Rhodopsin-like Proteins Research Articles

Expression analysis of microbial rhodopsin-like genes in Guillardia theta.

The Cryptomonad Guillardia theta has 42 genes encoding microbial rhodopsin-like proteins in their genomes. Light-driven ion-pump activity has been reported for some rhodopsins based on heterologous E. coli or mammalian cell expression systems. However, neither their physiological roles nor the expression of those genes in native cells are known. To reveal their physiological roles, we investigated the expression patterns of these genes under various growth conditions. Nitrogen (N) deficiency induced color change in exponentially growing G. theta cells from brown to green. The 29 rhodopsin-like genes were expressed in native cells. We found that the expression of 6 genes was induced under N depletion, while that of another 6 genes was reduced under N depletion.

A second rhodopsin-like protein in Cyanophora paradoxa: Gene sequence and protein expression in a cell-free system

Here we report the identification and expression of a second rhodopsin-like protein in the alga Cyanophora paradoxa (Glaucophyta), named Cyanophopsin_2. This new protein was identified due to a serendipity event, since the RACE reaction performed to complete the sequence of Cyanophopsin_1, (the first rhodopsin-like protein of C. paradoxa identified in 2009 by our group), amplified a 619bp sequence corresponding to a portion of a new gene of the same protein family. The full sequence consists of 1175bp consisting of 849bp coding DNA sequence and 4 introns of 326bp. The protein is characterized by an N-terminal region of 47 amino acids, followed by a region with 7 α-helices of 213 amino acids and a C-terminal region of 22 amino acids. This protein showed high identity with Cyanophopsin_1 and other rhodopsin-like proteins of Archea, Bacteria, Fungi and Algae. Cyanophosin_2 (CpR2) was expressed in a cell-free expression system, and characterized by means of absorption spectroscopy.

A rhodopsin-like protein in Cyanophora paradoxa: gene sequence and protein immunolocalization.

Here, we report the DNA sequence of the rhodopsin gene in the alga Cyanophora paradoxa (Glaucophyta). The primers were designed according to the conserved regions of prokaryotic and eukaryotic rhodopsin-like proteins deposited in the GenBank. The sequence consists of 1,272 bp comprised of 5 introns. The correspondent protein, named Cyanophopsin, showed high identity to rhodopsin-like proteins of Archea, Bacteria, Fungi, and Algae. At the N-terminal, the protein is characterized by a region with no transmembrane alpha-helices (80 aa), followed by a region with 7alpha-helices (219 aa) and a shorter 35-aa C-terminal region. The DNA sequence of the N-terminal region was expressed in E. coli and the recombinant purified peptide was used as antigen in hens to obtain polyclonal antibodies. Indirect immunofluorescence in C. paradoxa cells showed a marked labeling of the muroplast (aka cyanelle) membrane.

A rhodopsin immunoanalog in the related photosensitive protozoans Blepharisma japonicum and Stentor coeruleus

Immunoblotting of isolated cell membrane fractions from ciliates Blepharisma japonicum and Stentor coeruleus with a polyclonal antibody raised against rhodopsin revealed one strong protein band of about 36 kDa, thought to correspond to protozoan rhodopsin. Inspection of both ciliates labeled with the same antibody using a confocal microscope confirmed the immunoblotting result and demonstrated the presence of these rhodopsin-like molecules localized within the cell membrane area. Immunoblot analysis of the ciliate membrane fractions resolved by two-dimensional gel electrophoresis identified two distinct 36 kDa spots at pIs of 4.5 and 7.0 for Blepharisma, and three spots at pIs of 4.4, 5.0 and 6.0 for Stentor, indicating a possible mixture of phosphorylated rhodopsin species in these cells. The obtained results suggest that both Blepharisma and the related ciliate Stentor contain within the cell membrane the rhodopsin-like proteins, which may be involved as receptor molecules in the sensory transduction pathway mediating motile photoresponses in these protists as in other species of lower eukaryota.

