Remarkably enhanced photoelectrical efficiency of bacteriorhodopsin in quantum dot - Purple membrane complexes under two-photon excitation.

The photosensitive protein bacteriorhodopsin (bR) has been shown to be a promising material for optoelectronic applications, but it cannot effectively absorb and utilize light energy in the near-infrared (NIR) region of the optical spectrum. Semiconductor quantum dots (QDs) have two-photon absorption cross-sections two orders of magnitude larger than those of bR and can effectively transfer the up-converted energy of two NIR photons to bR via the Förster resonance energy transfer (FRET). In this study, we have engineered a photoelectrochemical cell based on a hybrid material consisting of QDs and bR-containing purple membranes (PMs) of Halobacterium salinarum and demonstrated that this cell can generate an electrical signal under the two-photon laser excitation. We have shown that the efficiency of light conversion by the PM-QD hybrid material under two-photon excitation is up to 4.3 times higher than the efficiency of conversion by PMs alone. The QD integration into the bR-containing PMs significantly improves the bR capacity for utilizing light upon two-photon laser excitation, thus paving the way to the engineering of biologically inspired hybrid NIR nonlinear optoelectronic elements. The nonlinear nature of two-photon excitation may provide considerable advantages, such as a sharp sensitivity threshold and the possibility of precise three-dimensional location of excitation in holography and optical computing.

Discovery of bacteriorhodopsins in Haloarchaeal species isolated from Indian solar salterns: deciphering the role of the N-terminal residues in protein folding and functional expression.

Interesting optical and photochemical properties make microbial rhodopsin a promising biological material suitable for various applications, but the cost-prohibitive nature of production has limited its commercialization. The aim of this study was to explore the natural biodiversity of Indian solar salterns to isolate natural bacteriorhodopsin (BR) variants that can be functionally expressed in Escherichia coli. In this study, we report the isolation, functional expression and purification of BRs from three pigmented haloarchaea, wsp3 (water sample Pondicherry), wsp5 and K1T isolated from two Indian solar salterns. The results of the 16S rRNA data analysis suggest that wsp3, wsp5 and K1T are novel strains belonging to the genera Halogeometricum, Haloferax and Haloarcula respectively. Overall, the results of our study suggest that 17 N-terminal residues, that were not included in the gene annotation of the close sequence homologues, are essential for functional expression of BRs. The primary sequence, secondary structural content, thermal stability and absorbance spectral properties of these recombinant BRs are similar to those of the previously reported Haloarcula marismortui HmBRI. This study demonstrates the cost-effective, functional expression of BRs isolated from haloarchaeal species using E. coli as an expression host and paves the way for feasibility studies for future applications.

X-ray structure analysis of bacteriorhodopsin at 1.3 Å resolution.

Bacteriorhodopsin (bR) of Halobacterium salinarum is a membrane protein that acts as a light-driven proton pump. bR and its homologues have recently been utilized in optogenetics and other applications. Although the structures of those have been reported so far, the resolutions are not sufficient for elucidation of the intrinsic structural features critical to the color tuning and ion pumping properties. Here we report the accurate crystallographic analysis of bR in the ground state. The influence of X-rays was suppressed by collecting the data under a low irradiation dose at 15 K. Consequently, individual atoms could be separately observed in the electron density map at better than 1.3 Å resolution. Residues from Thr5 to Ala233 were continuously constructed in the model. The twist of the retinal polyene was determined to be different from those in the previous models. Two conformations were observed for the proton release region. We discuss the meaning of these fine structural features.

Purification and biochemical characterization of photo-active membrane protein bacteriorhodopsin from Haloarcula marismortui, an extreme halophile from the Dead Sea.

