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Proteorhodopsin (PR) is the most abundant retinal protein on earth and functions as a light-driven proton pump. Despite extensive efforts, structural data for PR photointermediate states have not been obtained. On the basis of dynamic nuclear polarization (DNP)-enhanced solid-state NMR, we were able to analyze the retinal polyene chain between positions C10 and C15 as well as the Schiff base nitrogen in the ground state in comparison to light-induced, cryotrapped K- and M-states. A high M-state population could be achieved by preventing reprotonation of the Schiff base through a mutation of the primary proton donor (E108Q). Our data reveal unexpected large and alternating 13C chemical shift changes in the K-state propagating away from the Schiff base along the polyene chain. Furthermore, two different M-states have been observed reflecting the Schiff base reorientation after the deprotonation step. Our study provides novel insight into the photocycle of PR and also demonstrates the power of DNP-enhanced solid-state NMR to bridge the gap between functional and structural data and models. [ABSTRACT FROM AUTHOR]

Proteorhodopsin is a light-driven proton pumping membrane protein related to bacteriorhodopsin. It contains an all- trans retinal A1 chromophore covalently bound to a lysine residue via a protonated Schiff base. In this study, we exploited density functional theory (DFT) calculations to investigate the retinal binding pocket in the dark state and after mimicking photoisomerization. The model of the binding pocket is constructed incrementally by adding the residues near the retinal that are necessary to ensure a stable protonated Schiff base. The presence of a few water molecules near the Schiff base turns out to be an essential feature of the model. The absorption properties are then studied using time-dependent DFT (TDDFT) and compared to experimental data to further validate the structural model and to assess the accuracy of the computational setting. It is shown that TDDFT is able to reproduce the main absorption peak accurately and to quantitatively determine the spectral shift induced by substituting the native all- trans retinal A1 chromophore with different retinal analogues. Moreover, ab initio molecular dynamics simulations are performed to investigate the vibrational spectra of our models before and after isomerization. Specific differences in the vibrational spectra are identified that provide further insight into experimental FTIR difference spectra. [ABSTRACT FROM AUTHOR]

This study investigated the culturable aerobic phototrophic bacteria present in soil samples collected in the proximity of the Belgian Princess Elisabeth Station in the Sør Rondane Mountains, East Antarctica. Until recently, only oxygenic phototrophic bacteria ( Cyanobacteria ) were well known from Antarctic soils. However, more recent non-cultivation-based studies have demonstrated the presence of anoxygenic phototrophs and, particularly, aerobic anoxygenic phototrophic bacteria in these areas. Approximately 1000 isolates obtained after prolonged incubation under different growth conditions were studied and characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Representative strains were identified by sequence analysis of 16S rRNA genes. More than half of the isolates grouped among known aerobic anoxygenic phototrophic taxa, particularly with Sphingomonadaceae , Methylobacterium and Brevundimonas . In addition, a total of 330 isolates were tested for the presence of key phototrophy genes. While rhodopsin genes were not detected, multiple isolates possessed key genes of the bacteriochlorophyll synthesis pathway. The majority of these potential aerobic anoxygenic phototrophic strains grouped with Alphaproteobacteria ( Sphingomonas , Methylobacterium , Brevundimonas and Polymorphobacter ). [ABSTRACT FROM AUTHOR]

Marine Bacteroidetes constitute a very abundant bacterioplankton group in the oceans that plays a key role in recycling particulate organic matter and includes several photoheterotrophic members containing proteorhodopsin. Relatively few marine Bacteroidetes species have been described and, moreover, they correspond to cultured isolates, which in most cases do not represent the actual abundant or ecologically relevant microorganisms in the natural environment. In this study, we explored the microdiversity of 98 Single Amplified Genomes (SAGs) retrieved from the surface waters of the underexplored North Indian Ocean, whose most closely related isolate is Kordia algicida OT-1. Using Multi Locus Sequencing Analysis (MLSA) we found no microdiversity in the tested conserved phylogenetic markers (16S rRNA and 23S rRNA genes), the fast-evolving Internal Transcribed Spacer and the functional markers proteorhodopsin and the beta-subunit of RNA polymerase. Furthermore, we carried out a Fragment Recruitment Analysis (FRA) with marine metagenomes to learn about the distribution and dynamics of this microorganism in different locations, depths and size fractions. This analysis indicated that this taxon belongs to the rare biosphere, showing its highest abundance after upwelling-induced phytoplankton blooms and sinking to the deep ocean with large organic matter particles. This uncultured Kordia lineage likely represents a novel Kordia species (Kordia sp. CFSAG39SUR) that contains the proteorhodopsin gene and has a widespread spatial and vertical distribution. The combination of SAGs and MLSA makes a valuable approach to infer putative ecological roles of uncultured abundant microorganisms. [ABSTRACT FROM AUTHOR]

