Identification, expression and characterization of archaeal-type opsins of Chlamydomonas reinhardtii

Phototaxis and photophobic responses of green algae are mediated by rhodopsin-based photoreceptors that use microbial-type chromophores (all-trans retinal). Analysis of stimuli-response curves of the C. reinhardtii photoreceptor current led to the suggestion that they are based on two photosystems, one of which is more active at low flash intensities, whereas the other dominates at high flash energies. Two cDNA sequences were identified in the EST database of the C.reinhardtii that encoded microbial-type opsins, which were named Chlamyopsin-3 and 4 (Cop-3 and Cop-4) respectively, based on their homology to the known microbial-type opsins. The seven-transmembrane helices at the N-terminus of these opsins showed homology to the light-activated proton pump, bacteriorhodopsin (BR). However, after functional expression in the oocytes of X. laevis these opsins were renamed as Channelopsin-1 and 2 (Chop-1 and Chop-2) based on their ion channel activity. The amino acids that form the H+-conducting network in BR are conserved in Chop-1 and Chop-2, whereas the rest of their sequences are different. Heterologoulsy expressed Channelopsins (Chop-1 and 2) in E.coli formed inclusion bodies, and thus recombinant proteins were not functional. Expression of Chop-1 in P. pastoris led to the production of non-functional protein, since it did not bind all-trans retinal. Functional expression of Chop-1 mRNA in the oocytes of X. laevis (Chop-1 + all-trans retinal =ChR1) showed a light-gated ion channel conductance, which was studied in detail using a two-electrode voltage clamp technique (Nagel et al., 2002). The observed transport activity was purely passive and directly dependent on the membrane potential and the proton concentration gradient in bath solution. Outward photocurrents could be observed at high exracellular pH or low intracellular pH. The conductance was highly selective for protons, and other monovalent or divalent ions were not found to be permeating. It was also observed by mutational analysis that the H173 residue of Chop-1 does not function as a proton donor of a deprotonated Schiff base. Therefore, it was suggested that in ChR1 the retinal Schiff base is not de-protonated during the photocycle. It is likely that such light sensitive ion channels are widely distributed in other phototactic microalgae, as well as in gametes and zoospores of the macroalgae. This claim is corroborated by the observation that Volvoxopsin-2 (Vop-2) a partial opsin like sequence was identified in the V. carteri genome project, which showed 75% identical amino acid residues to Channelopsins in the helices 5-7 of the opsin domain. Heterologous expression of Chop-2 was also carried out in E.coli to produce functional recombinant protein. It was observed that the expression characteristic of Chop-2 were similar to that of Chop-1. Therefore, Chop-2 was directly expressed in Xenopus oocytes, in the presence of all-trans retinal to produce functional Channelrhodopsin-2 (ChR2) (Nagel et al., 2003). Photocurrents were recorded from these oocytes using two-electrode voltage clamp method. However, the cells not only became conductive for protons but also, most surprisingly, for monvalent and divalent cations like Na+ K+ and Ca++. It was concluded that ChR-2 functions as a cation-selective channel. Surprisingly, and in contrast to ChR1, the light-gated conductance of ChR-2 inactivates in continuous light to a smaller steady-state level. Western blotting analysis with membrane fractions of C. reinhardtii using anti-Chop1 and Chop2 antibodies revealed that both proteins were abundant, when cells were grown in low light conditions, both are degraded under high light conditions and that ChR2 was degraded more rapidly than ChR1. In conclusion, it is likely that both channelrhodopsins control photophobic responses and only indirectly influence phototaxis. Very recently, three more protein sequences were found in C. reinhardtii genome database, which showed homology to the sensory opsin. Surprisingly, all three sequences were coupled to a transducer like protein (HtrI and II). We have provisionally named these sequences Cop5, Cop6 and Cop7. Isolation, sequencing and bioinformatic analysis of Cop-5 protein sequence revealed that it is a unique putative opsin, which has four modular domains (Opsin, HK, RR and CYCc) in one protein. The assumption that one of these new rhodopsins could be responsible for phototaxis movement in C. reinhardtii seems to be justified. Nevertheless, other functions like control of retinal biosynthesis or developmental processes should also be taken into account

Cellular oxido-reductive proteins of Chlamydomonas reinhardtii control the biosynthesis of silver nanoparticles

