The photosynthetic reaction center (RC) from the purple Rhodobacter (R.) sphaeroides bacterium is a protein with unique photoconversion capability that can be exploited in hybrid systems for energy conversion. We have developed a tailored aryleneethynylene organic fluorophore (AE750) acting as efficient light harvesting antenna and successfully bioconjugated it to the photosynthetic RC. We have also demonstrated that, under suitable conditions, the biohybrid AE750-RC system can outperform the energy photoconversion ability of the native protein.

Humankind is in great need of new energy sources. The use of solar radiation for powering the planet would fulfil the energy requirements of Earth's inhabitants as well as greatly mitigate tension flares arising from the uneven distribution of fossil fuels and environmental problems associated to their extraction procedures. How to proceed than? Easy to say! Mother Nature is inspiring: all life on earth is based on the conversion of the solar radiation into high energy molecules, including gas and oil human beings are consuming these days, by mean of the so-called primary photoconverters, i.e. the photosynthetic organisms, plants, algae and some kind of bacteria. So, let's learn from Nature and assemble in our laboratories artificial systems capable of exploiting solar energy for photocatalysis and electrical energy production, i.e. mimic photosynthesis. Not an easy task of course, but a large number of laboratory are heavily involved since the last 25 years in the field of artificial photosynthesis and are obtaining encouraging results. The photosynthetic apparatus used by photosynthetic organisms to convert solar energy and drive their metabolism is the photochemical core where photoconversion takes place, and is constituted by a protein portion allocating several pigments directly involved in the harvesting of solar light and in the subsequent sequence of electron transfer reactions which eventually lead to the formation of an electron-hole couple to be used for any energy requiring process. In artificial photosynthesis the role of the protein scaffold in often ignored and attention is devoted to assembly molecular system for optimising light harvest and electron-transfer reactions, focussing to the "less-complex" portion of the photosynthetic apparatus. What would be a different paradigm in artificial photosynthesis? Assemble artificial photoconverters using genuine natural components formed by hybrid organic-biologic systems. The hybrids have a central protein, the so-called photosynthetic reaction center (RC) that converts sunlight into a charge-separated state having a lifetime sufficient to allow ancillary chemistry to take place. The RCs can be eventually garnished with opportune organic moieties to be used for different applications.The state of the art of these hybrid organic-biologic photosynthetic assemblies will be reviewed.

The reconstitution of the integral membrane protein photosynthetic reaction center (RC) in polymersomes, i.e. artificial closed vesicles, was achieved by the micelle-to-vesicle transition technique, a very mild protocol based on size exclusion chromatography often used to drive the incorporation of proteins contemporarily to liposome formation. An optimized protocol was used to successfully reconstitute the protein in a fully active state in polymersomes formed by the tri-block copolymers PMOXA<inf>22</inf>-PDMS<inf>61</inf>-PMOXA<inf>22</inf>. The RC is very sensitive to its solubilizing environment and was used to probe the positioning of the protein in the vesicles. According to charge-recombination experiments and to the enzymatic activity assay, the RC is found to accommodate in the PMOXA<inf>22</inf> region of the polymersome, facing the water bulk solution, rather than in the PDMS<inf>61</inf> transmembrane-like region. Furthermore, polymersomes were found to preserve protein integrity efficiently as the biomimetic lipid bilayers but show a much longer temporal stability than lipid based vesicles.

Hexavalent chromium represents an outstanding risk for the environment and the health of human beings, as it is considerably involved in the genesis of cancer and other fatal diseases. Biological reduction of Cr(VI) to Cr(III) is a potentially useful mechanism to remediate chromium (VI) pollution and to detoxify contaminated wastes. The photosynthetic purple bacterium Rhodobacter sphaeroides is known for its ability to tolerate high concentrations of several heavy metal ions, to bioaccumulate nickel and cobalt, and to reduce oxyanions as tellurite, selenite and chromate. The response of the carotenoidless mutant R26 to chromate stress under phototrophic conditions has been recently investigated by biochemical and spectroscopic measurements, proteomic analysis and cell imaging, revealing good Cr(VI) reduction ability associated with morphological and compositional changes of the cell envelope, while no specific stress-induced chromate-reductase activity was found in the soluble proteome. Phototrophic biomass of Rhodobacter sphaeroides strain R26, harvested, washed, and stored at -20°C, was just thawed and used as Cr(VI) reduction catalyst. Chromate solutions, buffered at neutral pH and supplemented with a mixture of succinate, malonate and glucose as electron donors, have been employed for simulating the waste-water environment. The decrease of Cr(VI) concentration triggered by cells addition was evaluated by the diphenylcarbazide (DPC) assay. The analysis of reaction kinetics revealed that Rhodobacter sphaeroides resting biomass acts as an excellent bio-catalyst promoting chromate reduction by oxidizable carbon compounds. The role of abiotic variables such as pH, light, temperature and oxygen concentration was also assessed. Our data extend the information available about this phototrophic microorganism and elucidate its potential in Cr(VI) bioremediation applications.

The intensification of industrial technology increased heavy metal contamination in aquatic systems. Since inorganic pollutants cannot be degraded, an efficient removal system must be designed in order to detoxify heavy metal-contaminated wastewaters. Metal ion biosorption by microorganisms is an interesting mechanism which can be exploited for this purpose. The purple bacterium Rhodobacter sphaeroides is known for its ability to tolerate under phototrophic conditions high concentrations of several heavy metal ions and to bioaccumulate Ni2+ and Co2+ ions. In this work Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectroscopy and X-Ray Photoelectron Spectroscopy (XPS) have been employed for getting information about Ni2+ binding onto R. sphaeroides cell surface. The ability to bind nickel ions was evaluated both in free cells and in calcium alginate-immobilized biomass. Before Ni2+ exposure the bacterial biomass was washed thoroughly with KCl 0.1 M in order to fully saturate with K+ ions the negatively charged cell envelopes. XPS measurements revealed that treatment with Ni2+ resulted in full displacement of K+ ions from free R. sphaeroides cells, indicating high affinity between nickel ions and surface functional groups. Moreover ATR-FTIR measurements showed that Ni2+-treatment induce the shift of absorption bands arising from symmetric and asymmetric stretching modes of cell surface carboxylate groups, in agreement with their involvement in metal complexation. Calcium alginate beads entrapping bacterial biomass were prepared dropping a cell suspension supplemented with sodium alginate into 2% CaCl . XPS analysis of Ni2+-treated beads revealed that the exposure of cells to Ca2+ strongly inhibited Ni2+ uptake suggesting that displacement of Ca2+ by nickel ions does not occur. These data are of interest in order to identify optimal conditions for the efficient removal of Ni2+ by means of phototrophic bacterial biomass.