The ability of aerobic anoxygenic photoheterotrophs (AAPs) to gain additional energy from sunlight represents a competitive advantage, especially in conditions where light has easy access or under environmental conditions may change quickly, such as those in the world´s oceans. However, the knowledge about the metabolic consequences of aerobic anoxygenic photosynthesis is very limited. Combining transcriptome and metabolome analyses, isotopic labelling techniques, measurements of growth, oxygen uptake rates, flow cytometry, and a number of other biochemical analytical techniques we obtained a comprehensive overview on the complex adaption of the marine bacterium Dinoroseobacter shibae DFL12T during transition from heterotrophy to photoheterotrophy (growth on succinate). Growth in light was characterized by reduced respiration, a decreased metabolic flux through the tricarboxylic acid (TCA) cycle and the assimilation of CO2 via an enhanced flux through the ethylmalonyl-CoA (EMC) pathway, which was shown to be connected to the serine metabolism. Adaptation to photoheterotrophy is mainly characterized by metabolic reactions caused by a surplus of reducing potential and might depend on genes located in one operon, encoding branching point enzymes of the EMC pathway, serine metabolism and the TCA cycle. [ABSTRACT FROM AUTHOR]

The retinal protein proteorhodopsin is a homolog of the well-characterized light-driven proton pump bacteriorhodopsin. Basic mechanisms of proton transport seem to be conserved, but there are noticeable differences in the pH ranges of proton transport. Proton transport and protonation state of a carboxylic acid side chain, the primary proton acceptor, are correlated. In case of proteorhodopsin, the p Ka of the primary proton acceptor Asp-97 (p Ka ≈ 7.5) is unexpectedly close to environmental pH (pH ≈ 8). A significant fraction of proteorhodopsin is possibly inactive at natural pH, in contrast to bacteriorhodopsin. We investigated photoinduced kinetics of proteorhodopsin between pH 5 and pH 9 by time resolved UV/vis absorption spectroscopy. Kinetics is inhomogeneous within that pH region and can be considered as a superposition of two fractions. These fractions are correlated with the Asp-97 titration curve. Beside Asp-97, protonation equilibria of other groups influence kinetics, but the observations do not point toward major differences of primary proton acceptor function in proteorhodopsin and bacteriorhodopsin. The p Ka of proteorhodopsin and some of its variants is suspected to be an example of molecular adaptation to the physiology of the original organisms. [ABSTRACT FROM AUTHOR]

The trans-membrane protein – proteorhodopsin (pR) has been incorporated into supported lipid bilayers (SLB). In-plane electric fields have been used to manipulate the orientation and concentration of these proteins, within the SLB, through electrophoresis leading to a 25-fold increase concentration of pR. [ABSTRACT FROM AUTHOR]

Sunlight can be directly harvested by photoheterotrophic bacteria to create a pH gradient across the membrane, which can then be utilized to produce ATP. Despite the potential importance of this trophic strategy, when and where such organisms are found in the seas and oceans is poorly described. Here, we describe the abundance and taxonomy of bacteria with different trophic strategies (heterotrophs, phototrophs and photoheterotrophs) in contrasting water masses of the ultra-oligotrophic eastern Mediterranean Sea. These water bodies, an anticyclonic eddy and a high-chlorophyll patch resulting from transport of nutrient-rich coastal waters into offshore oligotrophic waters, each supported different microbial populations in surface waters. Based on infrared microscopy and metagenomics, aerobic anoxygenic photoheterotrophic (AAP) bacteria represented up to 10.4% of the microbial community. In contrast, the proteorhodopsin (PR) gene was found in 78.6%-118.8% of the bacterial genome equivalents, the highest abundance reported to date. These results suggest that PR-mediated photoheterotrophy may be especially important in oligotrophic, potentially phosphate-limited conditions. [ABSTRACT FROM AUTHOR]

Proteorhodopsins (PR) are phylogenetically diverse and highly expressed proton pumps in marine bacterial communities. The phylogenetic diversity and in situ expression of the main PR groups in polar off-shore, coastal and estuarine waters is poorly known and their abundance has not yet been reported. Here, we show that PR gene sequences of the southern Beaufort Sea including MacKenzie shelf and estuary are mainly affiliated to Gammaproteobacteria, Alphaproteobacteria and Bacteroidetes. Substantial overlap (78%) between DNA- and cDNA-based librairies indicated in situ PR transcription within a large fraction of PR-containing community. Sets of specific qPCR primers were designed to measure the absolute abundances of the major PR types. Spatial and depth profiles showed that PR-containing bacteria were abundant throughout the photic zone, comprising up to 45% of total bacteria. Although their abundance varied greatly with location and depth, Alphaproteobacteria predominated in the PR community in all water masses, with SAR11 as the major PR type. Low nutrient concentrations rather than light were the environmental drivers that best explained the abundance and distribution of arctic PR types. Together, our data suggests that PR-based phototrophy could be the major phototrophic prokaryotic process during the Arctic Ocean summer.

