Bestrhodopsins constitute a class of light-regulated pentameric ion channels that consist of one or two rhodopsins in tandem fused with bestrophin ion channel domains. Here, we report on the isomerization dynamics in the rhodopsin tandem domains of Phaeocystis antarctica bestrhodopsin, which binds all-trans retinal Schiff-base (RSB) absorbing at 661 nm and, upon illumination, converts to the meta-stable P540 state with an unusual 11-cis RSB. The primary photoproduct P682 corresponds to a mixture of highly distorted 11-cis and 13-cis RSB directly formed from the excited state in 1.4 ps. P673 evolves from P682 in 500 ps and contains highly distorted 13-cis RSB, indicating that the 11-cis fraction in P682 converts to 13-cis. Next, P673 establishes an equilibrium with P595 in 1.2 µs, during which RSB converts to 11-cis and then further proceeds to P560 in 48 µs and P540 in 1.0 ms while remaining 11-cis. Hence, extensive isomeric switching occurs on the early ground state potential energy surface (PES) on the hundreds of ps to µs timescale before finally settling on a metastable 11-cis photoproduct. We propose that P682 and P673 are trapped high up on the ground-state PES after passing through either of two closely located conical intersections that result in 11-cis and 13-cis RSB. Co-rotation of C11=C12 and C13=C14 bonds results in a constricted conformational landscape that allows thermal switching between 11-cis and 13-cis species of highly strained RSB chromophores. Protein relaxation may release RSB strain, allowing it to evolve to a stable 11-cis isomeric configuration in microseconds.

• Klíčová slova
• conical intersection, far-red absorbing rhodopsin, femtosecond stimulated Raman spectroscopy, femtosecond to millisecond spectroscopy, multiple retinal isomerization,
• MeSH
• diterpeny * MeSH
• isomerie MeSH
• konformace proteinů MeSH
• retinaldehyd * chemie   MeSH
• rodopsin * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 13-cis-retinal MeSH Prohlížeč
• diterpeny * MeSH
• retinaldehyd * MeSH
• rodopsin * MeSH

The recently discovered Neorhodopsin (NeoR) exhibits absorption and emission maxima in the near-infrared spectral region, which together with the high fluorescence quantum yield makes it an attractive retinal protein for optogenetic applications. The unique optical properties can be rationalized by a theoretical model that predicts a high charge transfer character in the electronic ground state (S0) which is otherwise typical of the excited state S1 in canonical retinal proteins. The present study sets out to assess the electronic structure of the NeoR chromophore by resonance Raman (RR) spectroscopy since frequencies and relative intensities of RR bands are controlled by the ground and excited state's properties. The RR spectra of NeoR differ dramatically from those of canonical rhodopsins but can be reliably reproduced by the calculations carried out within two different structural models. The remarkable agreement between the experimental and calculated spectra confirms the consistency and robustness of the theoretical approach.

• MeSH
• barvicí látky MeSH
• Ramanova spektroskopie MeSH
• retina MeSH
• rhodopsiny mikrobiální * chemie   MeSH
• rodopsin * chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• barvicí látky MeSH
• rhodopsiny mikrobiální * MeSH
• rodopsin * MeSH

Rhodopsin photosystems convert light energy into electrochemical gradients used by the cell to produce ATP, or for other energy-demanding processes. While these photosystems are widespread in the ocean and have been identified in diverse microbial taxonomic groups, their physiological role in vivo has only been studied in few marine bacterial strains. Recent metagenomic studies revealed the presence of rhodopsin genes in the understudied Verrucomicrobiota phylum, yet their distribution within different Verrucomicrobiota lineages, their diversity, and function remain unknown. In this study, we show that more than 7% of Verrucomicrobiota genomes (n = 2916) harbor rhodopsins of different types. Furthermore, we describe the first two cultivated rhodopsin-containing strains, one harboring a proteorhodopsin gene and the other a xanthorhodopsin gene, allowing us to characterize their physiology under laboratory-controlled conditions. The strains were isolated in a previous study from the Eastern Mediterranean Sea and read mapping of 16S rRNA gene amplicons showed the highest abundances of these strains at the deep chlorophyll maximum (source of their inoculum) in winter and spring, with a substantial decrease in summer. Genomic analysis of the isolates suggests that motility and degradation of organic material, both energy demanding functions, may be supported by rhodopsin phototrophy in Verrucomicrobiota. Under culture conditions, we show that rhodopsin phototrophy occurs under carbon starvation, with light-mediated energy generation supporting sugar transport into the cells. Overall, this study suggests that photoheterotrophic Verrucomicrobiota may occupy an ecological niche where energy harvested from light enables bacterial motility toward organic matter and supports nutrient uptake.