We demonstrate the direct bioconjugation of hydrogen-bonded organic semiconductors with two different complex functional proteins in an aqueous environment. The representative semiconductors are epindolidione and quinacridone, materials used in devices in the form of vacuum-evaporated polycrystalline films. First, these molecules in thin films react spontaneously with N-hydroxysuccinimide functionalized linkers: disuccinimidyl suberate and succinimidyl biotinate. The suberate linker is then used to covalently bind the Rhodobacter sphaeroides reaction centre (RC), the key photoenzyme for conversion of light into electrical charges in photosynthetic bacteria. Similarly, the biotin linker is used to bridge streptavidin to the surface of the hydrogen-bonded semiconductor film. Multiple-reflection infrared spectroscopy, water contact angle measurements, and atomic force microscopy are used to verify surface functionalization. The presence and functional integrity of the immobilized proteins are demonstrated by specific experiments: a charge recombination kinetics assay in the case of the RC, and photoluminescence measurements for quantum dot-labelled streptavidin. As key results of our work, we have shown that upon bioconjugation, the semiconductors preserve their favourable electrical properties: as evidenced by photoconductor devices operating under water sensitized by the RC, and thin film transistor measurements before and after bioconjugation. These are enabling steps for using hydrogen-bonded semiconductors as platforms for multifunctional bioelectronics devices.

Photosynthesis represents one of the most important biological reactions in the biosphere, since all life on Earth, directly or indirectly, depends on it as a source of energy. Nature performs the photosynthetic process using specialized protein - pigments complexes organized to ensure up to 98% conversion of the absorbed photons in stable, long - living charge separated states. A proper combination of the photosynthetic core , the reaction center (RC), with engineered materials, i.e. metals or inorganic semiconductor electrodes, has attracted great attention for the building of new versatile hybrid devices for solar energy conversion 1 . Here we propose a covalent approach able to stably anchor RCs onto evaporated thin films of the hydrogen - bonded pigments epindolidione (EPI) and quinacridone (QNC) 2 , a well - known class of organic colorants which have recently emerged as promising semiconductors, demonstrating hole mobility in the range of 0.1 - 1 cm 2 /Vs and outstanding operatio nal stability in both air and aqueous media with pH 3 - 10. Due to low - toxicity, biocom patibility and potential low - cost, EPI and QNC substrates are envisioned to go where other traditional semicond uctors simply cannot be applied, resulting to be amenable to direct surface bioconjuga tion. T he NH functional group of these molecules in thin film reacts spontaneously with N - hydroxysuccinimide functionalized linke rs as disuccinimidyl sub erate. The protruding linker s are then used to covalently bind the lysines residues of the Rhodobacter sphaeroides reaction center, by forming an amide linkage. Our pro tocol is shown to preserve the semiconducting properties of the pigment s while maintaini ng the protein's photoactivity. Multiple - ref lection infrared spectroscopy and atomic force microscopy demonstrated the effective covalent binding and the robustness of the protein anchoring even after buffer washing procedures compared to the weakness of the physisorbed RC interactions. Furthermore, RC charge recombination kinetic measurements confirmed the fully functionality of bioconjugated proteins and ruled out any possible hindering effect from the organic films. As key results of our work, we have shown that semiconductors preserve their favo rable electrical properties: the proposed photoconductor devices operate under water, before and after RCs anchoring . These are enabling steps for usin g hydrogen - bonded pigments as a platform for multifunct ional bioelectronics devices, paving the way in designing and realizing new photosynthetic protein - based hybrid systems.

The covalent functionalization of photosynthetic proteins with properly tailored organic molecular antennas represents a powerful approach to build a new generation of hybrid systems capable of exploiting solar energy. In this paper the strategy for the synthesis of the tailored aryleneethynylene organic fluorophore (AE) properly designed to act as light harvesting antenna is presented along with its successful bioconjugation to the photosynthetic reaction center RC from the bacterium Rhodobacter sphaeroides

Hydrogen-bonded pigments are a class of organic colorants, which features many natural-origin molecules that have been used for centuries, as well as numerous mass-produced industrial synthetic compounds used in applications as various as out- door paints, cosmetics, and printing inks. Their widespread use in the dye and pigment industry is motivated by three favorable properties: low-cost production, excellent stability, and low toxicity, with some of them considered less hazardous than even water-soluble food dyes. Recently, H-bonded pigments have emerged also as promising organic semiconductors, with epindolidione (EPI) and quinacridone (QNC) demonstrating hole mobility in the range of 0.1-1 cm2 V-1 s-1 and outstanding operational stability in both air and in aqueous environments with pH 3-10 [1]. This latter finding is motivating for deploying these materials in applications requiring direct interfacing with biological ''wet'' environments. Their N-H and C=O functional groups are the "chemical handles" that are in principle amenable for direct bioconjugation. A proper combination of the photosynthetic reaction center (RC), the pivotal protein in photosynthesis, with engineered materials such as metals or inorganic semiconductor electrodes, has attracted great attention for the building of new versatile hybrid devices for solar energy conversion. Here we propose a covalent approach able to stably anchor RCs onto evaporated thin films of EPI and QNC. The N-H functional group of these molecules in thin film reacts spontaneously with N-hydroxysuccinimide functionalized linkers as disuccinimidyl suberate. The protruding linkers are then used to covalently bind the lysines residues of the Rb. sphaeroides RC, by forming an amide linkage (right panel in the figure). Our protocol is shown to preserve the semiconducting properties of the pigments while maintaining the protein's photoactivity. Multiple reflection IR spectroscopy and AFM demonstrated the effective covalent binding and the robustness of the protein anchoring even after buffer washing. Furthermore, RC charge recombination kinetic measurements confirmed the full functionality of bioconjugated proteins ruling out any possible hindering effect from the organic films. As key results of our work, we show that semiconductors preserve their favorable electrical properties and the proposed photoconductor device operates under water, before and after RCs anchoring. These are enabling steps for using H-bonded pigments as a platform for multifunctional bioelectronics devices.