Bacteria that utilize sunlight to supplement metabolic activity are now being described in a range of ecosystems. While it is likely that phototrophy provides an important competitive advantage, the contribution that these microorganisms make to the bioenergetics of polar marine ecosystems is unknown. In this minireview, we discuss recent advances in our understanding of phototrophic bacteria and highlight the need for future research.

Proteorhodopsin (PR) and Gloeobacter rhodopsin (GR) are retinal-based light-driven proton pumps that absorb visible light (maxima at 520-540 nm). Shifting the action spectra of these proton pumps beyond 700 nm would generate new prospects in optogenetics, membrane sensor technology, and complementation of oxygenic phototrophy. We therefore investigated the effect of red-shifting analogues of retinal, combined with red-shifting mutations, on the spectral properties and pump activity of the resulting pigments. We investigated a variety of analogues, including many novel ones. One of the novel analogues we tested, 3-methylamino-16-nor-1,2,3,4-didehydroretinal (MMAR), produced exciting results. This analogue red-shifted all of the rhodopsin variants tested, accompanied by a strong broadening of the absorbance band, tailing out to 850-950 nm. In particular, MMAR showed a strong synergistic effect with the PR-D212N,F234S double mutant, inducing an astonishing 200 nm red shift in the absorbance maximum. To our knowledge, this is by far the largest red shift reported for any retinal protein. Very importantly, all MMAR-containing holoproteins are the first rhodopsins retaining significant pump activity under near-infrared illumination (730 nm light-emitting diode). Such MMAR-based rhodopsin variants present very promising opportunities for further synthetic biology modification and for a variety of biotechnological and biophysical applications. [ABSTRACT FROM AUTHOR]

Proteorhodopsins (PR) are phylogenetically diverse and highly expressed proton pumps in marine bacterial communities. The phylogenetic diversity and in situ expression of the main PR groups in polar off-shore, coastal and estuarine waters is poorly known and their abundance has not yet been reported. Here, we show that PR gene sequences of the southern Beaufort Sea including MacKenzie shelf and estuary are mainly affiliated to Gammaproteobacteria, Alphaproteobacteria, and Bacteroidetes. Substantial overlap (78%) between DNA- and cDNA-based librairies indicated in situ PR transcription within a large fraction of PR-containing community. Sets of specific qPCR primers were designed to measure the absolute abundances of the major PR types. Spatial and depth profiles showed that PR-containing bacteria were abundant throughout the photic zone, comprising up to 45% of total bacteria. Although their abundance varied greatly with location and depth, Alphaproteobacteria predominated in the PR community in all water masses, with SAR11 as the major PR type. Low nutrient concentrations rather than light were the environmental drivers that best explained the abundance and distribution of arctic PR types. Together, our data suggests that PR-based phototrophy could be the major phototrophic prokaryotic process during the Arctic Ocean summer. [ABSTRACT FROM AUTHOR]

For applications in synthetic biology, for example, the bottom-up assembly of biomolecular nanofactories, modules of specific and controllable functionalities are essential. Of fundamental importance in such systems are energizing modules, which are able to establish an electrochemical gradient across a vesicular membrane as an energy source for powering other modules. Light-driven proton pumps like proteorhodopsin (PR) are excellent candidates for efficient energy conversion. We have extended the versatility of PR by implementing an on/off switch based on reversible chemical modification of a site-specifically introduced cysteine residue. The position of this cysteine residue in PR was identified by structure-based cysteine mutagenesis combined with a proton-pumping assay using E. coli cells overexpressing PR and PR proteoliposomes. The identified PR mutant represents the first light-driven proton pump that can be chemically switched on/off depending on the requirements of the molecular system. [ABSTRACT FROM AUTHOR]

The sequenced genome of the poly-extremophile Exiguobacterium sp. S17, isolated from modern stromatolites at Laguna Socompa (3,570 m), a High-Altitude Andean Lake (HAAL) in Argentinean Puna revealed a putative proteorhodopsin-encoding gene. The HAAL area is exposed to the highest UV irradiation on Earth, making the microbial community living in the stromatolites test cases for survival strategies under extreme conditions. The heterologous expressed protein E17R from Exiguobacterium (248 amino acids, 85% sequence identity to its ortholog ESR from E. sibiricum) was assembled with retinal displaying an absorbance maximum at 524 nm, which makes it a member of the green-absorbing PR-subfamily. Titration down to low pH values (eventually causing partial protein denaturation) indicated a pK value between two and three. Global fitting of data from laser flash-induced absorption changes gave evidence for an early red-shifted intermediate (its formation being below the experimental resolution) that decayed (τ1 = 3.5 μs) into another red-shifted intermediate. This species decayed in a two-step process (τ2 = 84 μs, τ3 = 11 ms), to which the initial state of E17-PR was reformed with a kinetics of 2 ms. Proton transport capability of the HAAL protein was determined by BLM measurements. Additional blue light irradiation reduced the proton current, clearly identifying a blue light absorbing, M-like intermediate. The apparent absence of this intermediate is explained by closely matching formation and decay kinetics. [ABSTRACT FROM AUTHOR]

