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
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
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
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
A model of the helical part of the human MT2 melatonin (hMT2) receptor, a member of the G protein-coupled receptors superfamily has been generated, based on the structure of bovine rhodopsin. Modeling has been combined with site-directed mutagenesis to investigate the role of the specific amino acid residues within the transmembrane domains (TM) numbers V, VI and VII of hMT2 receptor in the interaction with 2-iodomelatonin. Saturation binding assays with 2-iodomelatonin demonstrated that the substitution V204A (TMV) resulted in total loss of binding while the mutation V205A had no effect. The replacement of F209 with alanine led to a significant decrease in the Bmax value of receptor binding while mutations V205A and F209A also within TM V did not significantly change binding properties of the hMT2 receptor. In the case of TM VI, the substitution G271T caused substantial decrease in 2-iodomelatonin binding to the hMT2 receptor. The change L272A (TM VI) as well as mutation Y298A within TM VII completely abolished ligand binding to the receptor. These data suggest that several new amino acid residues within TM V, VI and VII are involved in ligand-MT2 receptor interaction.
- MeSH
- buněčné linie MeSH
- kompetitivní vazba MeSH
- lidé MeSH
- ligandy MeSH
- melatonin analogy a deriváty metabolismus farmakokinetika MeSH
- molekulární modely * MeSH
- mutageneze cílená MeSH
- počítačová simulace MeSH
- receptor melatoninový MT1 chemie MeSH
- receptor melatoninový MT2 chemie genetika metabolismus MeSH
- rodopsin chemie MeSH
- sekundární struktura proteinů MeSH
- skot MeSH
- substituce aminokyselin MeSH
- vazba proteinů genetika fyziologie MeSH
- vazebná místa genetika fyziologie MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 2-iodomelatonin MeSH Prohlížeč
- ligandy MeSH
- melatonin MeSH
- receptor melatoninový MT1 MeSH
- receptor melatoninový MT2 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
