Bacteriorhodopsin Molecules Research Articles

Reversible Conjugation of Non-ionic Detergent Micelles Promotes Partitioning of Membrane Proteins under Non-denaturing Conditions.

In the decades’-long quest for high-quality membrane protein (MP) crystals, non-ionic detergent micelles have primarily served as a passive shield against protein aggregation in aqueous solution and/or as a conformation stabilizing environment. We have focused on exploiting the physical chemistry of detergent micelles in order to direct intrinsic MP/detergent complexes to assemble via conjugation under ambient conditions, thereby permitting finely tuned control over the micelle cloud point. In the current work, three commercially available amphiphilic, bipyridine chelators in combination with Fe2+ or Ni2+ were tested for their ability to conjugate non-ionic detergent micelles both in the presence and absence of an encapsulated bacteriorhodopsin molecule. Water-soluble chelators were added, and results were monitored with light microscopy and dynamic light scattering (DLS). [Bipyridine:metal] complexes produced micellar conjugates, which appeared as oil-rich globules (10–200 μm) under a light microscope. DLS analysis demonstrated that micellar conjugation is complete 20 min after the introduction of the amphiphilic complex, and that the conjugation process can be fully or partially reversed with water-soluble chelators. This process of controlled conjugation/deconjugation under nondenaturing conditions provides broader flexibility in the choice of detergent for intrinsic MP purification and conformational flexibility during the crystallization procedure.

Abstract P-5: Neural Network Approaches to Classify 3D Protein Structures from the Data of X-ray Laser Radiation Diffraction from Single Particles

Background: Protein structure determination using X-ray free-electron laser (XFEL) includes analysis and merging a large number of snapshot diffraction patterns. Convolutional neural networks are widely used to solve numerous computer vision problems, e.g. image classification, and can be used for diffraction pattern analysis. But the task of protein structure determination with the use of CNNs only is not yet solved. Methods: We simulated the diffraction patterns using the Condor software library and obtained more than 1000 diffraction patterns for each structure with simulation parameters resembling real ones. To classify diffraction patterns, we tried two approaches, which are widely known in the area of image classification: a classic VGG network and residual networks. Results: 1. Recognition of a protein class (GPCRs vs globins). Globins and GPCR-like proteins are typical α-helical proteins. Each of these protein families has a large number of representatives (including those with known structure) but we used only 8 structures from every family. 12,000 of diffraction patterns were used for training and 4,000 patterns for testing. Results indicate that all considered networks are able to recognize the protein family type with high accuracy. 2. Recognition of the number of protein molecules in the liposome. We considered the usage of lyposomes as carriers of membrane or globular proteins for sample delivery in XFEL experiments in order to improve the X-ray beam hit rate. Three sets of diffractograms for liposomes of various radius were calculated, including diffractograms for empty liposomes, liposomes loaded with 5 bacteriorhodopsin molecules, and liposomes loaded with 10 bacteriorhodopsin molecules. The training set consisted of 23625 diffraction patterns, and test set of 7875 patterns. We found that all networks used in our study were able to identify the number of protein molecules in liposomes independent of the liposome radius. Our findings make this approach rather promising for the usage of liposomes as protein carriers in XFEL experiments. Conclusion: Thus, the performed numerical experiments show that the use of neural network algorithms for the recognition of diffraction images from single macromolecular particles makes it possible to determine changes in the structure at the angstrom scale.

Culturable bacteriorhodopsin-producing haloarchaea of Tuz Lake (Turkey)

Lake Tuz, the largest hypersaline lake in Turkey, has a great variety of microbial communities that adapted to its extreme environment and produce many industrially important compounds such as photosensitive bacteriorhodopsins. So far, the information about the bacteriorhodopsin-producing haloarchaea species of the lake is still limited. In the present study, archaeal bacteriorhodopsin producers were isolated from three locations of the lake. Their bacteriorhodopsin-producing capability was validated by the purification and SDS-PAGE analysis of the delipidated bacteriorhodopsin molecule. The active isolates were identified by the sequencing of partial 16S rDNA gene regions. According to the results, 11 bacteriorhodopsin-producing isolates grouped in Halobacterium salinarum (4), Halobacterium sp. (3), Haloarcula salaria (2), Haloarcula sp. (1) and Halorubrum sp. (1). Our research demonstrated that Lake Tuz is an important natural source of bacteriorhodopsin-producing haloarchaea and the isolates can be valuable for the related technological applications.

