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Bacteriorhodopsin (bR) of purple membrane (PM) has increasing technical interests, particularly in photonic devices and bioelectronics. The present work has concerned with monitoring the temperature dependence of passive electric responses in-plane and out-of-plane of the membranes. Based on thermal properties observed orthogonally here for PM, a high-temperature intermediate of bR has been suggested to populate at around 60 °C, which may be ascribed to a molten globule-like state. This intermediate has been found to be enclosed between two reversible thermal transitions for PM. Large-scale turnover in the energy of activation, for these two thermal transitions, occurs steeply at such state at 60 °C, above which does bR reverse the sign of dielectric anisotropy (i.e. crossover) provided the operating frequency should be above the crossover frequency, at which the reversal occurs. No such crossover was found to occur below the crossover frequency, even above the crossover temperature (i.e. 60 °C). Likewise, no such crossover was found to occur below the crossover temperature, even above the crossover frequency. Relying on this reasoning, a logic gate operation may be declared implicating bR for bioelectronics and sense technological relevance. In addition, the results specify "dual frequency" as well as "dual temperature" characteristics to bacteriorhodopsin.

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Disc-like lipid nanoparticles stabilized by saponin biosurfactants display fascinating properties, including their temperature-driven re-organization. ß-Aescin, a saponin from seed extract of the horse chestnut tree, shows strong interactions with lipid membranes and has gained interest due to its beneficial therapeutic implications as well as its ability to decompose continuous lipid membranes into size-tuneable discoidal nanoparticles. Here, we characterize lipid nanoparticles formed by aescin and the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine. We present site-resolved insights into central molecular interactions and their modulations by temperature and aescin content. Using the membrane protein bacteriorhodopsin, we additionally demonstrate that, under defined conditions, aescin-lipid discs can accommodate medium-sized transmembrane proteins. Our data reveal the general capability of this fascinating system to generate size-tuneable aescin-lipid-protein particles, opening the road for further applications in biochemical, biophysical and structural studies.

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Liquid crystal (LC) biosensors have received significant attention for their potential applications for point-of-care devices due to their sensitivity, low cost, and easy read-out. They have been employed to detect a wide range of important biological molecules. However, detecting the function of membrane proteins has been extremely challenging due to the difficulty of integrating membrane proteins, lipid membranes, and LCs into one system. In this study, we addressed this challenge by monitoring the proton-pumping function of bacteriorhodopsin (bR) using a pH-sensitive LC thin film biosensor. To achieve this, we deposited purple membranes (PMs) containing a 2D crystal form of bRs onto an LC-aqueous interface. Under light, the PM patches changed the local pH at the LC-aqueous interface, causing a color change in the LC thin film that is observable through a polarizing microscope with crossed polarizers. These findings open up new opportunities to study the biofunctions of membrane proteins and their induced local environmental changes in a solution using LC biosensors.

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The photoinduced all-trans to 13-cis isomerization of the retinal Schiff base represents the ultrafast first step in the reaction cycle of bacteriorhodopsin (BR). Extensive experimental and theoretical work has addressed excited-state dynamics and isomerization via a conical intersection with the ground state. In conflicting molecular pictures, the excited state potential energy surface has been modeled as a pure S[Formula: see text] state that intersects with the ground state, or in a 3-state picture involving the S[Formula: see text] and S[Formula: see text] states. Here, the photoexcited system passes two crossing regions to return to the ground state. The electric dipole moment of the Schiff base in the S[Formula: see text] and S[Formula: see text] state differs strongly and, thus, its measurement allows for assessing the character of the excited-state potential. We apply the method of ultrafast terahertz (THz) Stark spectroscopy to measure electric dipole changes of wild-type BR and a BR D85T mutant upon electronic excitation. A fully reversible transient broadening and spectral shift of electronic absorption is induced by a picosecond THz field of several megavolts/cm and mapped by a 120-fs optical probe pulse. For both BR variants, we derive a moderate electric dipole change of 5 [Formula: see text] 1 Debye, which is markedly smaller than predicted for a neat S[Formula: see text]-character of the excited state. In contrast, S[Formula: see text]-admixture and temporal averaging of excited-state dynamics over the probe pulse duration gives a dipole change in line with experiment. Our results support a picture of electronic and nuclear dynamics governed by the interaction of S[Formula: see text] and S[Formula: see text] states in a 3-state model.