Subpicosecond Protein Backbone Changes Detected during the Green-Absorbing Proteorhodopsin Primary Photoreaction

Recent studies demonstrate that photoactive proteins can react within several picoseconds to photon absorption by their chromophores. Faster subpicosecond protein responses have been suggested to occur in rhodopsin-like proteins where retinal photoisomerization may impulsively drive structural changes in nearby protein groups. Here, we test this possibility by investigating the earliest protein structural changes occurring in proteorhodopsin (PR) using ultrafast transient infrared (TIR) spectroscopy with approximately 200 fs time resolution combined with nonperturbing isotope labeling. PR is a recently discovered microbial rhodopsin similar to bacteriorhodopsin (BR) found in marine proteobacteria and functions as a proton pump. Vibrational bands in the retinal fingerprint (1175-1215 cm(-1)) and ethylenic stretching (1500-1570 cm(-1)) regions characteristic of all-trans to 13-cis chromophore isomerization and formation of a red-shifted photointermediate appear with a 500-700 fs time constant after photoexcitation. Bands characteristic of partial return to the ground state evolve with a 2.0-3.5 ps time constant. In addition, a negative band appears at 1548 cm(-1) with a time constant of 500-700 fs, which on the basis of total-15N and retinal C15D (retinal with a deuterium on carbon 15) isotope labeling is assigned to an amide II peptide backbone mode that shifts to near 1538 cm(-1) concomitantly with chromophore isomerization. Our results demonstrate that one or more peptide backbone groups in PR respond with a time constant of 500-700 fs, almost coincident with the light-driven retinylidene chromophore isomerization. The protein changes we observe on a subpicosecond time scale may be involved in storage of the absorbed photon energy subsequently utilized for proton transport.

Light Recognition Mechanism of Archaea through Phoborhodopsin.

Many organisms including human beings have rhodopsin as photoreceptor. In cell membranes of archaeal Halobacterium salinarum, there exist four rhodopsin-like proteins (so-called archaeal rhodopsins). Two of these work as light-driven ion pumps. The other two work as photosensors and are named sensory rhodopsin (sR) and phoborhodopsin (pR). pR mediates the negative phototaxis. Recently, X-ray crystallographic structure of pR derived from Natronobacterium pharaonis is reported, and allows us to investigate the function-structure relationship at atomic level. In this review, we describe our results on the photochemistry and the signal transduction mechanism of pR.

Presence of Rhodopsin-like Proteins in Sorghum bicolor and Pisum sativum

Summary Antiserum raised against squid ( Loligo forbesi ) rhodopsin cross-reacted with proteins from microsomal and plasma membranes isolated from Sorghum bicolor and Pisum sativum . In S. bicolor the main crossreacting protein (SbR) presented a molecular mass of 38 kDa. SbR appeared to be more abundant in mesocotyl microsomal membranes than in leaf or root membranes. SbR showed similarities to G-protein coupled receptors (GPCRs) in that it required a high detergent concentration for extraction and exhibited diffuse antibody staining on blots similar to that of squid rhodopsin. In P. sativum hypocotyl membranes the strongest immunological signal corresponded to a 35 kDa polypeptide (PsR) that was shown to be enriched in plasma membranes. A P. sativum glycoprotein bearing the same Mr and membrane association behaviour as PsR could be purified by Con A affinity chromatography and elution with α methyl manoside. The chromatographic fractions containing the glycoprotein were shown to possess high and specific GTPγS binding activity, indicating association to G-proteins. The glycoprotein showed weak cross-reaction to anti-rhodopsin serum. N-terminal sequencing of the glycoprotein did not show high homology to other proteins in the databases but indicated a likely amphipathic character.

Rhodopsin: A Photopigment for Phototaxis in Euglena gracilis

For over 100 years, a major focus of photobiological studies has been the unicellular flagellate, Euglena gracilis, an organism well suited for such investigations by its special complement of organelles that may be considered an ancient, yet complete “visual” system. The possible photoreceptive roles of the cytoplasmic stigma and the photoreceptor (paraflagellar swelling) of E. gracilis are still under debate, because of conflicting interpretations of the results produced so far by the different research groups working on this microorganism. This article deals with our hypothesis, first put forward in the late 1980s, that rhodopsin-like proteins are responsible for photo-detection and that the paraxial rod is involved in the control of flagellar movements. This hypothesis uses oriented dipole and electroconformational coupling mechanisms as the physical phenomena that produce signal transduction. A model for phototaxis is presented.