<p>Abstract</p> <p>Background</p> <p>Elucidation of molecular mechanism of silver nanoparticles (SNPs) biosynthesis is important to control its size, shape and monodispersity. The evaluation of molecular mechanism of biosynthesis of SNPs is of prime importance for the commercialization and methodology development for controlling the shape and size (uniform distribution) of SNPs. The unicellular algae <it>Chlamydomonas reinhardtii </it>was exploited as a model system to elucidate the role of cellular proteins in SNPs biosynthesis.</p> <p>Results</p> <p>The <it>C. reinhardtii </it>cell free extract (<it>in vitro</it>) and <it>in vivo </it>cells mediated synthesis of silver nanoparticles reveals SNPs of size range 5 ± 1 to 15 ± 2 nm and 5 ± 1 to 35 ± 5 nm respectively. <it>In vivo </it>biosynthesized SNPs were localized in the peripheral cytoplasm and at one side of flagella root, the site of pathway of ATP transport and its synthesis related enzymes. This provides an evidence for the involvement of oxidoreductive proteins in biosynthesis and stabilization of SNPs. Alteration in size distribution and decrease of synthesis rate of SNPs in protein-depleted fractions confirmed the involvement of cellular proteins in SNPs biosynthesis. Spectroscopic and SDS-PAGE analysis indicate the association of various proteins on <it>C. reinhardtii </it>mediated <it>in vivo </it>and <it>in vitro </it>biosynthesized SNPs. We have identified various cellular proteins associated with biosynthesized (<it>in vivo </it>and <it>in vitro) </it>SNPs by using MALDI-MS-MS, like ATP synthase, superoxide dismutase, carbonic anhydrase, ferredoxin-NADP⁺reductase, histone etc. However, these proteins were not associated on the incubation of pre-synthesized silver nanoparticles <it>in vitro</it>.</p> <p>Conclusion</p> <p>Present study provides the indication of involvement of molecular machinery and various cellular proteins in the biosynthesis of silver nanoparticles. In this report, the study is mainly focused towards understanding the role of diverse cellular protein in the synthesis and capping of silver nanoparticles using <it>C. reinhardtii </it>as a model system.</p

Photodynamics of photo-activated BLUF coupled Endonuclease III mutant RmPAE from mesophilic, pigmented bacterium Rubellimicrobium mesophilum strain MSL-20T

The pink to light reddish-pigmented bacterium Rubellimicrobium mesophilum strain MSL-20T contains a BLUF coupled endonuclease III of unknown function. A purified recombinant triple mutated sample of the BLUF coupled endonuclease III (F5Y, N27H, A87W) named RmPAE (Rubellimicrobium mesophilum Photo-Activated Endonuclease) was produced and characterized by optical spectroscopic methods. The BLUF domain photo-cycling dynamics occurred with high efficient blue-light induced signaling state formation (quantum yield of signaling state formation φs ≈ 0.6), small spectral red-shift (δλs,r ≈ 5.4 nm), and slow thermal activated dark recovery to the receptor state (τrec ≈ 20 min at room temperature). An apparent RmPAE melting temperature of ϑm ≈ 63 °C was determined by stepwise sample heating and absorption spectrum analysis. The photo-degradation of RmPAE in the signaling state was determined by prolonged intense blue-light exposure. An irreversible flavin photo-degradation occurred with quantum yield of φD ≈ 2.6×10-5. Schemes of the photo-cycling and the photo-degradation dynamics are presented. Engineered RmPAE may find application as light guided DNA cutter in optogenetic applications

Disulphide Bridges of Phospholipase C of Chlamydomonas reinhardtii Modulates Lipid Interaction and Dimer Stability

BACKGROUND: Phospholipase C (PLC) is an enzyme that plays pivotal role in a number of signaling cascades. These are active in the plasma membrane and triggers cellular responses by catalyzing the hydrolysis of membrane phospholipids and thereby generating the secondary messengers. Phosphatidylinositol-PLC (PI-PLC) specifically interacts with phosphoinositide and/or phosphoinositol and catalyzes specific cleavage of sn-3- phosphodiester bond. Several isoforms of PLC are known to form and function as dimer but very little is known about the molecular basis of the dimerization and its importance in the lipid interaction. PRINCIPAL FINDINGS: We herein report that, the disruption of disulphide bond of a novel PI-specific PLC of C. reinhardtii (CrPLC) can modulate its interaction affinity with a set of phospholipids and also the stability of its dimer. CrPLC was found to form a mixture of higher oligomeric states with monomer and dimer as major species. Dimer adduct of CrPLC disappeared in the presence of DTT, which suggested the involvement of disulphide bond(s) in CrPLC oligomerization. Dimer-monomer equilibrium studies with the isolated fractions of CrPLC monomer and dimer supported the involvement of covalent forces in the dimerization of CrPLC. A disulphide bridge was found to be responsible for the dimerization and Cys7 seems to be involved in the formation of the disulphide bond. This crucial disulphide bond also modulated the lipid affinity of CrPLC. Oligomers of CrPLC were also captured in in vivo condition. CrPLC was mainly found to be localized in the plasma membrane of the cell. The cell surface localization of CrPLC may have significant implication in the downstream regulatory function of CrPLC. SIGNIFICANCE: This study helps in establishing the role of CrPLC (or similar proteins) in the quaternary structure of the molecule its affinities during lipid interactions

Light-evoked Somatosensory Perception of Transgenic Rats That Express Channelrhodopsin-2 in Dorsal Root Ganglion Cells