Bacteriorhodopsin (BR) is an exciting photo-active retinal protein with many potential industrial applications. In this study, BR from the extremely halophilic archaeon Haloarcula marismortui (HmBR) was purified successfully using aqueous two phase extraction method. Absorption spectroscopy analysis showed maximum absorption peak of HmBR retinal protein (λmax) at 415 nm. The purified HmBR was visualized by SDS-PAGE, with a subunit molecular mass of 27 kDa, and its identity was confirmed by resonance Raman spectroscopy, Fourier transform infrared spectroscopy and atomic force microscopy. The effect of pH and salt concentration on the absorption spectrum of HmBR was evaluated. Red-shifted in λmax of HmBR was recorded at acidic condition (pH 5) and HmBR showed remarkable optical activity under high salinity condition. The photoelectric activity of HmBR was evaluated by measuring the DC-voltage generated from HmBR coated on indium tin oxide (ITO) glass when light illumination was applied.

Primary Transfer Step in the Light-Driven Ion Pump Bacteriorhodopsin: An Irreversible U-Turn Revealed by Dynamic Nuclear Polarization-Enhanced Magic Angle Spinning NMR.

Despite much attention, the path of the highly consequential primary proton transfer in the light-driven ion pump bacteriorhodopsin (bR) remains mysterious. Here we use DNP-enhanced magic angle spinning (MAS) NMR to study critical elements of the active site just before the Schiff base (SB) deprotonates (in the L intermediate), immediately after the SB has deprotonated and Asp85 has become protonated (in the Mo intermediate), and just after the SB has reprotonated and Asp96 has deprotonated (in the N intermediate). An essential feature that made these experiments possible is the 75-fold signal enhancement through DNP. 15N(SB)-1H correlations reveal that the newly deprotonated SB is accepting a hydrogen bond from an alcohol and 13C-13C correlations show that Asp85 draws close to Thr89 before the primary proton transfer. Concurrently, 15N-13C correlations between the SB and Asp85 show that helices C and G draw closer together just prior to the proton transfer and relax thereafter. Together, these results indicate that Thr89 serves to relay the SB proton to Asp85 and that creating this pathway involves rapprochement between the C and G helices as well as chromophore torsion.

Photonic Potential of Haloarchaeal Pigment Bacteriorhodopsin for Future Electronics: A Review.

Haloarchaea are known for its adaptation in extreme saline environment. Halophilic archaea produces carotenoid pigments and proton pumps to protect them from extremes of salinity. Bacteriorhodopsin (bR) is a light-driven proton pump that resides in the membrane of haloarchaea Halobacterium salinarum. The photocycle of Bacteriorhodopsin passes through several states from K to O, finally liberating ATP for host's survival. Extensive studies on Bacteriorhodopsin photocycle has provided in depth knowledge on their sequential mechanism of converting solar energy into chemical energy inside the cell. This ability of Bacteriorhodopsin to harvest sunlight has now been experimented to exploit the unexplored and extensively available solar energy in various biotechnological applications. Currently, bacteriorhodopsin finds its importance in dye-sensitized solar cell (DSSC), logic gates (integrated circuits, IC's), optical switching, optical memories, storage devices (random access memory, RAM), biosensors, electronic sensors and optical microcavities. This review deals with the optical and electrical applications of the purple pigment Bacteriorhodopsin.

Engineered-membranes and engineered-micelles as efficient tools for purification of halorhodopsin and bacteriorhodopsin.

We describe two alternative and complementary purification methods for halorhodopsin and bacteriorhodopsin. The first relies on a unique form of detergent micelles which we have called engineered-micelles. These are specifically conjugated in the presence of [hydrophobic chelator:Fe(2+)] complexes and form detergent aggregates into which membrane proteins partition, but hydrophilic water-soluble proteins do not. The approach was tested on the membrane protein, bacteriorhodopsin (bR), with five non-ionic detergents (OG, OTG, NG, DM, and DDM), commonly used in purification and crystallization of membrane proteins, in combination with the commercially available bathophenanthroline or with one of the three synthesized phenanthroline derivatives (Phen-C10, Phen-C8 and Phen-C6). Our results show that bR is extracted efficiently (60-86%) and directly from its native membrane into diverse detergent aggregates with preservation of its native conformation, while 90-95% of an artificial contaminating background is excluded. For implementation of the second method, based on engineered-membranes, the use of detergents, which in some cases may produce protein denaturation, is not required at all. Protein-containing membranes are conjugated via the same hydrophobic [chelator:metal ion] complexes but maintain the membrane protein in its native bilayer environment throughout the process. This method is demonstrated on the membrane protein halorhodopsin from Natronomonas pharaonis (phR) and leads to good recovery yields (74-89%) and removal of >85% of artificial background impurities while preserving the native state of phR. The detailed structure of the hydrophobic chelator used has been found to have a marked effect on the purity and yield of both methods.

