proteins because of their capacity to generate passive ionic currents following photoactivation. Owing to that, channelrhodopsins are widely used in neuroscience and cardiology as instruments for optogenetic manipulation of the activity of excitable cells. Photocurrents generated by channelrhodopsins were first discovered in the cells of green algae in the 1970s. In this review we describe this discovery and discuss the current state of research in the field. [ABSTRACT FROM AUTHOR]

Chloride ion-pumping rhodopsin (ClR) in some marine bacteria utilizes light energy to actively transport Cl- into cells. How the ClR initiates the transport is elusive. Here, we show the dynamics of ion transport observed with time-resolved serial femtosecond (fs) crystallography using the Linac Coherent Light Source. X-ray pulses captured structural changes in ClR upon flash illumination with a 550 nm fs-pumping laser. High-resolution structures for five time points (dark to 100 ps after flashing) reveal complex and coordinated dynamics comprising retinal isomerization, water molecule rearrangement, and conformational changes of various residues. Combining data from time-resolved spectroscopy experiments and molecular dynamics simulations, this study reveals that the chloride ion close to the Schiff base undergoes a dissociation-diffusion process upon light-triggered retinal isomerization.

The protonation state of embedded charged residues in transmembrane proteins (TMPs) can control the onset of protein function. It is understood that interactions between an embedded charged residue and other charged or polar residues in the moiety would influence its pKa, but how the surrounding environment in which the TMP resides affects the pKa of these residues is unclear. Proteorhodopsin (PR), a light-responsive proton pump from marine bacteria, was used as a model to examine externally accessible factors that tune the pKa of its embedded charged residue, specifically its primary proton acceptor D97. The pKa of D97 was compared between PR reconstituted in liposomes with different net headgroup charges and equilibrated in buffer with different ion concentrations. For PR reconstituted in net positively charged compared to net negatively charged liposomes in low-salt buffer solutions, a drop of the apparent pKa from 7.6 to 5.6 was observed, whereas intrinsic pKa modeled with surface pH calculated from Gouy-Chapman predictions found an opposite trend for the pKa change, suggesting that surface pH does not account for the main changes observed in the apparent pKa. This difference in the pKa of D97 observed from PR reconstituted in oppositely charged liposome environments disappeared when the NaCl concentration was increased to 150 mM. We suggest that protein-intrinsic structural properties must play a role in adjusting the local microenvironment around D97 to affect its pKa, as corroborated with observations of changes in protein side-chain and hydration dynamics around the E-F loop of PR. Understanding the effect of externally controllable factors in tuning the pKa of TMP-embedded charged residues is important for bioengineering and biomedical applications relying on TMP systems, in which the onset of functions can be controlled by the protonation state of embedded residues.

Antisera were raised against the C-terminal amino acid sequences of the two rhodopsins ADY17806 and AEA49880 of Oxyrrhis marina. The antisera and affinity-purified antibodies thereof were used in western immunoblotting experiments of total cell protein fractions from cultures grown either in darkness or in white, red, green, or blue light. Furthermore, the rhodopsin abundances were profiled in cultures fed with yeast or the prasinophyte Pyramimonas grossii. The immunosignals of ADY17806 and AEA49880 were similar when O. marina was grown in white, green, or blue light. Signal intensities were lower under conditions of red light and lowest in darkness. Higher amounts were registered for both rhodopsins when O. marina was fed with yeast compared to P. grossii. Furthermore, total cell protein of cultures of O. marina grown under all cultivation conditions was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by tryptic in-gel digestion and mass spectrometric analysis of the 25-kDa protein bands. The rhodopsin ADY17809 was detected in all samples of the light quality experiments and in 14 of the 16 samples of the prey quality experiments. The rhodopsin ABV22427 was not detected in one sample of the light quality experiments. It was detected in 15 of the 16 samples of the prey quality experiments. Peptide fragments of the other rhodopsins were detected less often, and no clear distribution pattern was evident with respect to the applied light quality or offered prey, indicating that none of them was exclusively formed under a distinct light regime or when feeding on yeast or the prasinophyte. Fluorescence light microscopy using the affinity-purified antibodies revealed significant labeling of the cell periphery and cell internal structures, which resembled vacuoles, tiny vesicles, and rather compact structures. Immunolabeling electron microscopy strengthened these results and showed that the cytoplasmic membrane, putative lysosome membranes, membranes encircling the food vacuole, and birefringent bodies became labeled. [ABSTRACT FROM AUTHOR]