A high-throughput crystallographic investigation on several crystals of photosynthetic reaction center covalently bound to an ad-hoc synthesized artificial antenna (AE600) is presented. The investigation did not show a preferential binding site of the antenna molecule AE600 to the reaction center in the solid phase. An accurate crystallographic study allowed identifying a lysine residue sitting on periplasmic side of the protein as one of the bioconjugation sites. The residue sits on subunit M of the protein, in close proximity to the bacteriochlorophylls of the reaction center involved in the light absorption and conversion processes. Distances obtained from the crystallographic structure confirm that energy transfer between the antenna and the protein proceed with the Förster resonance mechanism.

The bacterial photosynthetic reaction centre (RC) is a membrane spanning protein that, upon illumination, promotes the reduction of a ubiquinone molecule withdrawing electrons from cytochrome c2. This photo-activated reaction has been often exploited, in suitably designed photoelectrochemical cells, to generate photocurrents sustained by the reduction at the working electrode of the photo-oxidized electron donor or by the oxidation of the electron acceptor. In this work we have explored in more detail the factors affecting the photocurrent generation in commercially available screen-printed electrochemical cells containing an electrolyte solution where RC proteins and suitable mediators are solubilized. In particular, the role of the applied potential and the influence of concentration and structure of acceptor and donor molecules have been assessed. We show that efficient generation of cathodic photocurrents in a three electrode configuration occurs at an applied potential of 0.0 V versus quasi-ref Ag (the open circuit potential of the system measured in the dark) in presence of ferrocenemethanol and decylubiquinone, which proved to guarantee high performances as electron donor and acceptor respectively. Moreover, we employed a set of differential equations, describing reaction and diffusion processes, for modelling with high accuracy the chronoamperometry profiles recorded at variable RC concentrations. This model allowed us to estimate the kinetic parameters relevant to the chemical and electrochemical reactions triggered by light and to get a snapshot of the electrolyte composition in the bulk and electrode surroundings at different times from the light exposure. The characteristic time course of the photocurrent, showing a fast rise to a peak value followed by a slower decay, has been therefore explained as the result of the strict interconnection between the dynamical processes involved.

The induction (sudden dark-to-light transition) of fluorescence of photosynthetic bacteria has proved to be sensitive tool for early detection of mercury (Hg2+) contamination of the culture medium. The major characteristics of the induction (dark, variable and maximum fluorescence levels together with rise time) offer an easier, faster and more informative assay of indication of the contamination than the conventional techniques. The inhibition of Hg2+ is stronger in the light than in the dark and follows complex kinetics. The fast component (in minutes) reflects the damage of the quinone acceptor pool of the RC and the slow component (in hours) is sensitive to the disintegration of the light harvesting system including the loss of the structural organization and of the pigments. By use of fluorescence induction, the dependence of the diverse pathways and kinetics of the mercury-induced effects on the age and the metabolic state of the bacteria were revealed.

Ultrasounds are used in many industrial, medical and research applications. Properties and function of proteins are strongly influenced by the interaction with the ultrasonic waves and their bioactivity can be lost because of alteration of protein structure. Surprisingly, to the best of our knowledge no study was carried out on Integral Membrane Proteins (IMPs), which are responsible for a variety of fundamental biological functions. In this work, the photosynthetic Reaction Center (RC) of the bacterium Rhodobacter sphaeroides has been used as a model for the study of the ultrasound-induced IMP denaturation. Purified RCs were suspended in i) detergent micelles, in ii) detergent-free buffer and iii) reconstituted in liposomes, and then treated with ultrasound at 30 W and 20 kHz at increasing times. The optical absorption spectra showed a progressive and irreversible denaturation in all cases, resulting from the perturbation of the protein scaffold structure, as confirmed by circular dichroism spectra that showed progressive alterations of the RC secondary structure. Charge recombination kinetics were studied to assess the protein photoactivity. The lifetime for the loss of RC photoactivity was 32 min in detergent micelles, ranged from 3.8 to 6.5 min in the different proteoliposomes formulations, and 5.5 min in detergent-free buffer. Atomic force microscopy revealed the formation of large RC aggregates related to the sonication-induced denaturation, in agreement with the scattering increase observed in solution.

Epidemiological studies associate whole-grain consumption with several health benefits and increasing evidence suggests whole-grain wheat polyphenols as healthy agents with anti-inflammatory properties (1). However, many studies demonstrated the impact, usually negative, of wheat bran, rich in polyphenols, on bread quality. We have previously evaluated the bread making ability of meals composed of re-milled semolina biofortified with selected durum wheat milling by-products (200 g/kg) that were: i) residuals of the second and third debranning steps of durum wheat (DB), ii) the micronized and air-classified thin fraction obtained from the same residuals (MB), or iii) coarse bran obtained from conventional roller milling of non-debranned durum wheat (B) (2). We showed that total soluble phenolic compounds and antioxidant activity were significantly higher in MB and DB than in B (2), with acceptable bread quality in particular for MB. However, their biological anti-inflammatory potential was unknown. The aim of this study was to analyse the vascular anti-inflammatory properties of the phenolic extracts obtained from different biofortified bread by evaluating endothelium-monocyte adhesion and endothelial and monocytic inflammatory gene expression. Cultured human endothelial and monocytic cells were incubated with increasing concentrations (1, 5 or 10 ?g/mL) of phenolic acids extracts from biofortified bread (B, DB, MB) before stimulation with inflammatory challenge lipopolysaccharide (LPS 1 ?g/mL). All phenolic acids extracts inhibited, in a concentration-dependent manner, the stimulated endothelial leukocyte adhesion, and the protein expression of endothelial adhesion molecules. By real time PCR, we found that B, DB and MB phenolic extracts down-regulated the mRNA levels of adhesion molecules as well as pro-inflammatory cytokines and chemoattractants in stimulated endothelial cells and monocytes. Our findings appreciate the bread biofortified with selected durum wheat milling by-products as a source of phenolic acids with multiple anti-inflammatory properties.

The present study aimed to develop and optimize liposome formulation for the colonicdelivery of biologically active compounds. A strategy to facilitate such targeting is to formulateliposomes with a polymer coating sensitive to the pH shifts in the gastrointestinal tract. To this end,liposomes encapsulating curcumin--chosen as the biologically active compound model--and coatedwith the pH-responsive polymer Eudragit S100 were prepared and characterized. Curcumin wasencapsulated into small unilamellar vesicles (SUVs) by the micelle-to-vesicle transition method (MVT)in a simple and organic solvent-free way. Curcumin-loaded liposomes were coated with EudragitS100 by a fast and easily scalable pH-driven method. The prepared liposomes were evaluated for size,surface morphology, entrapment efficiency, stability, in vitro drug release, and curcumin antioxidantactivity. In particular, curcumin-loaded liposomes displayed size lower than 100 nm, encapsulationefficiency of 98%, high stability at both 4 oC and 25 oC, high in vitro antioxidant activity, and acumulative release that was completed within 200 min. A good Eudragit S100 coating which didnot alter the properties of the curcumin-loaded liposomes was obtained. The present work thereforeprovides a fast and solvent-free method to prepare pH-responsive polymer-coated liposomes for thecolonic delivery of biologically active compounds.