High overexpression and purification of optimized bacterio-opsin from Halobacterium Salinarum R1 in E. coli.

The purple membrane of Halobacterium Salinarum carries out a protein, bacteriorhodopsin (bR), which is a model for structure-function studies of membrane proteins. The heterologous expression of integral membrane proteins (IMPS) is difficult. In this study, we reported the heterologous overexpression of bacterio-opsin (bO) in Escherichia coli BL21 (DE3). Bacterio-opsin expression is facilitated by using mistic, a membrane protein from Bacillus subtilis in E. coli BL21 (DE3) membranes. The optimized bO gene was cloned in fusion to the C-terminus of mistic in pET 30a (+) and contains an oct-histidine in C-terminal to facilitate purification. Different medium, temperature, and induction time were used to optimize protein overexpression. The highest expression was obtained from the Terrific Broth (TB) medium at 18 °C with an IPTG concentration of 0.1 mM. The final purified bR was 192 ± 1 mg/L which has an important value for the production of membrane proteins in E. coli.

Small-angle scattering gives direct structural information about a membrane protein inside a lipid environment.

Monomeric bacteriorhodopsin (bR) reconstituted into POPC/POPG-containing nanodiscs was investigated by combined small-angle neutron and X-ray scattering. A novel hybrid approach to small-angle scattering data analysis was developed. In combination, these provided direct structural insight into membrane-protein localization in the nanodisc and into the protein-lipid interactions. It was found that bR is laterally decentred in the plane of the disc and is slightly tilted in the phospholipid bilayer. The thickness of the bilayer is reduced in response to the incorporation of bR. The observed tilt of bR is in good accordance with previously performed theoretical predictions and computer simulations based on the bR crystal structure. The result is a significant and essential step on the way to developing a general small-angle scattering-based method for determining the low-resolution structures of membrane proteins in physiologically relevant environments.

Purification of a membrane protein with conjugated engineered micelles.

A novel method for purifying membrane proteins is presented. The approach makes use of engineered micelles composed of a nonionic detergent, ß-octylglucoside, and a hydrophobic metal chelator, bathophenanthroline. Via the chelators, the micelles are specifically conjugated, i.e., tethered, in the presence of Fe(2+) ions, thereby forming micellar aggregates which provide the environment for separation of lipid-soluble membrane proteins from water-soluble proteins. The micellar aggregates (here imaged by cryo-transmission electron microscopy) successfully purify the light driven proton pump, bacteriorhodopsin (bR), from E. coli lysate. Purification takes place within 15 min and can be performed both at room temperature and at 4 °C. More than 94% of the water-soluble macromolecules in the lysate are excluded, with recovery yields of the membrane protein ranging between 74% and 85%. Since this approach does not require precipitants, high concentrations of detergent to induce micellar aggregates, high temperature, or changes in pH, it is suggested that it may be applied to the purification of a wide variety of membrane proteins.

Facile isolation of purple membrane from Halobacterium salinarum via aqueous-two-phase system.