Retinal-based photosynthesis may contribute to the free energy conversion needed for growth of an organism carrying out oxygenic photosynthesis, like a cyanobacterium. After optimization, this may even enhance the overall efficiency of phototrophic growth of such organisms in sustainability applications. As a first step towards this, we here report on functional expression of the archetype proteorhodopsin in Synechocystis sp. PCC 6803. Upon use of the moderate-strength psbA2 promoter, holo-proteorhodopsin is expressed in this cyanobacterium, at a level of up to 10 5 molecules per cell, presumably in a hexameric quaternary structure, and with approximately equal distribution (on a protein-content basis) over the thylakoid and the cytoplasmic membrane fraction. These results also demonstrate that Synechocystis sp . PCC 6803 has the capacity to synthesize all -trans -retinal. Expressing a substantial amount of a heterologous opsin membrane protein causes a substantial growth retardation Synechocystis , as is clear from a strain expressing PROPS, a non-pumping mutant derivative of proteorhodopsin. Relative to this latter strain, proteorhodopsin expression, however, measurably stimulates its growth. [ABSTRACT FROM AUTHOR]

Many organisms use proton pump to earn energy for living. Some proton pumps start to work by light and one of the famous proteins are called proteorhodopsin (PR). From recent study it used not only protons but also mono-valent cations, divalent cations, or mono-valent anions during pumping activity. The goal of this study is to find new types of proton pumping proteins in the surface of the ocean. Metagenome samples were collected from the beach in Taean-gun and Incheon (Kkotji beach (36°30′0′′N, 126°19′56′′E), Kkotji mud (36°30′8′′N, 126°19′60′′E), Duegi beach (36°31′6′′N, 126°19′39′′E), Sorae salt pond (37°24′25′′N, 126°44′41′′E), swamp (37°24′59′′N, 126°44′54′′E) and reservoir (37°24′39′′N, 126°45′5′′E) in West Sea of Korea. Genomic DNA of each sample was isolated and used for PCR with specific primers for PR and sodium pumping rhodopsin. As a result, we obtained an unidentified PR in Duegi beach sample. The unidentified PR was expressed with chimeric expression system. It has 528 nm absorption maximum at pH 7. By the light differential spectrum measurement, putative M and O photo-intermediates were detected at around 400 and 600 nm, respectively. Similar to GPR, it has light driven outward proton transfer activity. [ABSTRACT FROM AUTHOR]

The effects of chromophoric group structures on the functional properties of bacteriorhodopsin (BR) and proteorhodopsin from E. sibiricum (ESRh) were compared. ESRh retinal binding site was found as preserving the similar stereo- and spatial restrictions on the chromophore structure during the retinal protein reconstitution process (except for C25-analog AR8). It was revealed that the structure peculiarities of the chromophore analog molecules affect the optical parameters of ESRh and BR pigment families in similar ways. [ABSTRACT FROM AUTHOR]

Proteorhodopsin (PR) is an outward light-driven proton pump observed in marine eubacteria. Despite many structural and functional similarities to bacteriorhodopsin (BR) in archaea, which also acts as an outward proton pump, the mechanism of the photoinduced proton release and uptake is different between two H+-pumps. In this study, we investigated the pH dependence of the photocycle and proton transfer in PR reconstituted with the phospholipid membrane under alkaline conditions. Under these conditions, as the medium pH increased, a blue-shifted photoproduct (defined as Ma), which is different from M, with a pKa of ca. 9.2 was produced. The sequence of the photoinduced proton uptake and release during the photocycle was inverted with the increase in pH. A pKa value of ca. 9.5 was estimated for this inversion and was in good agreement with the pKa value of the formation of Ma (~9.2). In addition, we measured the photoelectric current generated by PRs attached to a thin polymer film at varying pH. Interestingly, increases in the medium pH evoked bidirectional photocurrents, which may imply a possible reversal of the direction of the proton movement at alkaline pH. On the basis of these findings, a putative photocycle and proton transfer scheme in PR under alkaline pH conditions was proposed. [ABSTRACT FROM AUTHOR]