Fluorescence of the vast majority of natural opsin-based photoactive proteins is extremely low, in accordance with their functions that depend on efficient transduction of absorbed light energy. However, several recently proposed classes of engineered rhodopsins with enhanced fluorescence, along with the discovery of a new natural highly fluorescent rhodopsin, NeoR, opened a way to exploit these transmembrane proteins as fluorescent sensors and draw more attention to studies on this untypical rhodopsin property. Here, we review the available data on the fluorescence of the retinal chromophore in microbial and animal rhodopsins and their photocycle intermediates, as well as different isomers of the protonated retinal Schiff base in various solvents and the gas phase.

Optogenetics is a technique employing natural or genetically engineered photoreceptors in transgene organisms to manipulate biological activities with light. Light can be turned on or off, and adjusting its intensity and duration allows optogenetic fine-tuning of cellular processes in a noninvasive and spatiotemporally resolved manner. Since the introduction of Channelrhodopsin-2 and phytochrome-based switches nearly 20 years ago, optogenetic tools have been applied in a variety of model organisms with enormous success, but rarely in plants. For a long time, the dependence of plant growth on light and the absence of retinal, the rhodopsin chromophore, prevented the establishment of plant optogenetics until recent progress overcame these difficulties. We summarize the recent results of work in the field to control plant growth and cellular motion via green light–gated ion channels and present successful applications to light-control gene expression with single or combined photoswitches in plants. Furthermore, we highlight the technical requirements and options for future plant optogenetic research. [ABSTRACT FROM AUTHOR]

The vitamin A derivative 11-cis-retinaldehyde plays a pivotal role in vertebrate vision by serving as the chromophore of rod and cone visual pigments. In the initial step of vision, a photon is absorbed by this chromophore resulting in its isomerization to an all-trans state and consequent activation of the visual pigment and phototransduction cascade. Spent chromophore is released from the pigments through hydrolysis. Subsequent photon detection requires the delivery of regenerated 11-cis-retinaldehyde to the visual pigment. This trans-cis conversion is achieved through a process known as the visual cycle. In this review, we will discuss the enzymes, binding proteins and transporters that enable the visual pigment renewal process with a focus on advances made during the past decade in our understanding of their structural biology.

A versatile synthetic protocol is reported that allows high concentrations of functionally active membrane proteins to be incorporated in mesostructured silica materials. Judicious selections of solvent, surfactant, silica precursor species, and synthesis conditions enable membrane proteins to be stabilized in solution and during subsequent coassembly into silica-surfactant composites with nano- and mesoscale order. This was demonstrated by using a combination of nonionic ( n-dodecyl-β-d-maltoside or Pluronic P123), lipid-like (1,2-diheptanoyl- s n-glycero-3-phosphocholine), and perfluoro-octanoate surfactants under mild acidic conditions to coassemble the light-responsive transmembrane protein proteorhodopsin at concentrations up to 15 wt % into the hydrophobic regions of worm-like mesostructured silica materials in films. Small-angle X-ray scattering, electron paramagnetic resonance spectroscopy, and transient UV-visible spectroscopy analyses established that proteorhodopsin molecules in mesostructured silica films exhibited native-like function, as well as enhanced thermal stability compared to surfactant or lipid environments. The light absorbance properties and light-activated conformational changes of proteorhodopsin guests in mesostructured silica films are consistent with those associated with the native H+-pumping mechanism of these biomolecules. The synthetic protocol is expected to be general, as demonstrated also for the incorporation of functionally active cytochrome c, a peripheral membrane protein enzyme involved in electron transport, into mesostructured silica-cationic surfactant films.

