Aquatic bacterial rhodopsin proton pumps harvest light energy for photoheterotrophic growth and are known to contain hydroxylated carotenoids that expand the wavelengths of light utilized, but these have not been characterized in marine archaea. Here, by combining a marine chromophore extract with purified archaeal rhodopsins identified in marine metagenomes, we show light energy transfer from diverse hydroxylated carotenoids to heimdallarchaeial rhodopsins (HeimdallRs) from uncultured marine planktonic members of 'Candidatus Kariarchaeaceae' ('Candidatus Asgardarchaeota'). These light-harvesting antennas absorb in the blue-light range and transfer energy to the green-light-absorbing retinal chromophore within HeimdallRs, enabling the use of light that is otherwise unavailable to the rhodopsin. Furthermore, we show elevated proton pumping by the antennas in HeimdallRs under white-light illumination, which better simulates the light conditions encountered by these archaea in their natural habitats. Our results indicate that light-harvesting antennas in microbial rhodopsins exist in families beyond xanthorhodopsins and proteorhodopsins and are present in both marine bacteria and archaea.
- MeSH
- Archaea * metabolismus genetika chemie MeSH
- archeální proteiny * metabolismus chemie genetika MeSH
- fylogeneze MeSH
- karotenoidy metabolismus chemie MeSH
- metagenom MeSH
- mořská voda mikrobiologie MeSH
- přenos energie MeSH
- rhodopsiny mikrobiální * chemie metabolismus MeSH
- rodopsin * chemie metabolismus MeSH
- světlo MeSH
- světlosběrné proteinové komplexy * chemie metabolismus MeSH
- vodní organismy metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- archeální proteiny * MeSH
- karotenoidy MeSH
- rhodopsiny mikrobiální * MeSH
- rodopsin * MeSH
- světlosběrné proteinové komplexy * MeSH
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
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
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.
- MeSH
- Archaea genetika metabolismus MeSH
- buněčná membrána metabolismus MeSH
- fluorescenční protilátková technika MeSH
- gating iontového kanálu účinky záření MeSH
- konformace proteinů MeSH
- molekulární modely MeSH
- multigenová rodina MeSH
- mutace MeSH
- protonové pumpy chemie genetika metabolismus MeSH
- rodopsin chemie genetika metabolismus MeSH
- spektroskopie infračervená s Fourierovou transformací MeSH
- světlo MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- protonové pumpy MeSH
- rodopsin MeSH
Retinal disorders represent the main cause of decreased quality of vision and even blindness worldwide. The loss of retinal cells causes irreversible damage of the retina, and there are currently no effective treatment protocols for most retinal degenerative diseases. A promising approach for the treatment of retinal disorders is represented by stem cell-based therapy. The perspective candidates are mesenchymal stem cells (MSCs), which can differentiate into multiple cell types and produce a number of trophic and growth factors. In this study, we show the potential of murine bone marrow-derived MSCs to differentiate into cells expressing retinal markers and we identify the key supportive role of interferon-γ (IFN-γ) in the differentiation process. MSCs were cultured for 7 days with retinal extract and supernatant from T-cell mitogen concanavalin A-stimulated splenocytes, simulating the inflammatory site of retinal damage. MSCs cultured in such conditions differentiated to the cells expressing retinal cell markers such as rhodopsin, S antigen, retinaldehyde-binding protein, calbindin 2, recoverin, and retinal pigment epithelium 65. To identify a supportive molecule in the supernatants from activated spleen cells, MSCs were cultured with retinal extract in the presence of various T-cell cytokines. The expression of retinal markers was enhanced only in the presence of IFN-γ, and the supportive role of spleen cell supernatants was abrogated with the neutralization antibody anti-IFN-γ. In addition, differentiated MSCs were able to express a number of neurotrophic factors, which are important for retinal regeneration. Taken together, the results show that MSCs can differentiate into cells expressing retinal markers and that this differentiation process is supported by IFN-γ.