The microbiome of the largest environment on Earth has been gradually revealing its secrets over four decades of study. Despite the dispersed nature of substrates and the transience of surfaces, marine microbes drive essential transformations in all global elemental cycles. Much has been learned about the microbes that carry out key biogeochemical processes, but there are still plenty of ambiguities about the factors important in regulating activity, including the role of microbial interactions. Identifying the molecular "currencies" exchanged within the microbial community will provide key information on microbiome function and its vulnerability to environmental change. [ABSTRACT FROM AUTHOR]

Proteorhodopsin, a member of the microbial rhodopsin family, is a seven-transmembrane a-helical protein that functions as a light-driven proton pump. Understanding the proton-pumping mechanism of proteorhodopsin requires intimate knowledge of the proton transfer pathway via complex hydrogen-bonding networks formed by amino acid residues and internal water molecules. Here we conducted a series of microsecond time scale molecular dynamics simulations on both the dark state and the initial photoactivated state of blue proteorhodopsin to reveal the structural basis for proton transfer with respect to protein internal hydration. A complex series of dynamic hydrogen-bonding networks involving water molecules exists, facilitated by water channels and hydration sites within proteorhodopsin. High levels of hydration were discovered at each proton transfer site--the retinal binding pocket and proton uptake and release sites--underscoring the critical participation of water molecules in the proton-pumping mechanism. Water-bridged interactions and local water channels were also observed and can potentially mediate long-distance proton transfer between each site. The most significant phenomenon is after isomerization of retinal, an increase in water flux occurs that connects the proton release group, a conserved arginine residue, and the retinal binding pocket. Our results provide a detailed description of the internal hydration of the early photointermediates in the proteorhodopsin photocycle under alkaline pH conditions. These results lay the fundamental groundwork for understanding the intimate role that hydration plays in the structure-function relationship underlying the proteorhodopsin proton-pumping mechanism, as well as providing context for the relationship of hydration in proteorhodopsin to other microbial retinal proteins. [ABSTRACT FROM AUTHOR]

Proteorhodopsins (PR) are retinal-binding membrane proteins that function as light-driven proton pumps to generate energy for metabolism and growth. Recently PR-like genes have been identified in some marine eukaryotic protists, including diatoms, dinoflagellates, haptophytes and cryptophytes. These rhodopsins are homologous to green-light-absorbing, ATP-generating PRs present within bacteria. Here we show that in the oceanic diatom Pseudo-nitzschia granii, PR-like gene and protein expressions increase appreciably under iron limitation. In a survey of available transcriptomes, PR-like genes in diatoms are generally found in isolates from marine habitats where seasonal to chronic growth limitation by the micronutrient iron is prevalent, yet similar biogeographical patterns are not apparent in other phytoplankton taxa. We propose that rhodopsin-based phototrophy could account for a proportion of energy synthesis in marine eukaryotic photoautotrophs, especially when photosynthesis is compromised by low iron availability. This alternative ATP-generating pathway could have significant effects on plankton community structure and global ocean carbon cycling. [ABSTRACT FROM AUTHOR]

Protein trans-splicing using split inteins is well established as a useful tool for protein engineering. Here we show, for the first time, that this method can be applied to a membrane protein under native conditions. We provide compelling evidence that the heptahelical proteorhodopsin can be assembled from two separate fragments consisting of helical bundles A and B and C, D, E, F, and G via a splicing site located in the BC loop. The procedure presented here is on the basis of dual expression and ligation in vivo. Global fold, stability, and photodynamics were analyzed in detergent by CD, stationary, as well as time-resolved optical spectroscopy. The fold within lipid bilayers has been probed by high field and dynamic nuclear polarization-enhanced solid-state NMR utilizing a 13C-labeled retinal cofactor and extensively 13C-15N-labeled protein. Our data show unambiguously that the ligation product is identical to its non-ligated counterpart. Furthermore, our data highlight the effects of BC loop modifications onto the photocycle kinetics of proteorhodopsin. Our data demonstrate that a correctly folded and functionally intact protein can be produced in this artificial way. Our findings are of high relevance for a general understanding of the assembly of membrane proteins for elucidating intramolecular interactions, and they offer the possibility of developing novel labeling schemes for spectroscopic applications. [ABSTRACT FROM AUTHOR]

Recognition of the environmental role of photoheterotrophic bacteria has been one of the main themes of aquatic microbiology over the last 15 years. Aside from cyanobacteria and proteorhodopsin-containing bacteria, aerobic anoxygenic phototrophic (AAP) bacteria are the third most numerous group of phototrophic prokaryotes in the ocean. This functional group represents a diverse assembly of species which taxonomically belong to various subgroups of Alpha-, Beta- and Gammaproteobacteria. AAP bacteria are facultative photoheterotrophs which use bacteriochlorophyll-containing reaction centers to harvest light energy. The light-derived energy increases their bacterial growth efficiency, which provides a competitive advantage over heterotrophic species. Thanks to their enzymatic machinery AAP bacteria are active, rapidly growing organisms which contribute significantly to the recycling of organic matter. This chapter summarizes the current knowledge of the ecology of AAP bacteria in aquatic environments, implying their specific role in the microbial loop. [ABSTRACT FROM AUTHOR]