Light machine: The simplest photosynthetic protein able to convert sunlight into other energy forms is covalently functionalized with a tailored organic dye to obtain a fully functional hybrid complex that outperforms the natural system in light harvesting and conversion ability

Following a bottom-up synthetic biology approach it is shown that vesicle-based cell-like systems (shortly "synthetic cells") can be designed and assembled to perform specific function (for biotechnological applications) and for studies in the origin-of-life field. We recently focused on the construction of synthetic cells capable to converting light into chemical energy. Here we first present our approach, which has been realized so far by the reconstitution of photosynthetic reaction centre in the membrane of giant lipid vesicles. Next, the details of our ongoing research program are presented. It involves the use of the reaction centre, the coenzyme Q-cytochrome c oxidoreductase, and the ATP synthase for creating an autonomous synthetic cell. We show experimental results on the chemistry of the first two proteins showing that they can efficiently sustain light-driven chemical oscillations. Moreover, the cyclic pattern has been reproduced in silico by a minimal kinetic model.

Deep eutectic solvents (DESs) are emerging as a new class of green solvents with the potential to replace organic solvents in several fundamental and applied processes. In this work, we offer an unprecedented characterization of the behavior of the bacterial photosynthetic reaction center (RC) from Rhodobacter sphaeroides in a series of choline chloride based DESs. RC is a membrane-spanning three-subunit pigment protein complex that, upon illumination, is capable of producing a stable charge-separated state. Thus, it represents the ideal model for carrying out basic studies of protein solvent interactions. Herein, we first report that, in many DES mixtures investigated, RC (a) is stable, (b) is capable of generating the charge-separated state, and (c) is even able to perform its natural photocycle. It proved, indeed, to be effective in reducing quinone molecules to quinol by withdrawing electrons from cytochrome c. As an example of biotechnological application, a photoelectrochemical cell based on DES-dissolved RC has also been designed and successfully employed to generate photocurrents arising from the reduction of the electron-donor ferrocenemethanol.

The development of an amperometric biosensor for herbicide detection, using bacterial reaction centers (RC) as biorecognition element, is presented. RC immobilization on gold screen printed electrodes was achieved by LIFT, a powerful physisorption-based immobilization technique that enhances the intimate contact between the protein and the electrode surface. As a result, stable photocurrents driven by direct electron transfer at the donor side were observed, both in the presence and in the absence of a quinone substrate in solution. The addition of quinone UQ(0) increased the photocurrents, while the UQ(0)-free system showed higher sensitivity to the herbicide terbutryn, a model inhibitor, acting as photocurrent attenuator. In spite of its simple design, the performances achieved by our mediatorless device are comparable or superior to those reported for analogous RC-based photoelectrochemical cells, in terms of both terbutryn sensing and photocurrent generation. (C) 2016 Elsevier B.V. All rights reserved.

The photosynthetic reaction center is an extraordinarily efficient natural photoconverter, which can be ideally used in combination with conducting or semiconducting interfaces to produce electrical signals in response to absorption of photons. The actual applicability of this protein in bioelectronic devices critically depends on the finding of (a) suitable deposition methods enabling controlled addressing and precise orientation of the protein on electrode interfaces and (b) chemical manipulation protocols able to tune and enhance protein light absorption in specific or broader spectral regions. Literature reports several examples of approaches to fulfill these requirements, which have faced in different ways the fundamental issues of assembling the biological component and non-natural systems, such as electrode surfaces and artificial light harvesting components. Here we present a short overview of the main methods reported to accomplish both the objectives by properly "garnishing" the photosynthetic reaction center (RC) via chemical modifications.

The non-sulfur, purple, facultatively phototrophic bacterium Rhodobacter (R.) sphaeroides represents a unique model for the investigation of the structure, function and biosynthesis of the energy-transducing system in photosynthetic machineries. Photosynthetic units share a basic architecture, composed by an efficient light collecting system, which funnels light to the reaction center, where photons are converted into chemical potential energy with a quantum yield close to unity. In the R. sphaeroides wild type (strain 2.4.1), two distinct antenna complexes are present: the LH-II, absorbing at 800 and 850 nm, and the LH-I, with a maximum at 870 nm, appearing as a shoulder of the 850 nm LH-II band. Strong dependence of the absorption spectrum on changing growing conditions, i.e light intensity or heavy metal ions concentration, requires to find out a robust, non-destructive instrumental method of investigation which could help to quickly solve the complex structure of the absorption signals in the presence of different stress factors. In this work, we present second-order derivative spectroscopy as the ideal technique to tackle this issue. Peaks that originate from derivation correspond to a maximum in the original spectrum, but with the advantage of a sharpening effect, which enables the precise assessment of relevant wavelengths. Such effect was successfully tested on potassium permanganate solutions, whose composite band, presenting seven overlapped peaks located in the visible region of the electromagnetic spectrum, was separated in well-distinct signals, even at very low concentrations. Interestingly, Lambert-Beer Law is maintained, allowing an accurate calculation of peak ratios directly on the sharpened second derivative spectrum. This great advantage was exploited to investigate the effect of the presence of heavy metal ions on the LH complexes within cultures of R. sphaeroides 2.4.1 cells. This was possible through the precise individuation of absorption maxima and the evaluation of relative ratios between the three LH-I and LH-II peaks. Preliminary results clearly show a direct influence of tested metals on the biosynthesis of the LH-I complex, thus confirming the potentialities of the proposed technique as a promising tool for the evaluation of the chronic effect of exposure to pollutants during bacterial growth.

Nature solved the problem of converting solar light into chemical energy in a very elegant way through the photosynthesis. The natural photosynthetic process takes place thanks to a very precise protein scaffolding architecture and pigments arrangement that lead to convert 98% of the absorbed photons into a stable charge separated state with a lifetime in the second timescale. An intelligent way towards the design of artificial devices for solar conversion is to try to learn the lesson from Nature by mimicking the organization of the natural photosynthetic process. The interest of the scientific and industrial world towards a system capable of storing sunlight and convert it into a suitable form of energy has been increasing markedly in recent years. The idea is to focus on the natural candidates capable of performing these tasks, the photosynthetic reaction center proteins (RC), whose photochemical properties, selected by nature in the evolution course, however, are complicated to reproduce in artificial device. Hybrids have as central protein the photosynthetic reaction center from the purple no sulphur bacterium Rhodobacter sphaeroides strain R26, that converts solar light into a electron-hole couple state having an adequate lifetime to allow the ancillary chemistry to happen.