ABSTRACT 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.

The ultrastructure of the birefringent bodies of the dinoflagellate Oxyrrhis marina was investigated by transmission electron microscopy. Ultrathin sectioning revealed that the bodies consist of highly ordered and densely packed lamellae, which show a regular striation along their longitudinal axis. A lattice distance of 6.1 nm was measured for the densely packed lamellae by FFT (Fast Fourier Transformation) analysis. In addition, a rather faint and oblique running striation was registered. Lamellae sectioned rather oblique or almost close to the surface show a honeycombed structure with a periodicity of 7.2–7.8 nm. Freeze-fracture transmission electron microscopy revealed that the lamellae are composed of highly ordered, crystalline arrays of particles. Here, FFT analysis resulted in lattice distances of 7.0–7.6 nm. Freeze-fracture transmission electron microscopy further revealed that the bodies remained intact after cell rupture followed by ascending flotation of the membrane fractions on discontinuous sucrose gradients. The birefringent bodies most likely are formed by evaginations of membranes, which separate the cytoplasm from the food vacuoles. Distinct, slightly reddish-colored areas, which resembled the birefringent bodies with respect to size and morphology, were registered by bright field light microscopy within Oxyrrhis marina cells. An absorbance maximum at 540 nm was registered for these areas, indicating that they are composed of rhodopsins. This was finally proven by immuno-transmission electron microscopy, as antisera directed against the C-terminal amino acid sequences of the rhodopsins AEA49880 and ADY17806 intensely immunolabeled the birefringent bodies of Oxyrrhis marina. [ABSTRACT FROM AUTHOR]

In recent years, photoactive proteins such as rhodopsins have become a common target for cutting-edge research in the field of optogenetics. Alongside wet-lab research, computational methods are also developing rapidly to provide the necessary tools to analyze and rationalize experimental results and, most of all, drive the design of novel systems. The Automatic Rhodopsin Modeling (ARM) protocol is focused on providing exactly the necessary computational tools to study rhodopsins, those being either natural or resulting from mutations. The code has evolved along the years to finally provide results that are reproducible by any user, accurate and reliable so as to replicate experimental trends. Furthermore, the code is efficient in terms of necessary computing resources and time, and scalable in terms of both number of concurrent calculations as well as features. In this review, we will show how the code underlying ARM achieved each of these properties. [ABSTRACT FROM AUTHOR]

The present study describes for the first time the community composition and functional potential of the microbial mats found in the supratidal, gypsum-rich and hypersaline region of Blue Holes, Shark Bay. This was achieved via high-throughput metagenomic sequencing of total mat community DNA and complementary analyses using hyperspectral confocal microscopy. Mat communities were dominated by Proteobacteria (29%), followed by Bacteroidetes/Chlorobi group (11%) and Planctomycetes (10%). These mats were found to also harbour a diverse community of potentially novel microorganisms, including members from the DPANN, Asgard archaea and candidate phyla radiation, with highest diversity found in the lower regions (∼14–20 mm depth) of the mat. In addition to pathways for major metabolic cycles, a range of putative rhodopsins with previously uncharacterized motifs and functions were identified along with heliorhodopsins and putative schizorhodopsins. Critical microbial interactions were also inferred, and from 117 medium- to high-quality metagenome-assembled genomes, viral defence mechanisms (CRISPR, BREX and DISARM), elemental transport, osmoprotection, heavy metal resistance and UV resistance were also detected. These analyses have provided a greater understanding of these distinct mat systems in Shark Bay, including key insights into adaptive responses and proposing that photoheterotrophy may be an important lifestyle in Blue Holes. [ABSTRACT FROM AUTHOR]