Study of the detergent Triton X100 effect on the properties of purple membranes

The paper analyzes the literature data concerning the influence of the Triton-X100 detergent on the optical properties of purple membranes and the photocycle of bacteriorhodopsin molecules. The method of changing the sizes of purple membranes fragments by means of treating them with a detergent and subsequent centrifugation is proposed. The control of the detergent effect on the properties of purple membranes was carried out using spectral analysis and AFM microscopy. As a result of the spectral characteristics investigation of bacteriorhodopsin suspensions and films with the addition of Triton-X100, it was found that high concentrations (>4 mM) of detergent cause the destruction of purple membranes and irreversible changes in the structure of bacteriorhodopsin under the influence of temperature and optical radiation. At the same time, at lower concentrations partial solubilisation is observed in which there is a decrease in the size of purple membranes and a significant part of the bacteriorhodopsin molecules retains its unique optical properties. The atomic force microscopy study of fragments of purple membranes treated with detergent Triton-X100 with a concentration of 2.5 mM showed that large fragments are divided into smaller ones, the dimensions of which make up about 1 μm. The use of detergents can influence the size of fragments of purple membranes and thus increase the optical quality of film structures based on bacteriorhodopsin. The disadvantage of this method is the loss of material in the process of detergent treatment due to the complete solubilization of a large part of the purple membranes.

Stability of the two-dimensional lattice of bacteriorhodopsin reconstituted in partially fluorinated phosphatidylcholine bilayers

This study aims to investigate bacteriorhodopsin (bR) molecules reconstituted in lipid bilayers composed of di(nonafluorotetradecanoyl)-phosphatidylcholine (F4-DMPC), a partially fluorinated analogue of dimyristoyl-phosphatidylcholine (DMPC) to clarify the effects of partially fluorinated hydrophobic chains of lipids on protein's stability. Calorimetry measurements showed that the chain-melting transition of F4-DMPC/bR systems occurs at 3.5 °C, whereas visible circular dichroism (CD) and X-ray diffraction measurements showed that a two-dimensional (2D) hexagonal lattice formed by bR trimers in F4-DMPC bilayers remains intact even above 30 °C, similar to bR in a native purple membrane. Complete dissociation of the trimers into the monomers detected by visible CD almost coincides with the complete melting of 2D lattice observed by X-ray diffraction, in which both take place at around 65 °C (10 °C lower than that for bR in a native purple membrane). However, it is extremely high in comparison with the bR reconstituted in DMPC bilayers in which the dissociation of bR trimer in DMPC bilayers occurs near the chain-melting transition temperature of DMPC bilayers at approximately 18 °C. In order to explore the rationale behind the difference in stability, a further investigation of the detailed structural features of pure F4-DMPC bilayers was performed by analyzing the lamellar diffraction data using simple electron density models. The results suggested that the perfluoroalkyl groups do not exhibit any conformation change even if the chain-melting transition occurs, which is likely to contribute to the stability of the 2D hexagonal lattice formed by the bR trimers.

Improving AFM Reveals a Multitude of Hidden Dynamics in the Unfolding of a Membraneprotein

Protein folding occurs as a set of transitions between structural states within an energy landscape. An oversimplified view of the folding process emerges when transiently populated states are undetected because of limited instrumental resolution. Moreover, predicting the structure and stability of membrane proteins significantly lags success with globular protein. Using force spectroscopy optimized for 1-µs resolution, we reexamined the unfolding of individual bacteriorhodopsin (BR) molecules in native lipid bilayers with a 100-fold improvement in time resolution and a 10-fold improvement in force precision. The resulting data revealed the unfolding pathway in unprecedented detail. Numerous newly detected intermediates—many separated by as few as 2-3 amino acids—exhibited complex dynamics, including frequent refolding and state occupancies of <10 µs. To elucidate the detailed dynamics of individual membrane proteins under their most native-likeset of intra- and inter-helix contacts, we labeled BR at its c-terminal with a copper-free click chemistry reagent and then gently but covalently coupled BR to an azide-functionalized, PEG-coated tip. This process suppressed nonspecific adhesion that confounds analysis of the initial unfolding of BR. The resulting force-extension curves revealed rapid near-equilibrium folding dynamics between previously undetected intermediate states. We assigned the first state to the top of the G-helix with the second and third states corresponding to a total unfolding of 5 and 9 amino acids. Interestingly, this assignment placed the third state where BR's retinal binds to lysine 216. We verified this assignment by performing dynamic force spectroscopy on BR with and without its retinal. We also reconstructed the full 1D free-energy landscape underlying the initial 9 amino acid unfolding of BR. Looking forward, this reversible unfolding and refolding assay should provide a platform to precisely quantify the energetics of membrane folding under native-like conditions.