The bioconjugation of photosynthetic proteins with efficient organic light harvesting antennas is a very intriguing approach to build novel hybrid organic-biological machineries that, mimicking nature, employ solar energy to generate photocurrents or to drive thermodynamically unfavoured reactions, reaching efficiencies higher than those obtainable by their natural conterparts. Such hybrid systems are potentially useful as active materials in new generation devices for photovoltaics and biosensing. In the frame of our studies on organic-biological hybrids for solar energy conversion,[1] here we present the design, synthesis and preliminary characterization of a series of heptamethine cyanine dyes (such as Cian-1 in Figure 1a) particularly suitable as light harvesting antennas for the photosynthetic Reaction Center (RC) of the purple bacterium Rhodobacter sphaeroides strain R26. These molecules have been properly tailored to have efficient light absorption in the visible spectral range, where the RC absorbance is very low, and efficient emission in the near infrared region, in correspondence of the highest RC absorption peaks.Moreover, the charged sites within their molecular structure make these molecules highly soluble in detergent aqueous environment where the RC is stable, this allowing them to approach the bioconjugation sites of the protein. Finally, the synthesized cyanines are endowed with a carboxylic moiety useful for their covalent binding to the amino groups of the RC lysine residues. Our preliminary results show that the bioconjugation of these organic antennas to the RC is expected to be a very profitable strategy to afford highly efficient organic-biological hybrids for solar energy conversion.

Photosynthesis is responsible for the photochemical conversion of light into the chemical energy that fuels the planet Earth. The photochemical core of this process in all photosynthetic organisms is a transmembrane protein called the reaction center. In purple photosynthetic bacteria a simple version of this photoenzyme catalyzes the reduction of a quinone molecule, accompanied by the uptake of two protons from the cytoplasm. This results in the establishment of a proton concentration gradient across the lipid membrane, which can be ultimately harnessed to synthesize ATP. Herein we show that synthetic protocells, based on giant lipid vesicles embedding an oriented population of reaction centers, are capable of generating a photoinduced proton gradient across the membrane. Under continuous illumination, the protocells generate a gradient of 0.061 pH units per min, equivalent to a proton motive force of 3.6 mV.min(-1). Remarkably, the facile reconstitution of the photosynthetic reaction center in the artificial lipid membrane, obtained by the droplet transfer method, paves the way for the construction of novel and more functional protocells for synthetic biology.

Because of the growing potential of nanoparticles in biological and medical applications, tuning and directing their properties toward a high compatibility with the aqueous biological milieu is of remarkable relevance. Moreover, the capability to combine nanocrystals (NCs) with biomolecules, such as proteins, offers great opportunities to design hybrid systems for both nanobiotechnology and biomedical technology. Here we report on the application of the micelle-to-vesicle transition (MVT) method for incorporation of hydrophobic, red-emitting CdSe@ZnS NCs into the bilayer of liposomes. This method enabled the construction of a novel hybrid proteo-NC-liposome containing, as model membrane protein, the photosynthetic reaction center (RC) of Rhodobacter sphaeroides. Electron microscopy confirmed the insertion of NCs within the lipid bilayer without significantly altering the structure of the unilamellar vesicles. The resulting aqueous NC-liposome suspensions showed low turbidity and kept unaltered the wavelengths of absorbance and emission peaks of the native NCs. A relative NC fluorescence quantum yield up to 8% was preserved after their incorporation in liposomes. Interestingly, in proteo-NC-liposomes, RC is not denatured by Cd-based NCs, retaining its structural and functional integrity as shown by absorption spectra and flash-induced charge recombination kinetics. The outlined strategy can be extended in principle to any suitably sized hydrophobic NC with similar surface chemistry and to any integral protein complex. Furthermore, the proposed approach could be used in nanomedicine for the realization of theranostic systems and provides new, interesting perspectives for understanding the interactions between integral membrane proteins and nanoparticles, i.e., in nanotoxicology studies.

Squarebop I bacteriorhodopsin is a light-activated proton pump present in the membranes of the archeonHaloquadratum walsbyi, a square-shaped organism representing 50-60% of microbial population in thecrystallizer ponds of the coastal salterns.Here we describe: (1) the operating mode of a bioreactor designed to concentrate the saltern biomassthrough a microfiltration process based on polyethersulfone hollow fibers; (2) the isolation of SquarebopI bacteriorhodopsin from solubilized biomass by means of a single chromatographic step; (3) tightlybound lipids to the isolated and purified protein as revealed by MALDI-TOF/MS analysis; (4) the photoactivityof Squarebop I bacteriorhodopsin isolated from environmental samples by flash spectroscopy.Yield of the isolation process is 150 lg of Squarebop I bacteriorhodopsin from 1 l of 25-fold concentratedbiomass. The possibility of using the concentrated biomass of salterns, as renewable resourcefor the isolation of functional bacteriorhodopsin and possibly other valuable bioproducts, is brieflydiscussed.

The life cycle of the bacterium Rhodobacter sphaeroides was investigated by isothermal microcalorimetry using two different procedures based on the use of a static ampoule and a flow cell, respectively. In the static ampoule method it is possible to follow the growth phase and also the death phase which cannot be revealed by total biomass based techniques like turbidimetry. However, different cellular metabolisms, possibly due do the oxygen limitation occurring during the bacterial life cycle, produce complex behavior in the experimental curves. In the stop- flow cell mode this limitation is overcome as the bacteria are grown outside the calorimeter under well-defined aerobic conditions and aliquots of cell suspension are transferred in the calorimeter at different time intervals. The complex behavior shown in the static ampoule mode was successfully analyzed by a population evolution model based on a Fujikawa modified logistic equation which provides a quantitative description of the process.