The photoisomerization of retinal is a unifying primary event in the rhodopsin protein family. In vertebrate rhodopsins it is the first step in the vision process, while in the microbial rhodopsins it activates the transport of ions across the cell‐membrane. This reaction is highly optimized in the protein, which is ultrafast, selective, and efficient. A great effort was directed to elucidate the mechanism due to the overall complexity of the process inside the protein. The classical one‐bond‐flip is too demanding in space for the confined protein cavity. Therefore, various space saving mechanisms based on the rotation of multiple double bonds have been proposed. The hybrid quantum mechanics/molecular mechanics (QM/MM) method played an important role in the elucidation of the mechanism inside the tight protein environment. It allows to take the entire protein into account while describing the ground and excited states of retinal. The predicted mechanisms include full isomerization of two or three double bonds, a simultaneous isomerization of a single and a double bond as well as the partial rotation of bonds adjacent to the central isomerization. This review summarizes mechanistic studies in the literature and compares them. This article is categorized under:Structure and Mechanism > Computational Biochemistry and BiophysicsElectronic Structure Theory > Combined QM/MM MethodsSoftware > Molecular Modeling [ABSTRACT FROM AUTHOR]

ABSTRACT Rhodopsins are light-activated proteins displaying an enormous versatility of function as cation/anion pumps or sensing environmental stimuli and are widely distributed across all domains of life. Even with wide sequence divergence and uncertain evolutionary linkages between microbial (type 1) and animal (type 2) rhodopsins, the membrane orientation of the core structural scaffold of both was presumed universal. This was recently amended through the discovery of heliorhodopsins (HeRs; type 3), that, in contrast to known rhodopsins, display an inverted membrane topology and yet retain similarities in sequence, structure, and the light-activated response. While no ion-pumping activity has been demonstrated for HeRs and multiple crystal structures are available, fundamental questions regarding their cellular and ecological function or even their taxonomic distribution remain unresolved. Here, we investigated HeR function and distribution using genomic/metagenomic data with protein domain fusions, contextual genomic information, and gene coexpression analysis with strand-specific metatranscriptomics. We bring to resolution the debated monoderm/diderm occurrence patterns and show that HeRs are restricted to monoderms. Moreover, we provide compelling evidence that HeRs are a novel type of sensory rhodopsins linked to histidine kinases and other two-component system genes across phyla. In addition, we also describe two novel putative signal-transducing domains fused to some HeRs. We posit that HeRs likely function as generalized light-dependent switches involved in the mitigation of light-induced oxidative stress and metabolic circuitry regulation. Their role as sensory rhodopsins is corroborated by their photocycle dynamics and their presence/function in monoderms is likely connected to the higher sensitivity of these organisms to light-induced damage. IMPORTANCE Heliorhodopsins are enigmatic, novel rhodopsins with a membrane orientation that is opposite to all known rhodopsins. However, their cellular and ecological functions are unknown, and even their taxonomic distribution remains a subject of debate. We provide evidence that HeRs are a novel type of sensory rhodopsins linked to histidine kinases and other two-component system genes across phyla boundaries. In support of this, we also identify two novel putative signal transducing domains in HeRs that are fused with them. We also observe linkages of HeRs to genes involved in mitigation of light-induced oxidative stress and increased carbon and nitrogen metabolism. Finally, we synthesize these findings into a framework that connects HeRs with the cellular response to light in monoderms, activating light-induced oxidative stress defenses along with carbon/nitrogen metabolic circuitries. These findings are consistent with the evolutionary, taxonomic, structural, and genomic data available so far.

The plasma membrane is the crucial interface between the cell and its exterior, packed with embedded proteins experiencing simultaneous protein-protein and protein-membrane interactions. A prominent example of cell membrane complexity is the assembly of transmembrane proteins into oligomeric structures, with potential functional consequences that are not well understood. From the study of proteorhodopsin (PR), a prototypical seven-transmembrane light-driven bacterial proton pump, we find evidence that the inter-protein interaction modulated by self-association yields functional changes observable from the protein interior. We also demonstrate that the oligomer is likely a physiologically relevant form of PR, as crosslinking of recombinantly expressed PR reveals an oligomeric population within the Escherichia coli membrane (putatively hexameric). Upon chromatographic isolation of oligomeric and monomeric PR in surfactant micelles, the oligomer exhibits distinctly different optical absorption properties from monomeric PR, as reflected in a prominent decrease in the pKa of the primary proton acceptor residue (D97) and slowing of the light-driven conformational change. These functional effects are predominantly determined by specific PR-PR contacts over nonspecific surfactant interactions. Interestingly, varying the surfactant type alters the population of oligomeric states and the proximity of proteins within an oligomer, as determined by sparse electron paramagnetic resonance distance measurements. Nevertheless, the dynamic surfactant environment retains the key function-tuning property exerted by oligomeric contacts. A potentially general design principle for transmembrane protein function emerges from this work, one that hinges on specific oligomeric contacts that can be modulated by protein expression or membrane composition.

