Proton-pumping Rhodopsin Research Articles

A new class of marine Euryarchaeota group II from the Mediterranean deep chlorophyll maximum.

We have analyzed metagenomic fosmid clones from the deep chlorophyll maximum (DCM), which, by genomic parameters, correspond to the 16S ribosomal RNA (rRNA)-defined marine Euryarchaeota group IIB (MGIIB). The fosmid collections associated with this group add up to 4 Mb and correspond to at least two species within this group. From the proposed essential genes contained in the collections, we infer that large sections of the conserved regions of the genomes of these microbes have been recovered. The genomes indicate a photoheterotrophic lifestyle, similar to that of the available genome of MGIIA (assembled from an estuarine metagenome in Puget Sound, Washington Pacific coast), with a proton-pumping rhodopsin of the same kind. Several genomic features support an aerobic metabolism with diversified substrate degradation capabilities that include xenobiotics and agar. On the other hand, these MGIIB representatives are non-motile and possess similar genome size to the MGIIA-assembled genome, but with a lower GC content. The large phylogenomic gap with other known archaea indicates that this is a new class of marine Euryarchaeota for which we suggest the name Thalassoarchaea. The analysis of recruitment from available metagenomes indicates that the representatives of group IIB described here are largely found at the DCM (ca. 50 m deep), in which they are abundant (up to 0.5% of the reads), and at the surface mostly during the winter mixing, which explains formerly described 16S rRNA distribution patterns. Their uneven representation in environmental samples that are close in space and time might indicate sporadic blooms.

Light-promoted rhodopsin expression and starvation survival in the marine dinoflagellate Oxyrrhis marina.

The discovery of microbial rhodopsins in marine proteobacteria changed the dogma that photosynthesis is the only pathway to use the solar energy for biological utilization in the marine environment. Although homologs of these rhodopsins have been identified in dinoflagellates, the diversity of the encoding genes and their physiological roles remain unexplored. As an initial step toward addressing the gap, we conducted high-throughput transcriptome sequencing on Oxyrrhis marina to retrieve rhodopsin transcripts, rapid amplification of cDNA ends to isolate full-length cDNAs of dominant representatives, and quantitative reverse-transcription PCR to investigate their expression under varying conditions. Our phylogenetic analyses showed that O. marina contained both the proton-pumping type (PR) and sensory type (SR) rhodopsins, and the transcriptome data showed that the PR type dominated over the SR type. We compared rhodopsin gene expression for cultures kept under light: dark cycle and continuous darkness in a time course of 24 days without feeding. Although both types of rhodopsin were expressed under the two conditions, the expression levels of PR were much higher than SR, consistent with the transcriptomic data. Furthermore, relative to cultures kept in the dark, rhodopsin expression levels and cell survival rate were both higher in cultures grown in the light. This is the first report of light-dependent promotion of starvation survival and concomitant promotion of PR expression in a eukaryote. While direct evidence needs to come from functional test on rhodopsins in vitro or gene knockout/knockdown experiments, our results suggest that the proton-pumping rhodopsin might be responsible for the light-enhanced survival of O. marina, as previously demonstrated in bacteria.

Irreversible trimer to monomer transition of thermophilic rhodopsin upon thermal stimulation.

Assembly is one of the keys to understand biological molecules, and it takes place in spatial and temporal domains upon stimulation. Microbial rhodopsin (also called retinal protein) is a membrane-embedded protein that has a retinal chromophore within seven-transmembrane α-helices and shows homo-, di-, tri-, penta-, and hexameric assemblies. Those assemblies are closely related to critical physiological properties such as stabilizing the protein structure and regulating their photoreaction dynamics. Here we investigated the assembly and disassembly of thermophilic rhodopsin (TR), which is a novel proton-pumping rhodopsin derived from a thermophile living at 75 °C. TR was characterized using size-exclusion chromatography and circular dichroism spectroscopy, and formed a trimer at 25 °C, but irreversibly dissociated into monomers upon thermal stimulation. The transition temperature was estimated to be 68 °C. The irreversible nature made it possible to investigate the photochemical properties of both the trimer and the monomer independently. Compared with the trimer, the absorption maximum of the monomer is blue-shifted by 6 nm without any changes in the retinal composition, pKa value for the counterion or the sequence of the proton movement. The photocycling rate of the monomeric TR was similar to that of the trimeric TR. A similar trimer-monomer transition upon thermal stimulation was observed for another eubacterial rhodopsin GR but not for the archaeal rhodopsins AR3 and HwBR, suggesting that the transition is conserved in bacterial rhodopsins. Thus, the thermal stimulation of TR induces the irreversible disassembly of the trimer.

Thermal and Spectroscopic Characterization of a Proton Pumping Rhodopsin from an Extreme Thermophile

So far retinylidene proteins (∼rhodopsin) have not been discovered in thermophilic organisms. In this study we investigated and characterized a microbial rhodopsin derived from the extreme thermophilic bacterium Thermus thermophilus, which lives in a hot spring at around 75 °C. The gene for the retinylidene protein, named thermophilic rhodopsin (TR), was chemically synthesized with codon optimization. The codon optimized TR protein was functionally expressed in the cell membranes of Escherichia coli cells and showed active proton transport upon photoillumination. Spectroscopic measurements revealed that the purified TR bound only all-trans-retinal as a chromophore and showed an absorption maximum at 530 nm. In addition, TR exhibited both photocycle kinetics and pH-dependent absorption changes, which are characteristic of rhodopsins. Of note, time-dependent thermal denaturation experiments revealed that TR maintained its absorption even at 75 °C, and the denaturation rate constant of TR was much lower than those of other proton pumping rhodopsins such as archaerhodopsin-3 (200 ×), Haloquadratum walsbyi bacteriorhodopsin (by 10-times), and Gloeobacter rhodopsin (100 ×). Thus, these results suggest that microbial rhodopsins are also distributed among thermophilic organisms and have high stability. TR should allow the investigation of the molecular mechanisms of ion transport and protein folding.

Dynamics of Voltage Profile in Enzymatic Ion Transporters, Demonstrated in Electrokinetics of Proton Pumping Rhodopsin

H +-pumping rhodopsins mediate a primordial conversion of light to metabolic energy. Bacteriorhodopsin from Halobacterium salinarium is the first identified and (biochemically) best-studied H +-pumping rhodopsin. The electrical properties of H +-pumping rhodopsins, however, are known in more detail for the homolog Acetabularia rhodopsin, isolated from the eukaryotic green alga Acetabularia acetabulum. Based on data from Acetabularia rhodopsin we present a general reaction kinetic model of H +-pumping rhodopsins with only seven independent parameters, which fits the kinetic properties of photocurrents as functions of light, transmembrane voltage, internal and external pH, and time. The model describes fast photoisomerization of retinal with simultaneous H + transfer to an H + acceptor, reprotonation of retinal from the intracellular face via an H + donor, and proton release to the extracellular space via an H + release complex. The voltage sensitivities of the individual reaction steps and their temporal changes are treated here by a novel approach, whereby—as in an Ohmic voltage divider—the effective portions of the total transmembrane voltage decrease with the relative velocities of the individual reaction steps. This analysis quantitatively infers dynamic changes of the voltage profile and of the pK values of the H +-binding sites involved.

1P-253 ロドプシンのカーボンナノチューブへの内包(光生物・視覚,光受容(1),第46回日本生物物理学会年会)

1P-253 ロドプシンのカーボンナノチューブへの内包(光生物・視覚,光受容(1),第46回日本生物物理学会年会)