Rhodopsin: A Photopigment for Phototaxis in Euglena gracilis

For over 100 years, a major focus of photobiological studies has been the unicellular flagellate, Euglena gracilis, an organism well suited for such investigations by its special complement of organelles that may be considered an ancient, yet complete “visual” system. The possible photoreceptive roles of the cytoplasmic stigma and the photoreceptor (paraflagellar swelling) of E. gracilis are still under debate, because of conflicting interpretations of the results produced so far by the different research groups working on this microorganism. This article deals with our hypothesis, first put forward in the late 1980s, that rhodopsin-like proteins are responsible for photo-detection and that the paraxial rod is involved in the control of flagellar movements. This hypothesis uses oriented dipole and electroconformational coupling mechanisms as the physical phenomena that produce signal transduction. A model for phototaxis is presented.

Rhodopsin-like proteins in planarian eye and auricle: detection and functional analysis

The presence of rhodopsin-like proteins in the eyes and auricles of the freshwater planarian Dugesia japonica was confirmed using anti-frog-rhodopsin rabbit IgG. The apparent relative molecular masses of these proteins were 65x10(3) and 62x10(3), and positive reactions to IgG were localized to the microvilli of the photoreceptor cells in the eyes and to the sensory cilia, rootlets and microvilli in the auricles. Eye- or head-excised planarians showed no negative phototaxis, whereas intact or auricle-excised planarians did. During regeneration in head-excised planarians, the appearance of rhodopsin-like proteins in the regenerating eyes corresponded to the recovery of negative phototaxis behaviour. Head or auricle excision enhanced asexual fission under continuous illumination. However, eye excision had no such effect. These results suggest that the rhodopsin-like proteins in the eyes work as photoreceptors for negative phototaxis behaviour and that, in the auricles, they are involved in asexual fission originating from the circadian rhythm.

Retinal Identification inPelvetia fastigiata

Unidirectional blue light directs the rhizoid-thallus axis in the apolar zygote of the brown algaPelvetia fastigiata.This effect is mediated by an increase in the intracellular concentration of cGMP. Here, we show the extraction, purification and identification of 1 μg of all-transretinal from 1.2 × 106Pelvetiazygotes. The number of retinal molecules per cell was about 4∗109. Since retinal, wherever present, is exclusively associated with an opsin to form a light sensitive complex (rhodopsin-like proteins), and since the physiological response originated by this protein produces a variation of cGMP concentration, this new finding suggests that a rhodopsin-like protein could be the photoreceptor in this brown alga.

Identification of retinal in higher plants: Is a rhodopsinlike protein the blue light receptor?

We here report the purification and identification of all- trans-retinal by column chromatography, HPLC and GC-MS in a tomato mutant ( Lycopersion esculentum Mill. aurea mutant). Since retinal, wherever present, is almost exclusively associated to an opsin to form a light-sensitive complex (rhodopsin-like proteins), this finding suggests that a rhodopsin-like protein could function as a blue light photoreceptor in higher plants.

Presence of rhodopsin-like proteins in the planarian head

Presence of rhodopsin-like proteins in the planarian head

Phototaxis and rhodopsinlike proteins in Halobacterium halobium.

An outline of the evidence was given that the receptor molecules for two photorepellent sensory systems in Halobacterium halobium were the two rhodopsinlike proteins found recently in this extremely halophilic archaebacterium. One is a photointermediate of sensory rhodopsin. The name phoborhodopsin was proposed for the other.

Retinal migration during dark reduction of bacteriorhodopsin.

When the retinal Schiff base in chymotryptically cleaved bacteriorhodopsin is reduced to a secondary retinylamine by prolonged exposure to 10% (wt/vol) sodium cyanoborohydride, at pH 10, in the absence of light, approximately 45% of the retinal is found linked to Lys-41 and 22% to Lys-40, and the remainder is scattered over various sites on the large chymotryptic fragment, including the physiological site at Lys-216. The retinal-binding site is destroyed or blocked by the reduction conditions, but the bacteriorhodopsin lattice remains intact. The results demonstrate that artifactual linkage to Lys-40/41 is possible under special conditions. Under these conditions, the epsilon-amino groups of Lys-40/41 show an enhanced ability to form retinylidene linkages with the retinal released by the physiological linkage site at Lys-216, due to some combination of close proximity to the normal linkage site, and increased reactivity with respect to other lysine epsilon-amino groups. The results are of interest for the characterization of the two newly discovered rhodopsin-like proteins, halorhodopsin and slow rhodopsin.