- Klíčová slova
- differentiation, interferon-γ, mesenchymal stem cell, neurotrophic factor, retina, rhodopsin,
- MeSH
- buněčná diferenciace * MeSH
- cis-trans-isomerasy genetika metabolismus MeSH
- interferon gama farmakologie MeSH
- kalbindin 2 genetika metabolismus MeSH
- kultivované buňky MeSH
- mezenchymální kmenové buňky cytologie účinky léků metabolismus MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- rekoverin genetika metabolismus MeSH
- retina cytologie metabolismus MeSH
- rodopsin genetika metabolismus MeSH
- transportní proteiny genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- 11-cis-retinal-binding protein MeSH Prohlížeč
- cis-trans-isomerasy MeSH
- interferon gama MeSH
- kalbindin 2 MeSH
- rekoverin MeSH
- retinoid isomerohydrolase MeSH Prohlížeč
- rodopsin MeSH
- transportní proteiny MeSH
Generation of an electrochemical proton gradient is the first step of cell bioenergetics. In prokaryotes, the gradient is created by outward membrane protein proton pumps. Inward plasma membrane native proton pumps are yet unknown. We describe comprehensive functional studies of the representatives of the yet noncharacterized xenorhodopsins from Nanohaloarchaea family of microbial rhodopsins. They are inward proton pumps as we demonstrate in model membrane systems, Escherichia coli cells, human embryonic kidney cells, neuroblastoma cells, and rat hippocampal neuronal cells. We also solved the structure of a xenorhodopsin from the nanohalosarchaeon Nanosalina (NsXeR) and suggest a mechanism of inward proton pumping. We demonstrate that the NsXeR is a powerful pump, which is able to elicit action potentials in rat hippocampal neuronal cells up to their maximal intrinsic firing frequency. Hence, inwardly directed proton pumps are suitable for light-induced remote control of neurons, and they are an alternative to the well-known cation-selective channelrhodopsins.
- MeSH
- Archaea metabolismus MeSH
- buněčné linie MeSH
- Escherichia coli metabolismus MeSH
- koncentrace vodíkových iontů MeSH
- konformace proteinů MeSH
- lidé MeSH
- liposomy MeSH
- molekulární modely MeSH
- optogenetika * metody MeSH
- protonové pumpy metabolismus MeSH
- protony MeSH
- retina metabolismus MeSH
- rodopsin chemie metabolismus MeSH
- spektrální analýza MeSH
- světlo MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- liposomy MeSH
- protonové pumpy MeSH
- protony MeSH
- rodopsin MeSH
The protein-protein interactions that underlie shut-off of the light-activated rhodopsin were studied using synthetic peptides derived from C-terminal region of the rhodopsin. The photoresponses were recorded in whole-cell voltage clamp from rod outer segments (ROS) that were internally dialyzed with an intracellular solution containing the synthetic peptides. This was the first time that synthetic peptides have been used in functionally intact ROS. None of the tested peptides promoted the shut-off of the photolyzed rhodopsin (R) by stimulating the binding of an activated arrestin to non-phosphorylated R, contrary to what was expected from in vitro experiments (Puig et al. FEBS Lett. 362: 185-188, 1995).
- MeSH
- adenosintrifosfát farmakologie MeSH
- arrestin metabolismus MeSH
- fosforylace MeSH
- fotolýza MeSH
- ještěři MeSH
- kalmodulin farmakologie MeSH
- kinetika MeSH
- molekulární sekvence - údaje MeSH
- peptidové fragmenty chemie farmakologie MeSH
- rodopsin analogy a deriváty chemie metabolismus farmakologie MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie MeSH
- skot MeSH
- světlo * MeSH
- zevní segment tyčinky fyziologie účinky záření MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Research Support, U.S. Gov't, P.H.S. MeSH
- Názvy látek
- adenosintrifosfát MeSH
- arrestin MeSH
- kalmodulin MeSH
- metarhodopsins MeSH Prohlížeč
- peptidové fragmenty MeSH
- rodopsin MeSH