• MeSH
• Bacteria * genetika   MeSH
• biologický transport MeSH
• fototrofní procesy MeSH
• fylogeneze MeSH
• rhodopsiny mikrobiální genetika   metabolismus   MeSH
• RNA ribozomální 16S genetika   metabolismus   MeSH
• rodopsin * genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• rhodopsiny mikrobiální MeSH
• RNA ribozomální 16S MeSH
• rodopsin * MeSH

The ability to optically image cellular transmembrane voltages at millisecond-timescale resolutions can offer unprecedented insight into the function of living brains in behaving animals. Here, we present a point mutation that increases the sensitivity of Ace2 opsin-based voltage indicators. We use the mutation to develop Voltron2, an improved chemigeneic voltage indicator that has a 65% higher sensitivity to single APs and 3-fold higher sensitivity to subthreshold potentials than Voltron. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, although with lower baseline fluorescence. In multiple in vitro and in vivo comparisons with its predecessor across multiple species, we found Voltron2 to be more sensitive to APs and subthreshold fluctuations. Finally, we used Voltron2 to study and evaluate the possible mechanisms of interneuron synchronization in the mouse hippocampus. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability.

• Klíčová slova
• biosensors, fluorescence imaging, fluorescent proteins, genetically encoded indicators, voltage imaging,
• MeSH
• akční potenciály fyziologie   MeSH
• angiotensin-konvertující enzym 2 * MeSH
• mutace genetika   MeSH
• myši MeSH
• neurony fyziologie   MeSH
• rodopsin * genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• angiotensin-konvertující enzym 2 * MeSH
• rodopsin * MeSH

Vertebrates have four visual cone opsin classes that mediate sensitivity from ultraviolet to red wavelengths of light. The rhodopsin-like 2 (RH2) opsin is sensitive to the central mostly green part of the spectrum. While lost in some terrestrial vertebrates (mammals), the RH2 opsin gene has proliferated during the evolution of teleost fishes. Here, we investigated the genomes of 132 extant teleosts and found between zero and eight RH2 gene copies per species. The RH2 gene shows a dynamic evolutionary history with repeated gene duplications, gene losses, and gene conversions affecting entire orders, families, and species. At least four ancestral duplications provided the substrate for today's RH2 diversity, with duplications occurring in the common ancestors of Clupeocephala (twice), Neoteleostei, and likely Acanthopterygii as well. Despite these evolutionary dynamics, we identified conserved RH2 synteny in two main gene clusters; the slc6A13/synpr cluster is highly conserved within Percomorpha and also present across most teleosts, including Otomorpha, Euteleostei and in parts in tarpons (Elopomorpha), and the mutSH5 cluster, which is specific for Otomorpha. When comparing the number of visual opsin genes (SWS1, SWS2, RH2, LWS, and total cone opsins) with habitat depth, we found that deeper-dwelling species had less (or none) long-wavelength-sensitive opsins. Using retinal/eye transcriptomes in a phylogenetic representative dataset of 32 species, we show that if present in the genome, RH2 is expressed in most fishes except for some species within the tarpons, characins, and gobies (and Osteoglossomorpha and some other characin species have lost the gene). Those species instead express a green-shifted long-wavelength-sensitive LWS opsin. Our study applies modern genomic and transcriptomic tools within a comparative framework to elucidate the evolutionary history of the visual sensory system in teleost fishes.

• Klíčová slova
• Colour vision, Deep sea, Evolution, Fish, Gene duplication, Opsin,
• MeSH
• čípky retiny - opsiny * genetika   MeSH
• fylogeneze MeSH
• opsiny genetika   MeSH
• rodopsin * genetika   MeSH
• ryby genetika   MeSH
• savci MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• čípky retiny - opsiny * MeSH
• opsiny MeSH
• rodopsin * MeSH

The functional properties of some biological ion channels and membrane transport proteins are proposed to exploit anion-hydrophobic interactions. Here, we investigate a chloride-pumping rhodopsin as an example of a membrane protein known to contain a defined anion binding site composed predominantly of hydrophobic residues. Using molecular dynamics simulations, we explore Cl- binding to this hydrophobic site and compare the dynamics arising when electronic polarization is neglected (CHARMM36 [c36] fixed-charge force field), included implicitly (via the prosECCo force field), or included explicitly (through the polarizable force field, AMOEBA). Free energy landscapes of Cl- moving out of the binding site and into bulk solution demonstrate that the inclusion of polarization results in stronger ion binding and a second metastable binding site in chloride-pumping rhodopsin. Simulations focused on this hydrophobic binding site also indicate longer binding durations and closer ion proximity when polarization is included. Furthermore, simulations reveal that Cl- within this binding site interacts with an adjacent loop to facilitate rebinding events that are not observed when polarization is neglected. These results demonstrate how the inclusion of polarization can influence the behavior of anions within protein binding sites and can yield results comparable with more accurate and computationally demanding methods.

• MeSH
• anionty MeSH
• chloridy * chemie   MeSH
• elektronika MeSH
• rodopsin * MeSH
• simulace molekulární dynamiky MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• anionty MeSH
• chloridy * MeSH
• rodopsin * MeSH

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.