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Membrane protein folding is distinct from folding of soluble proteins. Conformational acquisition in major membrane protein subclasses can be delineated into insertion and folding processes. An exception to the "two stage" folding, later developed to "three stage" folding, is observed within the last two helices in bacteriorhodopsin (BR), a system that serves as a model membrane protein. We employ a reductionist approach to understand interplay of molecular factors underlying the apparent defiance. Leveraging available solution NMR structures, we construct, sample in silico, and analyze partially (PIn) and fully inserted (FIn) BR membrane states. The membrane lateral C-terminal helix (CH) in PIn is markedly prone to transient structural distortions over microsecond timescales; a disorder prone region (DPR) is thereby identified. While clear transmembrane propensities are not acquired, the distortions induce alterations in local membrane curvature and area per lipid. Importantly, energetic decompositions reveal that overall, the N-terminal helix (NH) is thermodynamically more stable in the PIn. Higher overall stability of the FIn arises from favorable interactions between the NH and the CH. Our results establish lack of spontaneous transition of the PIn to the FIn, and attributes their partitioning to barriers that exceed those accessible with thermal fluctuations. This work paves the way for further detailed studies aimed at determining the thermo-kinetic roles of the initial five helices, or complementary external factors, in complete helical folding and insertion in BR. We comment that complementing such efforts with the growing field of machine learning assisted energy landscape searches may offer unprecedented insights.

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Controlling the pH at the microliter scale can be useful for applications in research, medicine, and industry, and therefore represents a valuable application for synthetic biology and microfluidics. The presented vesicular system translates light of different colors into specific pH changes in the surrounding solution. It works with the two light-driven proton pumps bacteriorhodopsin and blue light-absorbing proteorhodopsin Med12, that are oriented in opposite directions in the lipid membrane. A computer-controlled measuring device implements a feedback loop for automatic adjustment and maintenance of a selected pH value. A pH range spanning more than two units can be established, providing fine temporal and pH resolution. As an application example, a pH-sensitive enzyme reaction is presented where the light color controls the reaction progress. In summary, light color-controlled pH-adjustment using engineered proteoliposomes opens new possibilities to control processes at the microliter scale in different contexts, such as in synthetic biology applications.

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Boundary lipids surrounding membrane proteins play an essential role in protein function and structure. These protein-lipid interactions are mainly divided into electrostatic interactions between the polar amino acids of proteins and polar heads of phospholipids, and hydrophobic interactions between protein transmembrane sites and phospholipid acyl chains. Our previous report (Kawatake et al., Biochim. Biophys. Acta 1858 [2016] 2106-2115) covered a method for selectively analyzing boundary lipid interactions and showed differences in membrane protein-peripheral lipid interactions due to differences in their head group. Interactions in the hydrophobic acyl chains of phospholipids are relatively consistent among proteins, but the details of these interactions have not been elucidated. In this study, we reconstituted bacteriorhodopsin as a model protein into phospholipid membranes labeled with 2H and 13C for solid-state NMR measurement to investigate the depth-dependent effect of the head group structure on the lipid bilayer. The results showed that the position of the phospholipid near the carbonyl carbon was affected by the head group in terms of selectivity for protein surfaces, whereas in the deep interior of the bilayer near the leaflet interface, there was little difference between the head groups, indicating that the dependence of their interactions on the head group was much reduced.

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Over the last decade, the development of time-resolved serial crystallography (TR-SX) at X-ray free-electron lasers (XFELs) and synchrotrons has allowed researchers to study phenomena occurring in proteins on the femtosecond-to-minute timescale, taking advantage of many technical and methodological breakthroughs. Protein crystals of various sizes are presented to the X-ray beam in either a static or a moving medium. Photoactive proteins were naturally the initial systems to be studied in TR-SX experiments using pump-probe schemes, where the pump is a pulse of visible light. Other reaction initiations through small-molecule diffusion are gaining momentum. Here, selected examples of XFEL and synchrotron time-resolved crystallography studies will be used to highlight the specificities of the various instruments and methods with respect to time resolution, and are compared with cryo-trapping studies.