A group of microbial retinal proteins most closely related to the proton pump xanthorhodopsin has a novel sequence motif and a novel function. Instead of, or in addition to, proton transport, they perform light-driven sodium ion transport, as reported for one representative of this group (KR2) from Krokinobacter. In this paper, we examine a similar protein, GLR from Gillisia limnaea, expressed in Escherichia coli, which shares some properties with KR2 but transports only Na(+). The absorption spectrum of GLR is insensitive to Na(+) at concentrations of ≤3 M. However, very low concentrations of Na(+) cause profound differences in the decay and rise time of photocycle intermediates, consistent with a switch from a "Na(+)-independent" to a "Na(+)-dependent" photocycle (or photocycle branch) at ∼60 μM Na(+). The rates of photocycle steps in the latter, but not the former, are linearly dependent on Na(+) concentration. This suggests that a high-affinity Na(+) binding site is created transiently after photoexcitation, and entry of Na(+) from the bulk to this site redirects the course of events in the remainder of the cycle. A greater concentration of Na(+) is needed for switching the reaction path at lower pH. The data suggest therefore competition between H(+) and Na(+) to determine the two alternative pathways. The idea that a Na(+) binding site can be created at the Schiff base counterion is supported by the finding that upon perturbation of this region in the D251E mutant, Na(+) binds without photoexcitation. Binding of Na(+) to the mutant shifts the chromophore maximum to the red like that of H(+), which occurs in the photocycle of the wild type.

Visual rhodopsins are recognized members of the large and diverse family of G protein-coupled receptors (GPCRs), but their evolutionary origin and relationships to other proteins are not known. In a previous paper [Shlykov MA, Zheng WH, Chen JS & Saier MH Jr (2012) Biochim Biophys Acta 1818, 703-717], we characterized the 4-toluene sulfonate uptake permease (TSUP) family of transmembrane proteins, and showed that these 7-transmembrane segment (TMS) or 8-TMS proteins arose by intragenic duplication of a gene encoding a 4-TMS protein, sometimes followed by loss of a terminal TMS. In this study, we show that the TSUP, GPCR and microbial rhodopsin families are related to each other and to six other currently recognized transport protein families. We designate this superfamily the transporter/opsin/G protein-coupled receptor (TOG) superfamily. Despite their 8-TMS origins, the members of most constituent families exhibit 7-TMS topologies that are well conserved, and these arose by loss of either the N-terminal TMS (more frequent) or the C-terminal TMS (less frequent), depending on the family. Phylogenetic analyses revealed familial relationships within the superfamily and protein relationships within each of the nine families. The results of the statistical analyses leading to the conclusion of homology were confirmed using hidden Markov models, Pfam and 3D superimpositions. Proteins functioning by dissimilar mechanisms (channels, primary active transporters, secondary active transporters, group translocators and receptors) are interspersed on a phylogenetic tree of the TOG superfamily, suggesting that changes in the transport and energy-coupling mechanisms occurred multiple times during evolution of this superfamily.

Artificial membrane systems allow researchers to study the structure and function of membrane proteins in a matrix that approximates their natural environment and to integrate these proteins in ex vivo devices such as electronic biosensors, thin-film protein arrays, or biofuel cells. Given that most membrane proteins have vectorial functions, both functional studies and applications require effective control over protein orientation within a lipid bilayer. In this work, we explored the role of the bilayer surface charge in determining transmembrane protein orientation and functionality during formation of proteoliposomes. We reconstituted a model vectorial ion pump, proteorhodopsin, in liposomes of opposite charges and varying charge densities and determined the resultant protein orientation. Antibody-binding assay and proteolysis of proteoliposomes showed physical evidence of preferential orientation, and functional assays verified the vectorial nature of ion transport in this system. Our results indicate that the manipulation of lipid composition can indeed control orientation of an asymmetrically charged membrane protein, proteorhodopsin, in liposomes.