The fabrication of low-cost, stable, sensitive and selective biosensors for herbicides has gained considerable attention in recent years as modern agriculture makes massive use of pesticides that are known to be harmful for human health. The use of photosynthetic proteins for herbicide detection is the most straightforward strategy for this aim, as these are the natural target of this class of chemicals. Among the possible biosensor devices, the most successful that have led to commercial products are based on amperometric detection. In particular, in this type of biosensors, the monitored photocurrent is progressively attenuated in the presence of increasing amount of analyte, minimizing the mathematical treatment of the data. In the present work, we show the functionalization of screen-printed electrodes with an ordered film of photoactive biological material, by means of laser induced forward transfer (LIFT) technique, in order to fabricate a biohybrid device for energy conversion and/or biosensing. LIFT is an advanced tool for achieving the direct immobilization of biosystems [1] with high spatial resolution, due to the high impact pressure of the transferred droplets, at the receiver substrate. As a result, it enhances physical adsorption onto the electrode surface and high photocurrents can be attained by using extremely low quantities of deposited samples. The biomaterial used is the photosynthetic reaction center (RC) from the bacterium Rhodobacter (Rb.) sphaeroides. Cathodic or anodic photocurrents are detected depending on the potential applied to the working electrode: in particular, cathodic photocurrents are detected at the donor side under reducing potentials and anodic ones at the acceptor side under oxidizing potentials. Rb. sphaeroides RC is classified as Q-type and resembles the more evolved photosystem II (PSII) found in plants, algae and cyanobacteria, sharing the sensitivity to the same class of herbicides. However, bacterial RC has a simpler architecture, is more stable and is more selective to a particular class of herbicides, the triazinic ones, while PSII is sensitive to different classes of pesticides [2]. In the present case, thanks to the intimate contact between the RC and the electrode, we could detect cathodic photocurrents in the absence of any mediator at both acceptor and donor side, bringing about several advantages: simpler biosensor architecture, ideal square-wave shape of the photocurrents, and independence on the mediator concentration. Most importantly, the absence of quinone, often used as mediator at the acceptor side makes the biosensor particularly sensitive to the herbicide tested, due to the competitive nature of the binding reaction. We tested the herbicide terbutryn which, although is no longer permitted in the EU, has the strongest binding affinity to the RC and is more suitable to show the potentiality of the final device. A limit of detection for the terbutryn was found in the range 8-30 nM w

Photosynthetic reaction center (RC) is the minimal nanoscopic photoconverter in the photosynthetic membrane that catalyzes the conversion of solar light to energy readily usable for the metabolism of the living organisms. After electronic excitation the energy of light is converted into chemical potential by the generation of a charge separated state accompanied by intraprotein and ultimately transmembrane proton movements. We designed a system which fulfills the minimum structural and functional requirements to investigate the physico/chemical conditions of the processes: RCs were reconstituted in closed lipid vesicles made of selected lipids entrapping a pH sensitive indicator, and electron donors (cytochrome c(2) and K-4[Fe(CN)(6)]) and acceptors (decylubiquinone) were added to sustain the photocycle. Thanks to the low proton permeability of our preparations, we could show the formation of a transmembrane proton gradient under illumination and low buffering conditions directly by measuring proton-related signals simultaneously inside and outside the vesicles. The effect of selected ionophores such as gramicidin, nigericin and valinomycin was used to gain more information on the transmembrane proton gradient driven by the RC photochemistry.

This work demonstrates that lipid-detergent mixed micelles can be employed successfully inorder to achieve and modulate the transfer of bio-active hydrophobic compounds into lipidcarriers by means of a simple and bio-safe procedure. In our specific investigation, liposomepreparations incorporating mixture

Titanium and its alloys are widely employed materials for implants in orthopedic or dental surgery due to their mechanical properties, resistance to corrosion and osseointegration capability. However adverse reactions at the tissue/implant interface may occur, which limit the success of the osseointegration process. Therefore, different strategies have to be used to overcome these drawbacks. In this work, we developed two different liposome-based coatings on titanium surfaces as drug or bioactive molecule deposits for dental/orthopedic implant applications. The first one is a supported vesicular layer (SVL), obtained by liposome adhesion on passivated Ti surface, the second one is a covalently bonded vesicular layer (CBVL) grafted on properly functionalized Ti. Photoluminescence spectroscopy and atomic force microscopy investigations demonstrated the effective anchoring of intact liposomes in both systems. Cytotoxicity assays, performed after 48h, showed a MG63 cell viability higher than 75% and 70% on SVLs and CBVLs, respectively. Scanning electron microscopy investigation revealed numerous and spread MG63 cells after 48h on SVL modified Ti surface and a lower cell adhesion on samples coated with CBVL. The cellular uptake capability of liposome content was proved by fluorescence microscopy using carboxyfluorescein loaded SVLs and CBVLs. Finally, we demonstrated that these liposome-modified Ti surfaces were able to deliver a model bioactive molecule (phosphatidylserine) to adherent cells, confirming the potentiality of developed systems in bone related prosthetic applications.

The use of fluorescent nanocrystals (NCs) as probes for bioimaging applications has emerged as an advantageous alternative to conventional organic fluorescent dyes. Therefore their toxicological evaluation and intracellular delivery are currently a primary field of research. In this work, hydrophobic and highly fluorescent CdSe@ZnS NCs were encapsulated into the lipid bilayer of liposomes by the micelle-to-vesicle transition (MVT) method. The obtained aqueous NC-liposome suspensions preserved the spectroscopic characteristics of the native NCs. A systematic study of the in vitro toxicological effect on HeLa cells of these red emitting NC-liposomes was then carried out and compared to that of empty liposomes. By using liposomes of different phospholipid composition, we evaluated the effect of the lipid carrier on the cytotoxicity towards HeLa cells. Surprisingly, a cell proliferation and death study along with the MTT test on HeLa cells treated with NC-liposomes have shown that the toxic effects of NCs, at concentrations up to 20 nM, are negligible compared to those of the lipid carrier, especially when this is constituted by the cationic phospholipid DOTAP. In particular, obtained data suggest that DOTAP has a dose- and time-dependent toxic effect on HeLa cells. In contrast, the addition of PEG to the liposomes does not alter significantly the viability of the cells. In addition, the ability of NC-liposomes to penetrate the HeLa cells was assessed by fluorescence and confocal microscopy investigation. Captured images show that NC-liposomes are internalized into cells through the endocytic pathway, enter early endosomes and reach lysosomes in 1 h. Interestingly, red emitting NCs co-localized with endosomes and were positioned at the limiting membrane of the organelles. The overall results suggest that the fluorescent system as a whole, NCs and their carrier, should be considered for the development of fully safe biological applications of CdSe@ZnS NCs, and provide essential indications to define the optimal experimental conditions to use the proposed system as an optical probe for future in vivo experiments.