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There is a need to shift the absorbance of biomolecules to the optical transparency window of tissue for applications in optogenetics and photo-pharmacology. There are a few strategies to achieve the so-called red shift of the absorption maxima. Herein, a series of 11 merocyanine dyes were synthesized and employed as chromophores in place of retinal in bacteriorhodopsin (bR) to achieve a bathochromic shift of the absorption maxima relative to bR's [Formula: see text] of 568 nm. Assembly with the apoprotein bacterioopsin (bO) led to stable, covalently bound chromoproteins with strongly bathochromic absorbance bands, except for three compounds. Maximal red shifts were observed for molecules 9, 2, and 8 in bR where the [Formula: see text] was 766, 755, and 736 nm, respectively. While these three merocyanines have different end groups, they share a similar structural feature, namely, a methyl group which is located at the retinal equivalent position 13 of the polyene chain. The absorption and fluorescence data are also presented for the retinal derivatives in their aldehyde, Schiff base (SB), and protonated SB (PSB) forms in solution. According to their hemicyanine character, the PSBs and their analogue bRs exhibited fluorescence quantum yields (Φf) several orders of magnitude greater than native bR (Φf 0.02 to 0.18 versus 1.5 × 10-5 in bR) while also exhibiting much smaller Stokes shifts than bR (400 to 1000 cm-1 versus 4030 cm-1 in bR). The experimental results are complemented by quantum chemical calculations where excellent agreement between the experimental [Formula: see text] and the calculated [Formula: see text] was achieved with the second-order algebraic-diagrammatic construction [ADC(2)] method. In addition, quantum mechanics/molecular mechanics (QM/MM) calculations were employed to shed light on the origin of the bathochromic shift of merocyanine 2 in bR compared with native bR.

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The technique of time-resolved macromolecular crystallography (TR-MX) has recently been rejuvenated at synchrotrons, resulting in the design of dedicated beamlines. Using pump-probe schemes, this should make the mechanistic study of photoactive proteins and other suitable systems possible with time resolutions down to microseconds. In order to identify relevant time delays, time-resolved spectroscopic experiments directly performed on protein crystals are often desirable. To this end, an instrument has been built at the icOS Lab (in crystallo Optical Spectroscopy Laboratory) at the European Synchrotron Radiation Facility using reflective focusing objectives with a tuneable nanosecond laser as a pump and a microsecond xenon flash lamp as a probe, called the TR-icOS (time-resolved icOS) setup. Using this instrument, pump-probe spectra can rapidly be recorded from single crystals with time delays ranging from a few microseconds to seconds and beyond. This can be repeated at various laser pulse energies to track the potential presence of artefacts arising from two-photon absorption, which amounts to a power titration of a photoreaction. This approach has been applied to monitor the rise and decay of the M state in the photocycle of crystallized bacteriorhodopsin and showed that the photocycle is increasingly altered with laser pulses of peak fluence greater than 100 mJ cm-2, providing experimental laser and delay parameters for a successful TR-MX experiment.

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Membrane proteins (MPs) are affected by binding of specific lipids. We previously developed a methodology for systematically analyzing MP-lipid interactions leveraging surface plasmon resonance (SPR). In this method, the gold sensor chip surface was modified with a self-assembled monolayer (SAM), which allowed for a larger amount of MP-immobilization. However, the laborious lipid purification step remained a bottleneck. To address this issue, a new strategy has been developed utilizing gold nanoparticles (AuNPs) instead of the gold sensor chip. AuNPs were coated with SAM, on which MP was covalently anchored. The MP-immobilized AuNPs were mixed with a lipid mixture, and the recovered lipids were quantified by LC-MS. Bacteriorhodopsin (bR) was used as an MP to demonstrate this concept. We optimized immobilization conditions and confirmed the efficient immobilization of bR by dynamic light scattering and electron micrographs. Washing conditions for pulldown experiments were optimized to efficiently remove non-specific lipids. A new binding index was introduced to qualitatively reproduce the known affinity of lipids for bR. Consequently, the low-abundant and least-studied lipid S-TeGD was identified as a candidate for bR-specific lipids. This technique can skip the laborious lipid purification process, accelerating the screening of MP-specific lipids from complex lipid mixtures.

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Bacteriorhodopsin, isolated from a halophilic bacterium, is a photosynthetic protein with a structure and function similar to those of the visual pigment rhodopsin. A voltaic cell with bacteriorhodopsin sandwiched between two transparent electrodes exhibits a time-differential response akin to that observed in retinal ganglion cells. It is intriguing as a means to emulate excitation and inhibition in the neural response. Here, we present a neuromorphic device emulating the retinal ganglion cell receptive field fabricated by patterning bacteriorhodopsin onto two transparent electrodes and encapsulating them with an electrolyte solution. This protein-based artificial ganglion cell receptive field is characterized as a bandpass filter that simultaneously replicates excitatory and inhibitory responses within a single element, successfully detecting image edges and phenomena of brightness illusions. The device naturally emulates the highly interacting ganglion cell receptive fields by exploiting the inherent properties of proteins without the need for electronic components, bias power supply, or an external operating circuit.