Hidden dynamics in the unfolding of individual bacteriorhodopsin proteins

Protein folding occurs as a set of transitions between structural states within an energy landscape. An oversimplified view of the folding process emerges when transiently populated states are undetected because of limited instrumental resolution. Using force spectroscopy optimized for 1-microsecond resolution, we reexamined the unfolding of individual bacteriorhodopsin molecules in native lipid bilayers. The experimental data reveal the unfolding pathway in unprecedented detail. Numerous newly detected intermediates-many separated by as few as two or three amino acids-exhibited complex dynamics, including frequent refolding and state occupancies of <10 μs. Equilibrium measurements between such states enabled the folding free-energy landscape to be deduced. These results sharpen the picture of the mechanical unfolding of membrane proteins and, more broadly, enable experimental access to previously obscured protein dynamics.

The effect of plasmon silver and exiton semiconductor nanoparticles on the bacteriorhodopsin photocycle in Halobacterium salinarum membranes

The interaction of semiconductor quantum dots and silver nanoparticles (AgNPs) with bacteriorhodopsin (BR), a membrane protein contained in the purple membrane (PM) of Halobacterium salinarum, is studied. It is shown that both types of nanoparticles are adsorbed efficiently on the surface of the purple membranes, modulating the parameters of the bacteriorhodopsin photocycle. Electrostatic interactions are found to be the main cause of the effect of nanoparticles on the bacteriorhodopsin photocycle. These results explain our earlier data on the “fixation” of the bacteriorhodopsin photocycle for protein molecules trapped after incubation of the purple membranes with silver nanoparticles near the location of the “hot spots” of the effect of surface-enhanced Raman scattering (SERS). It is demonstrated that exposure of silver nanoparticles with bacteriorhodopsin in SERS-active regions lowers the amount of bacteriorhodopsin molecules involved in phototransformations.

Functional 2D nanomaterials for optoelectronics based on langmuir bacteriorhodopsin films

We have tested the possibility of using monomolecular layers of bacteriorhodopsin (BR) for the synthesis of highly sensitive and highly selective sensors based on second harmonic generation and surface plasma waves. We have used various methods to study the optical and nonlinear optical properties of Langmuir-Blodgett films of BR in order to clarify the extent to which specific properties of BR molecules are retained during their transfer from the surface of water to a solid substrate. We show that the second harmonic generation method is efficient for analyzing the molecular orientation and quality of Langmuir-Blodgett films. The experimental nonlinear optical susceptibility of second order BR molecules is 3.4·10−11m/V. The relative change in the resonant wave vector is (3.6±0.1)·10−2 at an excitation light wavelength of 630nm. We have obtained a BR spectrum with the effective excitation by incident radiation of surface plasma waves. On the basis of these studies, we have proposed new schemes of biosensors operating on the basis of second harmonic generation and surface plasma resonance caused by fundamental frequency reflection from BR monomolecular layers. This scheme was tested for a model device and demonstrated the possibility of obtaining sensitivities of the order of 1011 molecules/cm3.

Functional 2D nanomaterials for optoelectronic equipment on the basis of langmuir–blodgett bacteriorhodopsin films

We have tested the possibility of using monomolecular layers of bacteriorhodopsin (BR) for the synthesis of highly sensitive and highly selective sensors based on second harmonic generation and surface plasma waves. We have used various methods to study the optical and nonlinear optical properties of Langmuir-Blodgett films of BR in order to clarify the extent to which specific properties of BR molecules are retained during their transfer from the surface of water to a solid substrate. We show that the second harmonic generation method is efficient for analyzing the molecular orientation and quality of Langmuir-Blodgett films. The experimental nonlinear optical susceptibility of second order BR molecules is 3.4 · 10-11m/V. The relative change in the resonant wave vector is (3.6 ± 0.1)·10-2at an excitation light wavelength of 630 nm. We have obtained a BR spectrum with the effective excitation by incident radiation of surface plasma waves. On the basis of these studies, we have proposed new schemes of biosensors operating on the basis of second harmonic generation and surface plasma resonance caused by fundamental frequency reflection from BR monomolecular layers. This scheme was tested for a model device and demonstrated the possibility of obtaining sensitivities of the order of 1011molecules/cm3.