The functionalization of screen-printed electrodes (SPEs) with a thin film of reaction centre (RC) proteins from the phototrophic bacterium Rhodobacter (R.) sphaeroides, by means of laser induced forward transfer (LIFT) technique, allowed the fabrication of robust and sensitive bio-hybrid devices for terbutryn detection and analysis. The optimal wiring between RCs and the gold electrode surface, achieved by LIFT, led to the generation of cathodic photocurrents sustained by a direct electron transfer (DET) mechanism, which were attenuated by addition of the herbicide inhibitor. (C) 2016 The Authors. Published by Elsevier Ltd.

The photosynthetic reaction center (RC) is an integral membrane protein that, upon absorption of photons, generates a hole-electron couple with a yield close to one. This energetic state has numerous possible applications in several biotechnological fields given that its lifetime is long enough to allow non-metabolic ancillary redox chemistry to take place. Here we focus on RCs reconstituted in liposomes, formed with sole phospholipids or in blends with other lipids, and show that the electrical charge sitting on the polar head of such hydrophobic molecules does play an important role on the stability of the hole-electron couple. More specifically this study shows that the presence of negative charges in the surrounding of the protein stabilizes the charge-separated state while positive charges have a strong opposite effect.

Molecular nanoelectronics is attracting much attention, because of the possibility to add functionalities to silicon-based electronics by means of intrinsically nanoscale biological or organic materials. The contact point between active molecules and electrodes must present, besides nanoscale size, a very low resistance. To realize Metal-Molecule-Metal junctions it is, thus, mandatory to be able to control the formation of useful nanometric contacts. The distance between the electrodes has to be of the same size of the molecule being put in between. Nanogaps technology is a perfect fit to fulfill this requirement. In this work, nanogaps between gold electrodes have been used to develop optoelectronic devices based on photoactive proteins. Reaction Centers (RC) and Bacteriorhodopsin (BR) have been inserted in nanogaps by drop casting. Electrical characterizations of the obtained structures were performed. It has been demonstrated that these nanodevices working principle is based on charge separation and photovoltage response. The former is induced by the application of a proper voltage on the RC, while the latter comes from the activation of BR by light of appropriate wavelengths.

Osmotic shock was used as a tool to obtain cardiolipin (CL) enriched chromatophores of Rhodobacter sphaeroides. After incubation of cells in iso- and hyper-osmotic buffers both chromatophores with a physiological lipid profile (Control) and with an almost doubled amount of CL (CL enriched) were isolated. Spectroscopic properties, reaction centre (RC) and reducible cytochrome (cyt) contents in Control and CL enriched chromatophores were the same. The oxidoreductase activity was found higher for CL enriched than for Control chromatophores, raising from 60 ± 2 to 93 ± 3 mol cyt c s-1 (mol total cyt c)-1. Antymicin and myxothiazol were tested to prove that oxidoreductase activity thus measured was mainly attributable to the cyt bc 1 complex. The enzyme was then purified from BH6 strain yielding a partially delipidated and almost inactive cyt bc 1 complex, although the protein was found to maintain its structural integrity in terms of subunit composition. The ability of CL in restoring the activity of the partially delipidated cyt bc 1 complex was proved in micellar systems by addition of exogenous CL. Results here reported indicate that CL affects oxidoreductase activity in the bacterium Rhodobacter sphaeroides both in chromatophore and in purified cyt bc 1 complex.

The design of new materials as active layers is important for electrochemical sensor and biosensor development. Among the techniques for the modification and functionalization of electrodes, the laser induced forward transfer (LIFT) has emerged as a powerful physisorption method for the deposition of various materials (even labile materials like enzymes) that results in intimate and stable contact with target surface. In this work, Pt, Au, and glassy carbon screen printed electrodes (SPEs) treated by LIFT with phosphate buffer have been characterized by scanning electron microscopy and atomic force microscopy to reveal a flattening effect of all surfaces. The electrochemical characterization by cyclic voltammetry shows significant differences depending on the electrode material. The electroactivity of Au is reduced while that of glassy carbon and Pt is greatly enhanced. In particular, the electrochemical behavior of a phosphate LIFT treated Pt showed a marked enrichment of hydrogen adsorbed layer, suggesting an elevated electrocatalytic activity towards glucose oxidation. When Pt electrodes modified in this way were used as an effective glucose sensor, a 1-10 mM linear response and a 10 µM detection limit were obtained. A possible role of phosphate that was securely immobilized on a Pt surface, as evidenced by XPS analysis, enhancing the glucose electrooxidation is discussed.

Photosynthetic Reaction Center (RC) is a transmembrane photoenzyme capable of converting absorbed photons into electron-hole pairs with almost unitary efficiency. The unique properties of this natural photoconverter attract considerable interest for its use as functional component in nanomaterials and bioelectronics devices. Implementation of RC into nanostructures or anchoring on devices' electrode surfaces require the development of suitable chemical manipulation. Here we report our methods to embed this protein in soft nanostructures or to covalently attach it on surfaces without denaturating it or altering its chemical properties. © (2016) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

A critical selection of the recent literature reports on the use of photosynthetic and photoresponsive bacteria as a source of materials for optoelectronics and photonic devices is discussed, together with the applications foreseen in solar energy conversion and storage and light information technologies.The use of both photoactive cellular components and entire living cells is reviewed, aiming to highlight the great conceptual impact of these studies.These studies point out possible deep changes in the paradigm of design, and synthesis of materials and devices for optoelectronics. Although the possible technological impact of this technology is still hard to be predicted, these studies advance the understanding of photonics of living organisms and develop new intriguing concepts in biomaterials research.

Photosynthetic reaction centres are membrane-spanning proteins, found in several classes of autotroph organisms, where a photoinduced charge separation and stabilization takes place with a quantum efficiency close to unity. The protein remains stable and fully functional also when extracted and purified in detergents thereby biotechnological applications are possible, for example, assembling it in nano-structures or in optoelectronic systems. Several types of bio-nanocomposite materials have been assembled by using reaction centres and different carrier matrices for different purposes in the field of light energy conversion (e. g., photovoltaics) or biosensing (e. g., for specific detection of pesticides). In this review we will summarize the current status of knowledge, the kinds of applications available and the difficulties to be overcome in the different applications. We will also show possible research directions for the close future in this specific field.