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In the bioenergetics studies, the direct electrometric method played an important role. This method is based on measuring the electrical potential difference (Δψ) between two compartments of the experimental cell generated by some membrane proteins. These proteins are incorporated into closed lipid-protein membrane vesicles associated with an artificial lipid membrane that separates the compartments. The very existence of such proteins able to generate Δψ was one of the consequences of Peter Mitchell's chemiosmotic concept. The discovery and investigation of their functioning contributed to the recognition of this concept and, eventually the well-deserved awarding of the Nobel Prize to P. Mitchell. Lel A. Drachev (1926-2022) was one of the main authors of the direct electrometrical method. With his participation, key studies were carried out on the electrogenesis of photosynthetic and respiratory membrane proteins, including bacteriorhodopsin, visual rhodopsin, photosynthetic bacterial reaction centers, cytochrome oxidase and others.

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The diversity of the retinal-containing proteins (rhodopsins) in nature is extremely large. Fundamental similarity of the structure and photochemical properties unites them into one family. However, there is still a debate about the origin of retinal-containing proteins: divergent or convergent evolution? In this review, based on the results of our own and literature data, a comparative analysis of the similarities and differences in the photoconversion of the rhodopsin of types I and II is carried out. The results of experimental studies of the forward and reverse photoreactions of the bacteriorhodopsin (type I) and visual rhodopsin (type II) rhodopsins in the femto- and picosecond time scale, photo-reversible reaction of the octopus rhodopsin (type II), photovoltaic reactions, as well as quantum chemical calculations of the forward photoreactions of bacteriorhodopsin and visual rhodopsin are presented. The issue of probable convergent evolution of type I and type II rhodopsins is discussed.

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An effective early diagnosis is important for rheumatoid arthritis (RA) management. This study reveals a novel RA detection method using bacteriorhodopsin as a photoelectric transducer, a light-driven proton pump in purple membranes (PMs). It was devised by covalently conjugating a PM monolayer-coated electrode with a citrullinated-inter-alpha-trypsin inhibitor heavy chain 3 (ITIH3)542-556 peptide that recognizes the serum RA-associated autoantibodies. The direct serum coating decreased the photocurrents in the biosensor, with the reduction in the photocurrent caused by coating with an RA-patient serum that is significantly larger than that with a healthy-control serum (38.1% vs. 20.2%). The difference in the reduction in the photocurrent between those two serum groups widened after the serum-coated biosensor was further labeled with gold nanoparticle (AuNP)-conjugated anti-IgA (anti-IgA-AuNP) (53.6% vs. 30.6%). Both atomic force microscopic (AFM) and Raman analyses confirmed the sequential peptide, serum, and anti-IgA-AuNP coatings on the PM-coated substrates. The reductions in the photocurrent measured in both the serum and anti-IgA-AuNPs coating steps correlated well with the results using commercial enzyme-linked immunosorbent assay kits (Spearman rho = 0.805 and 0.787, respectively), with both a sensitivity and specificity close to 100% in both steps. It was shown that an RA diagnosis can be performed in either a single- or two-step mode using the developed biosensor.

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Biomimetic visual sensors utilizing bacteriorhodopsin (bR) were fabricated by using an inkjet method. The inkjet printer facilitated the jetting of the bR suspension, allowing for the deposition of bR films. The resulting inkjet-printed bR film exhibited time-differential photocurrent response characteristics similar to those of a dip-coated bR film. By adjusting the number of printed bR film layers, the intensity of the photocurrent could be easily controlled. Moreover, the inkjet printing technique enabled unconstrained patterning, facilitating the design of various visual information processing functions, such as visual filters. In this study, we successfully fabricated two visual filters, namely, a two-dimensional Difference of Gaussian (DOG) filter and a Gabor filter. The printed DOG filter demonstrated edge detection capabilities corresponding to contour recognition in visual receptive fields. On the other hand, the printed Gabor filter proved effective in detecting objects of specific sizes as well as their motion and orientation. The integration of bR and the inkjet method holds significant potential for the widespread implementation of highly functional biomaterial-based visual sensors. These sensors have the capability to provide real-time visual information while operating in an energy-efficient manner.