Inhibitory interneurons are essential components of the neural circuits underlying various brain functions. In the neocortex, a large diversity of GABA (γ-aminobutyric acid) interneurons has been identified on the basis of their morphology, molecular markers, biophysical properties and innervation pattern. However, how the activity of each subtype of interneurons contributes to sensory processing remains unclear. Here we show that optogenetic activation of parvalbumin-positive (PV+) interneurons in the mouse primary visual cortex (V1) sharpens neuronal feature selectivity and improves perceptual discrimination. Using multichannel recording with silicon probes and channelrhodopsin-2 (ChR2)-mediated optical activation, we found that increased spiking of PV+ interneurons markedly sharpened orientation tuning and enhanced direction selectivity of nearby neurons. These effects were caused by the activation of inhibitory neurons rather than a decreased spiking of excitatory neurons, as archaerhodopsin-3 (Arch)-mediated optical silencing of calcium/calmodulin-dependent protein kinase IIα (CAMKIIα)-positive excitatory neurons caused no significant change in V1 stimulus selectivity. Moreover, the improved selectivity specifically required PV+ neuron activation, as activating somatostatin or vasointestinal peptide interneurons had no significant effect. Notably, PV+ neuron activation in awake mice caused a significant improvement in their orientation discrimination, mirroring the sharpened V1 orientation tuning. Together, these results provide the first demonstration that visual coding and perception can be improved by increased spiking of a specific subtype of cortical inhibitory interneurons.

Bistable rhodopsins have two stable forms that can be interconverted by light. Due to their ability to act as photoswitches, these proteins are considered as ideal candidates for applications such as optogenetics. In this work, we analyze a recently crystalized bistable rhodopsin, namely the jumping spider rhodopsin-1 (JSR1). This rhodopsin exhibits identical absorption maxima for the parent and the photoproduct form, which impedes its broad application. We performed hybrid QM/MM simulations to study three isomers of the retinal chromophore: the 9-cis, 11-cis and all-trans configurations. The main aim was to gain insight into the specific interactions of each isomer and their impact on the absorption maximum in JSR1. The absorption spectra were computed using sampled snapshots from QM/MM molecular dynamics trajectories and compared to their experimental counterparts. The chromophore–protein interactions were analyzed by visualizing the electrostatic potential of the protein and projecting it onto the chromophore. It was found that the distance between a nearby tyrosine (Y126) residue plays a larger role in the predicted absorption maximum than the primary counterion (E194). Geometric differences between the isomers were also noted, including a structural change in the polyene chain of the chromophore, as well as changes in the nearby hydrogen bonding network.

Ion channels are membrane proteins that play important roles in a wide range of fundamental cellular processes. Studying membrane proteins at a molecular level becomes challenging in complex cellular environments. Instead, many studies focus on the isolation and reconstitution of the membrane proteins into model lipid membranes. Such simpler, in vitro, systems offer the advantage of control over the membrane and protein composition and the lipid environment. Rhodopsin and rhodopsin-like ion channels are widely studied due to their light-interacting properties and are a natural candidate for investigation with fluorescence methods. Here we review techniques for synthesizing liposomes and for reconstituting membrane proteins into lipid bilayers. We then summarize fluorescence assays which can be used to verify the functionality of reconstituted membrane proteins in synthetic liposomes.

We report the first record of albinism in the swell shark, Cephaloscyllium ventriosum (Garman, 1880). The specimen was caught off the southern part of the Baja California Peninsula, Mexico. The shark had no skin pigmentation and golden-coloured eyes. The pigmentation of its eyes may be related to the previously reported presence of ocular pigments that are different from melanin, and thus not affected by albinism.