The aim of the present investigation was to evaluate the influence of liposome formulation on the ability of vesicles to penetrate a pathological mucus model obtained from COPD affected patients in order to assess the potential of such vesicles for the treatment of chronic respiratory diseases by inhalation. Therefore, Small Unilamellar Liposomes (PLAIN-LIPOSOMEs), Pluronic (R) F127-surface modified liposomes (PF-LIPOSOMEs) and PEG 2000PE-surface modified liposomes (PEG-LIPOSOMEs) were prepared using the micelle-to-vesicle transition (MVT) method and beclomethasone dipropionate (BDP) as model drug. The obtained liposomes showed diameters in the range of 40-65 nm, PDI values between 0.25 and 0.30 and surface electric charge essentially close to zero. The encapsulation efficiency was found to be dependent on the BDP/lipid ratio used and, furthermore, BDP-loaded liposomes were stable in size both at 37 degrees C and at 4 degrees C. All liposomes were not cytotoxic on H441 cell line as assessed by the MTT assay. The liposome uptake was evaluated through a cytofluorimetric assay that showed a non-significant reduction in the internalization of PEG-LIPOSOMEs as compared with PLAIN-LIPOSOMEs. The penetration studies of mucus from COPD patients showed that the PEG-LIPOSOMEs were the most mucus-penetrating vesicles after 27 h. In addition, PEG-and PF-LIPOSOMEs did not cause any effect on bronchoalveolar lavage fluid proteins after aerosol administration in the mouse. The results highlight that PEG-LIPOSOMEs show the most interesting features in terms of penetration through the pathologic sputum, uptake by airway epithelial cells and safety profile.

The ability of microorganisms to adhere to abiotic surfaces and the potentialities of attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy have been exploited to study protonation and heavy metal binding events onto bacterial surfaces. This work represents the first attempt to apply on bacteria the recently developed method known as perfusion-induced ATR-FTIR difference spectroscopy.(1, 2) Such a technique allows measurement of even slight changes in the infrared spectrum of the sample, deposited as a thin layer on an ATR crystal, while an aqueous solution is perfused over its surface. Solutions at different pH have been used for inducing protonation/deprotonation of functional groups lying on the surface of Rhodobacter sphaeroides cells, chosen as a model system. The interaction of Ni2+ with surface protonable groups of this microorganism has been investigated with a double-difference approach exploiting competition between nickel cations and protons. Protonation-induced difference spectra of simple model compounds have been acquired to guide band assignment in bacterial spectra, thus allowing identification of major components involved in proton uptake and metal binding. The data collected reveal that carboxylate moieties on the bacterial surface of R. sphaeroides play a role in extracellular biosorption of Ni2+, establishing with this ion relatively weak coordinative bonds.

The photosynthetic reaction center (RC) from the Rhodobacter sphaeroides bacterium has been covalently bioconjugated with a NIR-emitting fluorophore (AE800) whose synthesis was specifically tailored to act as artificial antenna harvesting light in the entire visible region. AE800 has a broad absorption spectrum with peaks centered in the absorption gaps of the RC and its emission overlaps the most intense RC absorption bands, ensuring a consistent increase of the protein optical cross section. The covalent hybrid AE800-RC is stable and fully functional. The energy collected by the artificial antenna is transferred to the protein via FRET mechanism, and the hybrid system outperforms by a noteworthy 30% the overall photochemical activity of the native protein under the entire range of visible light. This improvement in the optical characteristic of the photoenzyme demonstrates the effectiveness of the bioconjugation approach as a suitable route to new biohybrid materials for energy conversion, photocatalysis, and biosensing. © 2016 American Chemical Society.

Liposomes represent a versatile biomimetic environment for studying the interaction between integral membrane proteins and hydrophobic ligands. In this paper, the quinone binding to the QB-site of the photosynthetic reaction centers (RC) from Rhodobacter sphaeroides has been investigated in liposomes prepared with either the zwitterionic phosphatidylcholine (PC) or the negatively charged phosphatidylglycerol (PG) to highlight the role of the different phospholipid polar heads. Quinone binding (K Q) and interquinone electron transfer (L AB) equilibrium constants in the two type of liposomes were obtained by charge recombination reaction of QB-depleted RC in the presence of increasing amounts of ubiquinone-10 over the temperature interval 6-35 °C. The kinetic of the charge recombination reactions has been fitted by numerically solving the ordinary differential equations set associated with a detailed kinetic scheme involving electron transfer reactions coupled with quinone release and uptake. The entire set of traces at each temperature was accurately fitted using the sole quinone release constants (both in a neutral and a charge separated state) as adjustable parameters. The temperature dependence of the quinone exchange rate at the QB-site was, hence, obtained. It was found that the quinone exchange regime was always fast for PC while it switched from slow to fast in PG as the temperature rose above 20 °C. A new method was introduced in this paper for the evaluation of constant K Q using the area underneath the charge recombination traces as the indicator of the amount of quinone bound to the QB-site.

We have isolated and characterized the light-driven proton pump Bop I from the ultrathin square archaeon Haloquadratum walsbyi, the most abundant component of the dense microbial community inhabiting hypersaline environments. The disruption of cells by hypo-osmotic shock yielded Bop I retinal protein highly enriched membranes, which contain one main 27 kDa protein band together with a high content of the carotenoid bacterioruberin. Light-induced pH changes were observed in suspensions of Bop I retinal protein-enriched membranes under sustained illumination. Solubilization of H. walsbyi cells with Triton X-100, followed by phenyl-Sepharose chromatography, resulted in isolation of two purified Bop I retinal protein bands; mass spectrometry analysis revealed that the Bop I was present as only protein in both the bands. The study of light/dark adaptations, M-decay kinetics, responses to titration with alkali in the dark and endogenous lipid compositions of the two Bop I retinal protein bands showed functional differences that could be attributed to different protein aggregation states. Proton-pumping activity of Bop I during the photocycle was observed in liposomes constituted of archaeal lipids. Similarities and differences of Bop I with other archaeal proton-pumping retinal proteins will be discussed. Haloquadratum walsbyi is a peculiar organism which often dominates the microbial communities of the hypersaline ecosystems, such as salt lakes and solar saltern crystallizer ponds. It is extremely thin and possesses a unique square-like shape, with sharp edges and acute straight corners. The flat cells form large sheets similar to solar panels, able to efficiently collect light as an energy source for metabolism. Like other archaeal extremely halophilic microorganisms, square cells encode light-activated retinal-proteins to survive in hypersaline environments. In this study we describe the biochemical properties and the photochemistry of the light-activated proton pump Bop I of H. walsbyi, grown in laboratory.