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A uniformly oriented purple membrane (PM) monolayer containing photoactive bacteriorhodopsin has recently been applied as a sensitive photoelectric transducer to assay color proteins and microbes quantitatively. This study extends its application to detecting small molecules, using adenosine triphosphate (ATP) as an example. A reverse detection method is used, which employs AuNPs labeling and specific DNA strand displacement. A PM monolayer-coated electrode is first covalently conjugated with an ATP-specific nucleic acid aptamer and then hybridized with another gold nanoparticle-labeled nucleic acid strand with a sequence that is partially complementary to the ATP aptamer, in order to significantly minimize the photocurrent that is generated by the PM. The resulting ATP-sensing chip restores its photocurrent production in the presence of ATP, and the photocurrent recovers more effectively as the ATP concentration increases. Direct and single-step ATP detection is achieved in 15 min, with detection limits of 5 nM and a dynamic range of 5 nM-0.1 mM. The sensing chip exhibits high selectivity against other ATP analogs and is satisfactorily stable in storage. The ATP-sensing chip is used to assay bacterial populations and achieves a detection limit for Bacillus subtilis and Escherichia coli of 102 and 103 CFU/mL, respectively. The demonstration shows that a variety of small molecules can be simultaneously quantified using PM-based biosensors.

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We have previously demonstrated the potential of gelatin films as a memory device, offering a novel approach for writing, reading, and erasing through the manipulation of gelatin structure and bound water content. Here, we discovered that incorporating a bacteriorhodopsin (BR)-lipid membrane into the gelatin devices can further increase the electron conductivity of the polypeptide-bound water network and the ON/OFF ratio of the device by two folds. Our photocurrent measurements show that the BR incorporated in the membrane sandwiched in a gelatin device can generate a net proton flow from the counter side to the deposited side of the membrane. This leads to the establishment of non-electroneutrality on the gelatin films adjacent to the BR-incorporated membrane. Our Raman spectroscopy results show that BR proton pumping in the ON state gelatin device increases the bound water presence and promotes polypeptide unwinding compared to devices without BR. These findings suggest that the non-electroneutrality induced by BR proton pumping can increase the extent of polypeptide unwinding within the gelatin matrix, consequently trapping more bound water within the gelatin-bound water network. The resulting rise in hydrogen bonds could expand electron transfer routes, thereby enhancing the electron conductivity of the memory device in the ON state.

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Bio-solar cells are studied as sustainable and biocompatible energy sources with significant potential for biomedical applications. However, they are composed of light-harvesting biomolecules with narrow absorption wavelengths and weak transient photocurrent generation. In this study, a nano-biohybrid-based bio-solar cell composed of bacteriorhodopsin, chlorophyllin, and Ni/TiO2 nanoparticles is developed to overcome the current limitations and verify the possibility of biomedical applications. Bacteriorhodopsin and chlorophyllin are introduced as light-harvesting biomolecules to broaden the absorption wavelength. As a photocatalyst, Ni/TiO2 nanoparticles are introduced to generate a photocurrent and amplify the photocurrent generated by the biomolecules. The developed bio-solar cell absorbs a broad range of visible wavelengths and generates an amplified stationary photocurrent density (152.6 nA cm-2 ) with a long lifetime (up to 1 month). Besides, the electrophysiological signals of muscle cells at neuromuscular junctions are precisely regulated by motor neurons excited by the photocurrent of the bio-solar cell, indicating that the bio-solar cell can control living cells by signal transmission through other types of living cells. The proposed nano-biohybrid-based bio-solar cell can be used as a sustainable and biocompatible energy source for the development of wearable and implantable biodevices and bioelectronic medicines for humans.

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Bacteriorhodopsin (bR) of purple membrane (PM) is a retinal protein that forms aggregates in the form of trimers constituting, together with archaeal lipids, the crystalline structure of PM. The rotary motion of bR inside PM may be pertinent in understanding the essence of the crystalline lattice. An attempt has been made to determine the rotation of bR trimers which has been found to be detected solely at thermal phase transitions of PM, namely lipid, crystalline lattice and protein melting phase transitions. The temperature dependences of dielectric versus electronic absorption spectra of bR have been determined. The results suggest that the rotation of bR trimers, together with concomitant bending of PM, are most likely brought by structural changes in bR which might be driven by retinal isomerization and mediated by lipid. The rupturing of the lipid-protein contact might consequently lead to rotation of trimers associated with bending, curling or vesicle formation of PM. So the retinal reorientation may underlie the concomitant rotation of trimers. Most importantly, rotation of trimers might play a role, in terms of the essence of the crystalline lattice, in the functional activity of bR and may serve physiological relevance.

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