Purple membrane (PM) is a part of cytoplasmic membrane in certain extreme halophilic microorganisms belonging to Domain Archaea. It transduces light energy to generate proton gradient for ATP synthesis in the microorganisms. Bacteriorhodopsin (BR) is the only protein in PM responsible for the generation of proton gradient. Generally, PM was purified from Halobacterium salinarum via a tedious and lengthy sucrose density gradient ultracentrifugation (SGU). In this work, a facile method based on polyethyleneglycol (PEG)-phosphate aqueous-two- phase extraction system (ATPS) was employed to purify PM from cell lysate of H. salinarum. The results showed that PM could be completely recovered from the interface of PEG-phosphate ATPS with BR purity ca 94.1% as measured by UV-visible absorption spectra. In comparison with PM obtained by SGU, the PM isolated by ATPS could achieve the same level of purity and photocurrent activity (ca 177.2nA/µgBR/cm(2)) as analyzed by SDS-PAGE and photocurrent measurement, respectively. The easily scalable and straightforward ATPS procedure demonstrated that PM can be purified and recovered more cost-effectively with a significantly reduced operation time that should lead to broader range applications of PM possible.

High-speed atomic force microscopy.

High-speed atomic force microscopy (HS-AFM) has been developed as a nano-dynamics visualization technique. This microscopy permits direct observation of structure dynamics and dynamic processes of biological molecules in physiological solutions, at a subsecond to sub-100 ms temporal resolution and an ∼2 nm lateral and a 0.1 nm vertical resolution. Importantly, tip-sample interactions do not disturb the biomolecules' functions. Various functioning proteins including myosin V walking on an actin filament and bacteriorhodopsin responding to light have been successfully visualized with HS-AFM. In the quest for understanding the functional mechanisms of proteins, inferences no longer have to be made from static snapshots of molecular structures and dynamic behavior of optical markers attached to proteins. High-resolution molecular movies obtained from HS-AFM observations reveal the details of molecules' dynamic behavior in action, without the need for intricate analyses and interpretations. In this review, I first describe the fundamentals behind the achieved high imaging rate and low invasiveness to samples, and then highlight recent imaging studies. Finally, future studies are briefly described.

Further studies with isolated absolute infrared spectra of bacteriorhodopsin photocycle intermediates: conformational changes and possible role of a new proton-binding center.

We recently published procedures describing the isolation of absolute infrared spectra for the intermediates of the bacteriorhodopsin (BR) photocycle and from these, obtaining transitional difference spectra between consecutive intermediates. In that work, we concentrated mainly on proton-binding centers and the route of proton transport across the membrane. In the current study, we used isolated spectra for the amide I, amide II, and amide III envelopes to obtain quantitative information on the extent of conformational change accompanying each transition in the photocycle. Our main finding was that most of the conformational changes occur in the conversion of the M(F) intermediate to N. In our earlier publication, a new proton acceptor, absorbing at 1650 cm(-1) was identified, which appeared to accept a proton from Asp96COOH during the transformation of BR† to L. Below, we present evidence that supports this interpretation and propose a possible role for this new component.

Surface chemical functionalization of single walled carbon nanotubes with a bacteriorhodopsin mutant.

In this work, single walled carbon nanotubes (SWNTs) have been chemically functionalized at their walls with a membrane protein, namely the mutated bacteriorhodopsin D96N, integrated in its native archaeal lipid membrane. The modification of the SWNT walls with the mutant has been carried out in different buffer solutions, at pH 5, 7.5 and 9, to investigate the anchoring process, the typical chemical and physical properties of the component materials being dependent on the pH. The SWNTs modified by interactions with bacteriorhodopsin membrane patches have been characterized by UV-vis steady state, Raman and attenuated total reflection Fourier transform infrared spectroscopy and by atomic force and transmission electron microscopy. The investigation shows that the membrane protein patches wrap the carbon walls by tight chemical interactions undergoing a conformational change; such chemical interactions increase the mechanical strength of the SWNTs and promote charge transfers which p-dope the nano-objects. The functionalization, as well as the SWNT doping, is favoured in acid and basic buffer conditions; such buffers make the nanotube walls more reactive, thus catalysing the anchoring of the membrane protein. The direct electron communication among the materials can be exploited for effectively interfacing the transport properties of carbon nanotubes with both molecular recognition capability and photoactivity of the cell membrane for sensing and photoconversion applications upon integration of the achieved hybrid materials in sensors or photovoltaic devices.