In vertebrate somatosensory systems, each mode of touch-pressure, temperature or pain is sensed by sensory endings of different dorsal root ganglion (DRG) neurons, which conducted to the specific cortical loci as nerve impulses. Therefore, direct electrical stimulation of the peripheral nerve endings causes an erroneous sensation to be conducted by the nerve. We have recently generated several transgenic lines of rat in which channelrhodopsin-2 (ChR2) transgene is driven by the Thy-1.2 promoter. In one of them, W-TChR2V4, some neurons were endowed with photosensitivity by the introduction of the ChR2 gene, coding an algal photoreceptor molecule. The DRG neurons expressing ChR2 were immunohistochemically identified using specific antibodies to the markers of mechanoreceptive or nociceptive neurons. Their peripheral nerve endings in the plantar skin as well as the central endings in the spinal cord were also examined. We identified that ChR2 is expressed in a certain population of large neurons in the DRG of W-TChR2V4. On the basis of their morphology and molecular markers, these neurons were classified as mechanoreceptive but not nociceptive. ChR2 was also distributed in their peripheral sensory nerve endings, some of which were closely associated with CK20-positive cells to form Merkel cell-neurite complexes or with S-100-positive cells to form structures like Meissner's corpuscles. These nerve endings are thus suggested to be involved in the sensing of touch. Each W-TChR2V4 rat showed a sensory-evoked behavior in response to blue LED flashes on the plantar skin. It is thus suggested that each rat acquired an unusual sensory modality of sensing blue light through the skin as touch-pressure. This light-evoked somatosensory perception should facilitate study of how the complex tactile sense emerges in the brain

Diversity and Evolution of Sensor Histidine Kinases in Eukaryotes

Histidine kinases (HKs) are primary sensor proteins that act in cell signaling pathways generically referred to as "two component systems" (TCSs). TCSs are among the most widely distributed transduction systems used by both prokaryotic and eukaryotic organisms to detect and respond to a broad range of environmental cues. The structure and distribution of HK proteins are now well documented in prokaryotes but information is still fragmentary for eukaryotes. Here, we have taken advantage of recent genomic resources to explore the structural diversity and the phylogenetic distribution of HKs in the prominent eukaryotic supergroups. Searches of the genomes of 67 eukaryotic species spread evenly throughout the phylogenetic tree of life identified 748 predicted HK proteins. Independent phylogenetic analyses of predicted HK proteins were carried out for each of the major eukaryotic supergroups. This allowed most of the compiled sequences to be categorised into previously described HK groups. Beyond the phylogenetic analysis of eukaryotic HKs, this study revealed some interesting findings: (i) characterisation of some previously undescribed eukaryotic HK groups with predicted functions putatively related to physiological traits; (ii) discovery of HK groups that were previously believed to be restricted to a single kingdom in additional supergroups and (iii) indications that some evolutionary paths have led to the appearance, transfer, duplication, and loss of HK genes in some phylogenetic lineages. This study provides an unprecedented overview of the structure and distribution of HKs in the Eukaryota and represents a first step towards deciphering the evolution of TCS signaling in living organisms

Localization and dimer stability of a newly identified microbial rhodopsin from a polar, non-motile green algae

Abstract Objective The eukaryotic plasma membrane localized light-gated proton-pumping rhodopsins possesses great optogenetic applications for repolarization (silencing) of the neuronal activity simply by light illumination. Very few plasma membrane localized proton-pumping rhodopsins of a eukaryotic origin are known that have optogenetic potential. Our objective was to identify and characterize microbial rhodopsin of an eukaryotic origin that expresses on plasma membrane. The plasma membrane localized light-gated proton pump of an eukaryotic origin hold great promise to be used as an optogenetic tools for the neurobiology. Results Here, we had characterized the cellular expression and membrane localization of a new rhodopsin in Antarctican algae Coccomyxa subellipsoidea. It is the first algal ion pumping rhodopsin that localizes to the plasma membrane of the eukaryotic cells. Coccomyxa subellipsoidea rhodopsin exists in the monomeric and dimeric state both the in vivo and in vitro. The dimeric form of the Coccomyxa subellipsoidea rhodopsin is resistant to heat and detergent denaturants

MOESM1 of Localization and dimer stability of a newly identified microbial rhodopsin from a polar, non-motile green algae

Additional file 1. Recombinant CsR expressed in E. coli were probed with anti-Penta His Ab and anti-CsR Ab

Photodynamics of optogenetic BLUF coupled photoactivated adenylyl cyclases (PACs)

Optogenetics is a fast developing science that combines gene biology and optics to new research fields in neuroscience and cell biology. In the introduction a short literature update of optogenetic tools is given with some emphasis on rhodopsin and flavin based optogenetic tools. Then the BLUF coupled photo activated adenylyl cyclases (PACs) are discussed (BLUF = Blue Light sensor Using FAD). The primary photo-dynamics of the BLUF domains is described. A BLUF domain photocycle scheme and a BLUF domain reaction coordinate scheme are presented. The adenylyl cyclase action of catalytic ATP to cAMP (a cellular second messenger) conversion is described. Light-induced cyclase activity of BLUF coupled photoactivated adenylyl cyclases (BLUF-PACs) is analyzed. Optogenetic BLUF-PAC applications are shortly reviewed. (C) 2016 Elsevier Ltd. All rights reserved