Anisotropic photoselection of the bacteriorhodopsin molecules in the purple membrane with hexagonal crystalline lattice of trimers

Anisotropic photoselection of the bacteriorhodopsin molecules in the purple membrane with hexagonal crystalline lattice of trimers

Rate Constant Change of Photo Reaction of Bacteriorhodopsin Observed in Trimeric Molecular System.

To elucidate the time evolution of photo reaction of bacteriorhodopsin in glycerol mixed purple membrane at around 196 K under irradiation by red light, a kinetic model was constructed. The change of absorption with irradiation at times of 560 nm and 412 nm was analyzed for the purpose of determining reaction rates of photo reaction of bacteriorhodopsin and its product M intermediate. In this study it is shown that reaction rates of conversion from bacteriorhodopsin to the M intermediate can be explained by a set of linear differential equations. This model analysis concludes that bacteriorhodopsin in which constitutes a trimer unit with other two bacteriorhodopsin molecules changes into M intermediates in the 1.73 of reaction rate, in the initial step, and according to the number of M intermediate in a trimer unit, from three to one, the reaction rate of bacteriorhodopsin into M intermediates smaller as 1.73, 0.80, 0.19 which caused by influence of inter-molecular interaction between bacteriorhodopsin.

Silver Nanoparticles Strongly Affect the Properties of Bacteriorhodopsin, a Photosensitive Protein of Halobacterium Salinarium Purple Membranes

Interaction of silver nanoparticles (AgNPs) with purple membranes (PM) has been studied using atomic force microscopy and optical spectroscopy. It was shown that AgNPs alter the photocycle of the bacteriorhodopsin (BRh) molecules located in SERS-active spots. AgNPs suppresses the photocycle by “freezing” the state of retinal in which BRh has bound AgNPs. Outside SERS-active spots, BRh molecules retain the capacity for carrying out the photocycle but with shorter lifetimes of its photointermediate states. Thus, the AgNPs may affect the BRh photocycle through its acceleration in the regions of weak AgNPs-PM interaction or by the photocycle freezing in the SERS-active spots.

Bacteriorhodopsin as a superior substitute for hydrazine in chemical reduction of single-layer graphene oxide sheets

Bacteriorhodopsin (bR) molecules were utilized as light-driven proton pumps for green as well as effective reduction of single-layer graphene oxide (GO) sheets. The bR molecules and graphene sheets were separated from each other in an aqueous environment by using a polytetrafluoroethylene membrane filter, in order to prevent their direct interactions (including attachment of the bR molecules onto the GO). Although reduction of GO using hydrazine or bR showed similar deoxygenation levels (based on X-ray photoelectron spectroscopy), the former resulted in formation of CN bonds which can substantially decrease the electrical conductivity of the reduced sheets. The electrical characteristics of the single-layer graphene sheets were studied by recording current–voltage curves of the sheets located between two Au electrodes on a SiO2 (300nm)/Si (100) substrate. The electrical conductivity of the bR-reduced graphene oxide (rGO) sheets was found about one order of magnitude better than that of hydrazine-rGO sheets. The excellent electrical conductivity of the bR-rGO sheets (with sheet resistance of ∼7.1×104Ω/sq) was assigned to the effective deoxygenation (without formation of any CN bonds) and better restoration of the graphitic structure of the GO sheets, using the protons pumped by the bR molecules.

Photonic spectral modulation in bacteriorhodopsin molecules and optimization of signal wavelength

In general, in excited-state absorption (ESA) based all-optical light modulator, maximum signal intensity modulation is achieved at the wavelength corresponding to maximum excited to ground state absorption cross-section ratio. However, theoretical analysis of photonic spectral modulation of broadband signal beam based on ESA in bacteriorhodopsin (BR) provides counterintuitive interesting results in the signal wavelength region 460–700nm. It is shown that the signal modulation is enhanced significantly at some particular wavelength values (optimum signal wavelength). Optimum signal wavelength in the region 450–700nm, varies with modulating intensity, and does not correspond to the peak absorption wavelength of the longer lived intermediate states. Present analysis is very useful in designing ESA based power efficient photonic devices.