Periodic mesoporous organosilica as a multifunctional nanodevice for large-scale characterization of membrane proteins.

A versatile protocol has been developed for large-scale characterization of hydrophobic membrane proteins based on the periodic mesoporous organosilica (PMO) acting as both an extractor for hydrophobic substrate capture and a nanoreactor for efficient in situ digestion. With introduction of organic groups in the pore frameworks and the presence of hydrophilic silanol groups on the surface, PMO can be well-dispersed into not only an organic solution to concentrate the dissolved membrane proteins but also an aqueous solution containing enzymes for sequential rapid proteolysis in the nanopores. The unique amphiphilic property of PMO ensures a facile switch in different solutions to realize the processes of substrate dissolution, enrichment, and digestion effectively. Furthermore, this novel PMO-assisted protocol has been successfully applied for identification of complex membrane proteins extracted from mouse liver as proof of general applicability.

Isolation of Squarebop I bacteriorhodopsin from biomass of coastal salterns.

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

Molecular scale conductance photoswitching in engineered bacteriorhodopsin.

Bacteriorhodopsin (BR) is a robust light-driven proton pump embedded in the purple membrane of the extremophilic archae Halobacterium salinarium . Its photoactivity remains in the dry state, making BR of significant interest for nanotechnological use. Here, in a novel configuration, BR was depleted from most of its endogenous lipids and covalently and asymmetrically anchored onto a gold electrode through a strategically located and highly responsive cysteine mutation; BR has no indigenous cysteines. Chemisorption on gold was characterized by surface plasmon resonance, reductive striping voltammetry, ellipsometry, and atomic force microscopy (AFM). For the first time, the conductance of isolated protein trimers, intimately probed by conducting AFM, was reproducibly and reversibly switched under wavelength-specific conditions (mean resistance of 39 ± 12 MΩ under illumination, 137 ± 18 MΩ in the dark), demonstrating a surface stability that is relevant to potential nanodevice applications.

Production of a novel biomacromolecule for nanodevices from glycerol as carbon source in different conditions.

The aim of this study was the investigation of producing cruxrhodopsin as a biomacromolecule with nanofunction from glycerol as carbon source using several process parameters. The optimum medium composition for cruxrhodopsin production was found to contain glycerol 1%, yeast extract 0.05% and K(2)HPO(4) 0.001%. The production of cruxrhodopsin in optimal conditions was 139.86 mg/l. In conclusion, halophilic microorganism Haloarcula sp. IRU1 could be a potential microorganism for production of cruxrhodopsin from glycerol in different conditions.

Efficient production and purification of functional bacteriorhodopsin with a wheat-germ cell-free system and a combination of Fos-choline and CHAPS detergents.

Cell-free translation is one potential approach to the production of functional transmembrane proteins. We have now examined various detergents as supplements to a wheat-germ cell-free system in order to optimize the production and subsequent purification of a functional model transmembrane protein, bacteriorhodopsin. We found that Fos-choline and CHAPS detergents counteracted each other's inhibitory effects on cell-free translation activity and thereby allowed the efficient production and subsequent purification of functional bacteriorhodopsin in high yield.

A new hybrid protein for production of recombinant bacteriorhodopsin in Escherichia coli.

Unique properties of bacteriorhodopsin, namely, photochromism and high thermal stability, make this protein an attractive target for physico-chemical studies, as well as for various biotechnological applications. Using Mistic as a suitable carrier for insertion of recombinant membrane proteins into cytoplasmic membrane of Escherichia coli, we developed a system for overexpression of bacteriorhodopsin and worked out an efficient procedure for its purification and renaturation with the final yield of 120 mg/l of refolded protein, which is the highest value reported to date for bacteriorhodopsin produced in E. coli. Functional activity of recombinant bacteriorhodopsin was confirmed by spectroscopic and electrochemical assays.