• Klíčová slova
• Alt-rhodopsins, AltRs, heliorhodopsins, metagenomics, optogenetics, rhodopsins,
• MeSH
• fylogeneze MeSH
• rhodopsiny mikrobiální * genetika   MeSH
• rodopsin * genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• rhodopsiny mikrobiální * MeSH
• rodopsin * MeSH

The visual sensory system is essential for animals to perceive their environment and is thus under strong selection. In aquatic environments, light intensity and spectrum differ primarily along a depth gradient. Rhodopsin (RH1) is the only opsin responsible for dim-light vision in vertebrates and has been shown to evolve in response to the respective light conditions, including along a water depth gradient in fishes. In this study, we examined the diversity and sequence evolution of RH1 in virtually the entire adaptive radiation of cichlid fishes in Lake Tanganyika, focusing on adaptations to the environmental light with respect to depth. We show that Tanganyikan cichlid genomes contain a single copy of RH1. The 76 variable amino acid sites detected in RH1 across the radiation were not uniformly distributed along the protein sequence, and 31 of these variable sites show signals of positive selection. Moreover, the amino acid substitutions at 15 positively selected sites appeared to be depth-related, including three key tuning sites that directly mediate shifts in the peak spectral sensitivity, one site involved in protein stability and 11 sites that may be functionally important on the basis of their physicochemical properties. Among the strongest candidate sites for deep-water adaptations are two known key tuning sites (positions 292 and 299) and three newly identified variable sites (37, 104 and 290). Our study, which is the first comprehensive analysis of RH1 evolution in a massive adaptive radiation of cichlid fishes, provides novel insights into the evolution of RH1 in a freshwater environment.

• Klíčová slova
• freshwater fish, opsin, photic environment, rod photoreceptor, spectral tuning, vision,
• MeSH
• cichlidy * genetika   MeSH
• fylogeneze MeSH
• jezera MeSH
• molekulární evoluce MeSH
• rodopsin genetika   MeSH
• ryby MeSH
• voda MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Tanzanie MeSH
• Názvy látek
• rodopsin MeSH
• voda MeSH

The ER membrane protein complex (EMC) is required for the biogenesis of a subset of tail anchored (TA) and polytopic membrane proteins, including Rhodopsin-1 (Rh1) and the TRP channel. To understand the physiological implications of EMC-dependent membrane protein biogenesis, we perform a bioinformatic identification of Drosophila TA proteins. From 254 predicted TA proteins, screening in larval eye discs identified two proteins that require EMC for their biogenesis: fan and Xport-A. Fan is required for male fertility in Drosophila and we show that EMC is also required for this process. Xport-A is essential for the biogenesis of both Rh1 and TRP, raising the possibility that disruption of Rh1 and TRP biogenesis in EMC mutants is secondary to the Xport-A defect. We show that EMC is required for Xport-A TMD membrane insertion and that EMC-independent Xport-A mutants rescue Rh1 and TRP biogenesis in EMC mutants. Finally, our work also reveals a role for Xport-A in a glycosylation-dependent triage mechanism during Rh1 biogenesis in the endoplasmic reticulum.

• Klíčová slova
• ER membrane protein complex, Rh1, TRP, Xport-A, tail anchored proteins,
• MeSH
• Drosophila genetika   metabolismus   MeSH
• endoplazmatické retikulum metabolismus   MeSH
• membránové proteiny genetika   metabolismus   MeSH
• molekulární chaperony * genetika   metabolismus   MeSH
• proteiny Drosophily * genetika   metabolismus   MeSH
• represorové proteiny * genetika   metabolismus   MeSH
• rodopsin * genetika   MeSH
• transkripční faktory bHLH * genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• emc protein, Drosophila MeSH Prohlížeč
• membránové proteiny MeSH
• molekulární chaperony * MeSH
• proteiny Drosophily * MeSH
• represorové proteiny * MeSH
• rodopsin * MeSH
• transkripční faktory bHLH * MeSH
• Xport-A protein, Drosophila MeSH Prohlížeč

Rhodopsins, most of which are proton pumps generating transmembrane electrochemical proton gradients, span all three domains of life, are abundant in the biosphere, and could play a crucial role in the early evolution of life on earth. Whereas archaeal and bacterial proton pumps are among the best structurally characterized proteins, rhodopsins from unicellular eukaryotes have not been well characterized. To fill this gap in the current understanding of the proton pumps and to gain insight into the evolution of rhodopsins using a structure-based approach, we performed a structural and functional analysis of the light-driven proton pump LR (Mac) from the pathogenic fungus Leptosphaeria maculans. The first high-resolution structure of fungi rhodopsin and its functional properties reveal the striking similarity of its membrane part to archaeal but not to bacterial rhodopsins. We show that an unusually long N-terminal region stabilizes the protein through direct interaction with its extracellular loop (ECL2). We compare to our knowledge all available structures and sequences of outward light-driven proton pumps and show that eukaryotic and archaeal proton pumps, most likely, share a common ancestor.

• MeSH
• fylogeneze MeSH
• iontový transport MeSH
• proteinové domény MeSH
• protonové pumpy chemie   MeSH
• rodopsin chemie   fyziologie   MeSH
• světlo MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protonové pumpy MeSH
• rodopsin MeSH