A typical feature of proteins from the rhodopsin family is the sensitivity of their absorption band maximum to protein amino acid composition. For this reason, studies of these proteins often require methodologies that determine spectral shift caused by amino acid substitutions. Generally, quantum mechanics/molecular mechanics models allow for the calculation of a substitution-induced spectral shift with high accuracy, but their application is not always easy and requires special knowledge. In the present study, we propose simple models that allow us to estimate the direct effect of a charged or polar residue substitution without extensive calculations using only rhodopsin three-dimensional structure and plots or tables that are provided in this article. The models are based on absorption maximum values calculated at the SORCI+Q level of theory for cis- and trans-forms of retinal protonated Schiff base in an external electrostatic field of charges and dipoles. Each value corresponds to a certain position of a charged or polar residue relative to the retinal chromophore. The proposed approach was evaluated against an example set consisting of twelve bovine rhodopsin and sodium pumping rhodopsin mutants. The limits of the applicability of the models are also discussed. The results of our study can be useful for the interpretation of experimental data and for the rational design of rhodopsins with required spectral properties.

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. [ABSTRACT FROM AUTHOR]

A halophilic archaeon, strain F20-122T, was isolated from a marine saltern of Isla Bacuta (Huelva, Spain). Cells were Gram-stain-negative, aerobic, and coccoid in morphology. It grew at 25–50 °C (optimum 37 °C), pH 6.5–9.0 (optimum pH 8.0), and 10–30% (w/v) total salts (optimum 25% salts). The phylogenetic analyses based on the 16S rRNA and rpoB’ genes showed its affiliation with the genus Natronomonas and suggested its placement as a new species within this genus. The in silico DNA–DNA hybridization (DDH) and average nucleotide identity (ANI) analyses of this strain against closely related species supported its placement in a new taxon. The DNA G + C content of this isolate was 63.0 mol%. The polar lipids of strain F20-122T were phosphatidylglycerol phosphate methyl ester (PGP-Me), phosphatidylglycerol (PG), and phosphatidylglycerol sulfate (PGS). Traces of biphosphatidylglycerol (BPG) and other minor phospholipids and unidentified glycolipids were also present. Based on the phylogenetic, genomic, phenotypic, and chemotaxonomic characterization, we propose strain F20-122T (= CCM 8891T = CECT 9564T = JCM 33320T) as the type strain of a new species within the genus Natronomonas, with the name Natronomonas salsuginis sp. nov. Rhodopsin-like sequence analysis of strain F20-122T revealed the presence of haloarchaeal proton pumps, suggesting a light-mediated ATP synthesis for this strain and a maximum wavelength absorption in the green spectrum.

Retinal-binding proteins, mainly known as rhodopsins, function as photosensors and ion transporters in a wide range of organisms. From halobacterial light-driven proton pump, bacteriorhodopsin, to bovine photoreceptor, visual rhodopsin, they have served as prototypical α-helical membrane proteins in a large number of biophysical studies and aided in the development of many cutting-edge techniques of structural biology and biospectroscopy. In the last decade, microbial and animal rhodopsin families have expanded significantly, bringing into play a number of new interesting structures and functions. In this review, we will discuss recent advances in biophysical approaches to retinal-binding proteins, primarily microbial rhodopsins, including those in optical spectroscopy, X-ray crystallography, nuclear magnetic resonance, and electron paramagnetic resonance, as applied to such fundamental biological aspects as protein oligomerization, folding, and structure. [ABSTRACT FROM AUTHOR]

Ion gradients are a universal form of energy, information storage and conversion in living cells. Advances in optogenetics inspire the development of novel tools towards control of different cellular processes with light. Rhodopsins are perspective tools for optogenetic manipulation of ion gradients in cells and subcellular compartments, controlling pH of the cytosol and intracellular organelles. The key step of the development of new optogenetic tools is evaluation of their efficiency. Here, we used a high-throughput quantitative method for comparing efficiency of proton-pumping rhodopsins in Escherichia coli cells. This approach allowed us to show that an inward proton pump xenorhodopsin from Nanosalina sp. (Ns XeR) is a powerful tool for optogenetic control of pH of mammalian subcellular compartments. Further, we demonstrate that Ns XeR can be used for fast optogenetic acidification of the cytosol of mammalian cells. This is the first evidence of optogenetic cytosol acidification by an inward proton pump at physiological pH values. Our approach offers unique opportunities to study cellular metabolism at normal and pathological conditions and might help to understand the role of pH dysregulation in cellular dysfunctions. [ABSTRACT FROM AUTHOR]