Microbial rhodopsins and G-protein coupled receptors (GPCRs, which include animal rhodopsins) are two distinct (super) families of heptahelical (7TM) membrane proteins that share obvious structural similarities but no significant sequence similarity. Comparison of the recently solved high-resolution structures of the sodium-translocating bacterial rhodopsin and various Na+-binding GPCRs revealed striking similarity of their sodium-binding sites. This similarity allowed us to construct a structure-guided sequence alignment for the two (super)families, which highlighted their evolutionary relatedness. Our analysis supports a common underlying molecular mechanism for both families that involves a highly conserved aromatic residue playing a pivotal role in rotation of the 6th transmembrane helix. Reviewers: This article was reviewed by Oded Beja, G. P. S. Raghava and L. Aravind. [ABSTRACT FROM AUTHOR]

The UV light absorbing species of proteorhodopsin with deprotonated Schiff base retinal was investigated using steady-state fluorescence and femtosecond pumpprobe spectroscopy. Compared to the all-trans retinal with protonated Schiff base, the deprotonated chromophore absorbs at 365 nm and exhibits a blue-shifted fluorescence spectrum. The unusually long-lived excited state decays bi-exponentially with time constants of 8 ps and 130 ps to form a deprotonated 13-cis retinal as the primary photo-product. [ABSTRACT FROM AUTHOR]

Fluorescence is not frequently used as a tool for investigating the photocycles of rhodopsins, largely because of the low quantum yield of the retinal chromophore. However, a new class of genetically encoded voltage sensors is based upon rhodopsins and their fluorescence. The first such sensor reported in the literature was the proteorhodopsin optical proton sensor (PROPS), which is capable of indicating membrane voltage changes in bacteria by means of changes in fluorescence. However, the properties of this fluorescence, such as its lifetime decay components and its origin in the protein photocycle, remain unknown. This paper reports steady-state and nanosecond time-resolved emission of this protein expressed in two strains of Escherichia coli, before and after membrane depolarization. The voltage-dependence of a particularly long lifetime component is established. Additional work to improve quantum yields and improve the general utility of PROPS is suggested. [ABSTRACT FROM AUTHOR]

The significance of specific lipids for proton pumping by the bacterial rhodopsin proteorhodopsin (pR) was studied. To this end, it was examined whether pR preferentially binds certain lipids and whether molecular properties of the lipid environment affect the photocycle. pR's photocycle was followed by microsecond flash-photolysis in the visible spectral range. It was fastest in phosphatidylcholine liposomes (soy bean lipid), intermediate in 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonate (CHAPS): 1,2-dioleoyl- sn -glycero-3-phosphocholine (DOPC) bicelles and in Triton X-100, and slowest when pR was solubilized in CHAPS. In bicelles with different lipid compositions, the nature of the head groups, the unsaturation level and the fatty acid chain length had small effects on the photocycle. The specific affinity of pR for lipids of the expression host Escherichia coli was investigated by an optimized method of lipid isolation from purified membrane protein using two different concentrations of the detergent N -dodecyl-β- d -maltoside (DDM). We found that 11 lipids were copurified per pR molecule at 0.1% DDM, whereas essentially all lipids were stripped off from pR by 1% DDM. The relative amounts of copurified phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin did not correlate with the molar percentages normally present in E. coli cells. The results indicate a predominance of phosphatidylethanolamine species in the lipid annulus around recombinant pR that are less polar than the dominant species in the cell membrane of the expression host E. coli . [ABSTRACT FROM AUTHOR]

Membrane proteins often form oligomeric complexes within the lipid bilayer, but factors controlling their assembly are hard to predict and experimentally difficult to determine. An understanding of protein--protein interactions within the lipid bilayer is however required in order to elucidate the role of oligomerization for their functional mechanism and stabilization. Here, we demonstrate for the pentameric, heptahelical membrane protein green proteorho-dopsin that solid-state NMR could identify specific interactions at the protomer interfaces, if the sensitivity is enhanced by dynamic nuclear polarization. For this purpose, differently labeled protomers have been assembled into the full pentamer complex embedded within the lipid bilayer. We show for this proof of concept that one specific salt bridge determines the formation of pentamers or hexamers. Data are supported by laser-induced liquid bead ion desorption mass spectrometry and by blue native polyacrylamide gel electrophoresis analysis. The presented approach is universally applicable and opens the door toward analyzing membrane protein interactions within homo-oligomers directly in the membrane. [ABSTRACT FROM AUTHOR]