Optoelectronic Logic Gates Based on Photovoltaic Response of Bacteriorhodopsin Polymer Composite Thin Films

We present designs of optoelectronic OR, AND, NOR, and NAND logic gates with multiple pulsed pump laser beams based on the photovoltaic response of bacteriorhodopsin (BR) molecules embedded in a polyvinyl matrix coated on ITO. A detailed experimental study of the photovoltaic response reveals that continuous pulsed exposure to 532 nm and 405 nm laser light results in a large photocurrent/photovoltage, due to rapid reprotonation and chromophore reisomerization, taking BR to the ground state in hundreds of nanoseconds. It also helps in sustaining the photovoltage at higher frequencies and in maintaining the shape of the photovoltage. It is shown experimentally that for a pulsed laser beam at 532 nm with peak pump intensity of 1.19 W/cm (2), a photovoltage of 50 mV is generated. A detailed numerical simulation of the photovoltaic response of BR has been carried out taking into account all the six states (B, K, L, M, N, and O) in the BR photocycle to ascertain the effect of various parameters such as lifetime of the M-state, the pump pulse-width, pump intensity, lifetime of excited protons, and rate constant of excited protons. Experimental results are in good agreement with theoretical simulations. The present study opens up new prospects for protein-based optoelectronic computing.

Structural Changes in Bacteriorhodopsin Caused by Two-Photon-Induced Photobleaching

Bacteriorhodopsin (BR) is the key protein of the halobacterial photosynthetic system. BR assembles into two-dimensional crystalline patches, the so-called purple membranes (PM), and acts as a light-driven proton pump converting light energy into the chemical energy of a proton gradient over the cell membrane. The two-photon absorption (TPA) of BR is so far not fully understood. Astonishingly high TPA cross sections have been reported, but the molecular mechanisms have not been elucidated. In this work, we address structural changes in BR and PM upon TPA, investigating its TPA photochemistry by spectroscopy, small-angle X-ray scattering, as well as electron and atomic force microscopy. We observe that TPA of BR leads to formation of an UV-absorbing N-retinyl-bacterioopsin state, which is accompanied by the loss of crystalline order in PM. FTIR and CD spectroscopy confirm that BR trimers as well as the secondary structure of the BR molecules are preserved. We demonstrate that excitation by TPA results in the photochemical reduction of the retinal Schiff base, which in turn causes a permanent asymmetric shape change of BR, similar to the one transiently observed during the photocycle-related opening and closing of the cytoplasmic proton half channel. This shape change causes PM sheets to merely roll up toward the extracellular side and causes the loss of crystallinity of PM. We present a model for the TPA photoresponse of BR, which also explains the irreversibility of the process in terms of a photochemical reduction of the Schiff base.

All-optical logic gates based on photoinduced anisotropy of bacteriorhodopsin film

All-optical logic gates based on photoinduced anisotropy of bacteriorhodopsin (BR) film are proposed. The photoinduced anisotropy in BR film, which arises from the selective absorption of BR molecules to polarized light, can be controlled by changing the amplitudes and polarizations of exiting beams. As a consequence, the polarization of the probe light passing through the BR film can be controlled by the polarization of the exiting beam. Based on this property, a novel scheme of all-optical logic gates, such as AND, OR, XOR and NOT, has been implemented via the pump-probe technique. A theoretical model for the all-optical logic gates is proposed, and the theoretical predictions are demonstrated with the experimental results.

Real-Time Optical Information Processing Through the Use of Low-Saturable Absorption in Bacteriorhodopsin Films

We have shown that the mechanism for a reversible anisotropic photoselection of bacteriorhodopsin (BR) molecules and a very small saturation intensity of a BR film allow for the blocking of any intensity feature from an image by just varying the intensity of a He-Ne laser beam without conventional frequency filters or rotation of an analyzer. It is found that the E204Q variant of BR offers additional valuable features. The photochemical cycle with a red-light absorbing O intermediate, B→M→O→B, and a distortion of the grating profile due to the low-saturation absorption are responsible for a large initial peak sharpness of the dynamic holographic recording kinetics.

Velocity-Dependent Mechanical Unfolding of Bacteriorhodopsin Is Governed by a Dynamic Interaction Network

Bacteriorhodopsin is a model system for membrane proteins. This seven transmembrane helical protein is embedded within a membrane structure called purple membrane. Its structural stability against mechanical stress was recently investigated by atomic force microscopy experiments, in which single proteins were extracted from the purple membrane. Here, we study this process by all-atom molecular dynamics simulations, in which single bacteriorhodopsin molecules were extracted and unfolded from an atomistic purple membrane model. In our simulations, key features from the experiments like force profiles and location of key residues that resist mechanical unfolding were reproduced. These key residues were seen to be stabilized by a dynamic network of intramolecular interactions. Further, the unfolding pathway was found to be velocity-dependent. Simulations in which the mechanical stress was released during unfolding revealed relaxation motions that allowed characterization of the nonequilibrium processes during fast extraction.