Light profoundly affects the circadian clock and the activity levels of animals. Along with the systematic changes in intensity and spectral composition, over the 24-h day, light shows considerable irregular fluctuations (noise). Using light as the Zeitgeber for the circadian clock is, therefore, a complex task and this might explain why animals utilize multiple photoreceptors to entrain their circadian clock. The fruit fly Drosophila melanogaster possesses light-sensitive Cryptochrome and seven Rhodopsins that all contribute to light detection. We review the role of Rhodopsins in circadian entrainment, and of direct light-effects on the activity, with a special emphasis on the newly discovered Rhodopsin 7 (Rh7). We present evidence that Rhodopsin 6 in receptor cells 8 of the compound eyes, as well as in the extra retinal Hofbauer-Buchner eyelets, plays a major role in entraining the fly’s circadian clock with an appropriate phase-to-light–dark cycles. We discuss recent contradictory findings regarding Rhodopsin 7 and report original data that support its role in the compound eyes and in the brain. While Rhodopsin 7 in the brain appears to have a minor role in entrainment, in the compound eyes it seems crucial for fine-tuning light sensitivity to prevent overshooting responses to bright light.

A review of physicochemical properties, photochemistry, functions, and evolution of retinal-containing proteins (microbial and of metazoan rhodopsins, mostly visual rhodopsins) is provided. Comparative physiology of visual rhodopsins is considered in detail, mainly the molecular mechanisms of their spectral tuning. [ABSTRACT FROM AUTHOR]

Rhodopsin 7 ( Rh7), a new invertebrate Rhodopsin gene, was discovered in the genome of Drosophila melanogaster in 2000 and thought to encode for a functional Rhodopsin protein. Indeed, Rh7 exhibits most hallmarks of the known Rhodopsins, except for the G-protein-activating QAKK motif in the third cytoplasmic loop that is absent in Rh7. Here, we show that Rh7 can partially substitute Rh1 in the outer receptor cells (R1-6) for rhabdomere maintenance, but that it cannot activate the phototransduction cascade in these cells. This speaks against a role of Rh7 as photopigment in R1-6, but does not exclude that it works in the inner photoreceptor cells. [ABSTRACT FROM AUTHOR]

We report the first record of albinism in the swell shark, Cephaloscyllium ventriosum (Garman, 1880). The specimen was caught off the southern part of the Baja California Peninsula, Mexico. The shark had no skin pigmentation and golden-coloured eyes. The pigmentation of its eyes may be related to the previously reported presence of ocular pigments that are different from melanin, and thus not affected by albinism. [ABSTRACT FROM AUTHOR]

Rhodopsins are the major photopigments in the fruit fly Drosophila melanogaster. Drosophila express six well-characterized Rhodopsins (Rh1–Rh6) with distinct absorption maxima and expression pattern. In 2000, when the Drosophila genome was published, a novel Rhodopsin gene was discovered: Rhodopsin 7 (Rh7). Rh7 is highly conserved among the Drosophila genus and is also found in other arthropods. Phylogenetic trees based on protein sequences suggest that the seven Drosophila Rhodopsins cluster in three different groups. While Rh1, Rh2 and Rh6 form a “vertebrate-melanopsin-type”–cluster, and Rh3, Rh4 and Rh5 form an “insect-type”-Rhodopsin cluster, Rh7 seem to form its own cluster. Although Rh7 has nearly all important features of a functional Rhodopsin, it differs from other Rhodopsins in its genomic and structural properties, suggesting it might have an overall different role than other known Rhodopsins.