Proteorhodopsin (PR) is a light harvesting protein widely distributed among bacterioplankton that plays an integral energetic role in a new pathway of marine light capture. The conversion of light into chemical energy in non-chlorophyll-based bacterial systems could contribute to overcoming thermodynamic and metabolic constraints in biofuels production. In an attempt to improve biohydrogen production yields, H 2 evolution catalyzed by endogenous hydrogenases, Hyd-3 and/or Hyd-4, was measured when recombinant proteorhodopsin (PR) was concomitantly expressed in Escherichia coli cells. Higher amounts of H 2 were obtained with recombinant cells in a light and chromophore dependent manner. This effect was only observed when HyfR, the specific transcriptional activator of the hyf operon encoding Hyd-4 was overexpressed in E. coli , suggesting that an excess of protons generated by PR activity could increase hydrogen production by Hyd-4 but not by Hyd-3. Although many of the subunits of Hyd-3 and Hyd-4 are very similar, Hyd-4 possesses three additional proton-translocating NADH-ubiquinone oxidoreductase subunits, suggesting that it is dependent upon Δμ H + . Altogether, these results suggest that protons generated by proteorhodopsin in the periplasm can only enhance hydrogen production by hydrogenases with associated proton translocating subunits. [ABSTRACT FROM AUTHOR]

A type of photoheterotrophic bacteria contain a transmembrane light-driven proton pump called proteorhodopsins (PRs). Due to the prevalence of these organisms in the upper water column of the World's Ocean, and their potential for light-driven ATP generation, they have been suggested to significantly influence energy and matter flows in the biosphere. To date, evidence for the significance of the light-driven metabolism of PR-containing prokaryotes has been obtained by comparing growth in batch culture, under light versus dark conditions, and it appears that responses to light are linked to unfavorable conditions, which so far have not been well parameterized. We studied light responses to carbon yields of the PR-containing Photobacterium angustum S14 using continuous culture conditions and light-dark cycles. We observed significant effects of light-dark cycles compared to dark controls, as well as significant differences between samples after 12 h illumination versus 12 h darkness. However, these effects were only observed under higher cell counts and lower pH associated with higher substrate concentrations. Under these substrate levels Pirt's maintenance coefficient was higher when compared to lower substrate dark controls, and decreased under light-dark cycles. It appears that light responses by P. angustum S14 are induced by the energetic status of the cells rather than by low substrate concentrations. [ABSTRACT FROM AUTHOR]

This review covers the properties of a retinal protein (ESR) from the psychrotrophic bacterium Exiguobacterium sibiricum that functions as a light-driven proton pump. The presence of a lysine residue at the position corresponding to intramolecular proton donor for the Schiff base represents a unique structural feature of ESR. We have shown that Lys96 successfully facilitates delivery of protons from the cytoplasmic surface to the Schiff base, thus acting as a proton donor in ESR. Since proton uptake during the photocycle precedes Schiff base reprotonation, we conclude that this residue is initially in the uncharged state and acquires a proton for a short time after Schiff base deprotonation and M intermediate formation. Involvement of Lys as a proton donor distinguishes ESR from the related retinal proteins - bacteriorhodopsin (BR), proteorhodopsin (PR), and xanthorhodopsin (XR), in which the donor function is performed by residues with a carboxyl side chain. Like other eubacterial proton pumps (PR and XR), ESR contains a histidine residue interacting with the proton acceptor Asp85. In contrast to PR, this interaction leads to shift of the acceptor's p K to more acidic pH, thus providing its ability to function over a wide pH range. The presence of a strong H-bond between Asp85 and His57, the structure of the proton-conducting pathways from cytoplasmic surface to the Schiff base and to extracellular surface, and other properties of ESR were demonstrated by solving its three-dimensional structure, which revealed several differences from known structures of BR and XR. The structure of ESR, its photocycle, and proton transfer reactions are discussed in comparison with homologous retinal proteins. [ABSTRACT FROM AUTHOR]

ABSTRACT Since solar light energy is the source of all renewable biological energy, the direct usage of light energy by bacterial cell factory has been a very attractive concept, especially using light energy to promote anaerobic fermentation growth and even recycle low-energy carbon source when energy is the limiting factor. Proteorhodopsin(PR), a light-driven proton pump proven to couple with ATP synthesis when expressed heterogeneously, is an interesting and simple option to enable light usage in engineered strains. However, although it was reported to influence fermentation in some cases, heterogeneous proteorhodopsin expression was never shown to support growth advantage or cause metabolic shift by photophosphorylation so far. Hereby, we presented the first experimental evidence that heterogeneously expressed proteorhodopsin can provide growth advantage and cause ATP-dependent metabolism shift of acetate and lactate changes in Escherichia coli at anaerobic condition. Those discoveries suggest further application potential of PR in anaerobic fermentation where energy is a limiting factor. Biotechnol. Bioeng. 2015;112: 947-956. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The gene encoding proteorhodopsin AEX55013 from Dokdonia sp. PRO95 was cloned and expressed in Escherichia coli cells. Illumination of the proteorhodopsin-producing E. coli cells in Na-containing media resulted in alkalinization of the media. This response was accelerated by uncoupler CCCP and inhibited by penetrating anion SCN. Illumination of the cells in a sodium-free medium (made by substituting Na with K) resulted in SCN-stimulated and CCCP-sensitive acidification of the medium. Illumination of the proteorhodopsin-containing E. coli cells caused CCCP-resistant transmembrane sodium export from these cells. We conclude that the proteorhodopsin from the marine flavobacterium Dokdonia sp. PRO95 is a primary light-driven Na-pump. A high level of the heterologous production in E. coli cells as well as stability and purity of the isolated protein makes this proteorhodopsin an attractive model for studying the mechanism of active sodium transmembrane translocation. [ABSTRACT FROM AUTHOR]

The plasma membrane is the crucial interface between the cell and its exterior, packed with embedded proteins experiencing simultaneous protein–protein and protein–membrane interactions. A prominent example of cell membrane complexity is the assembly of transmembrane proteins into oligomeric structures, with potential functional consequences that are not well understood. From the study of proteorhodopsin (PR), a prototypical seven-transmembrane light-driven bacterial proton pump, we find evidence that the inter-protein interaction modulated by self-association yields functional changes observable from the protein interior. We also demonstrate that the oligomer is likely a physiologically relevant form of PR, as crosslinking of recombinantly expressed PR reveals an oligomeric population within the Escherichia coli membrane (putatively hexameric). Upon chromatographic isolation of oligomeric and monomeric PR in surfactant micelles, the oligomer exhibits distinctly different optical absorption properties from monomeric PR, as reflected in a prominent decrease in the p K a of the primary proton acceptor residue (D97) and slowing of the light-driven conformational change. These functional effects are predominantly determined by specific PR–PR contacts over nonspecific surfactant interactions. Interestingly, varying the surfactant type alters the population of oligomeric states and the proximity of proteins within an oligomer, as determined by sparse electron paramagnetic resonance distance measurements. Nevertheless, the dynamic surfactant environment retains the key function-tuning property exerted by oligomeric contacts. A potentially general design principle for transmembrane protein function emerges from this work, one that hinges on specific oligomeric contacts that can be modulated by protein expression or membrane composition. [ABSTRACT FROM AUTHOR]

Nanoparticles composed of amphiphilic scaffold proteins and small lipid bilayers are valuable tools for reconstitution and subsequent functional and structural characterization of membrane proteins. In combination with cell-free protein production systems, nanoparticles can be used to cotranslationally and translocon independently insert membrane proteins into tailored lipid environments. This strategy enables rapid generation of protein/nanoparticle complexes by avoiding detergent contact of nascent membrane proteins. Frequently in use are nanoparticles assembled with engineered derivatives of either the membrane scaffold protein (MSP) or the Saposin A (SapA) scaffold. Furthermore, several strategies for the formation of membrane protein/nanoparticle complexes in cell-free reactions exist. However, it is unknown how these strategies affect functional folding, oligomeric assembly and membrane insertion efficiency of cell-free synthesized membrane proteins. We systematically studied membrane protein insertion efficiency and sample quality of cell-free synthesized proteorhodopsin (PR) which was cotranslationally inserted in MSP and SapA based nanoparticles. Three possible PR/nanoparticle formation strategies were analyzed: (i) PR integration into supplied preassembled nanoparticles, (ii) coassembly of nanoparticles from supplied scaffold proteins and lipids upon PR expression, and (iii) coexpression of scaffold proteins together with PR in presence of supplied lipids. Yield, homogeneity as well as the formation of higher PR oligomeric complexes from samples generated by the three strategies were analyzed. Conditions found optimal for PR were applied for the synthesis of a G-protein coupled receptor. The study gives a comprehensive guideline for the rapid synthesis of membrane protein/nanoparticle samples by different processes and identifies key parameters to modulate sample yield and quality. [Display omitted] • First report on implementing Salipro particles for cell-free production of membrane protein/nanoparticles. • First report on using the coassembly strategy for the cotranslational production of membrane protein/nanoparticles. • First direct quantitative comparison of the cotranslational membrane protein/nanoparticle formation with three different strategies. • First report analyzing MP oligomerization after cotranslational MP/nanoparticle formation with different strategies. [ABSTRACT FROM AUTHOR]