Search Results (11)

This study elucidates the structural determinants and optogenetic potential of Archaerhodopsin HeAR, a proton pump from Halorubrum sp. Ejinoor isolated from Inner Mongolian salt lakes. Through heterologous expression in E. coli BL21 (DE3) and integrative biophysical analyses, we demonstrate that HeAR adopts a stable trimeric architecture (129 kDa) with detergent-binding characteristics mirroring bacteriorhodopsin (BR); however, it exhibits a 10 nm bathochromic spectral shift (λmax = 550 nm) and elevated proton affinity (Asp-95 pKa = 3.5 vs. BR Asp-85 pKa = 2.6), indicative of evolutionary optimization in its retinal-binding electrostatic microenvironment. Kinetic profiling reveals HeAR’s prolonged photocycle (100 ms vs. BR’s 11 ms), marked by rapid M-state decay (3.3 ms) and extended dark-adaptation half-life (160 min), a bistable behavior attributed to enhanced hydrogen bond persistence (80%) and reduced conformational entropy (RMSD = 2.0 Å). Functional assays confirm light-driven proton extrusion (0.1 ng H⁺/mg·s) with DCCD-amplified flux (0.3 ng H⁺/mg·s) and ATP synthesis (0.3 nmol/mg·s), underscoring its synergy with H⁺-ATPase. Phylogenetic and structural analyses reveal 95% homology with Halorubrum AR4 and conservation of 11 proton-wire residues, despite divergent Trp/Tyr/Ser networks that redefine chromophore stabilization. AlphaFold-predicted models (TM-score > 0.92) and molecular docking identify superior retinoid-binding affinity (ΔG = −12.27 kcal/mol), while spectral specificity (550–560 nm) and acid-stable photoresponse highlight its adaptability for low-irradiance neuromodulation. These findings position HeAR as a precision optogenetic tool, circumventing spectral overlap with excitatory opsins and enabling sustained hyperpolarization with minimized phototoxicity. By bridging microbial energetics and optobioengineering, this work expands the archaeal rhodopsin toolkit and provides a blueprint for designing wavelength-optimized photoregulatory systems. Full article

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. Full article

Ferredoxin:NADP-oxidoreductase (FNR) catalyzes the reversible exchange of electrons between ferredoxin (Fd) and NADP(H). Reduction of NADP⁺ by Fd via FNR is essential in the terminal steps of photosynthetic electron transfer, as light-activated electron flow produces NADPH for CO₂ assimilation. FNR also catalyzes the reverse reaction in photosynthetic organisms, transferring electrons from NADPH to Fd, which is important in cyanobacteria for respiration and cyclic electron flow (CEF). The cyanobacterium Synechocystis sp. PCC6803 possesses two isoforms of FNR, a large form attached to the phycobilisome (FNR_L) and a small form that is soluble (FNR_S). While both isoforms are capable of NADPH oxidation or NADP⁺ reduction, FNR_L is most abundant during typical growth conditions, whereas FNR_S accumulates under stressful conditions that require enhanced CEF. Because CEF-driven proton pumping in the light–dark transition is due to NDH-1 complex activity and they are powered by reduced Fd, CEF-driven proton pumping and the redox state of the PQ and NADP(H) pools were investigated in mutants possessing either FNR_L or FNR_S. We found that the FNR_S isoform facilitates proton pumping in the dark–light transition, contributing more to CEF than FNR_L. FNR_L is capable of providing reducing power for CEF-driven proton pumping, but only after an adaptation period to illumination. The results support that FNR_S is indeed associated with increased cyclic electron flow and proton pumping, which is consistent with the idea that stress conditions create a higher demand for ATP relative to NADPH. Full article

Bacteriorhodopsin (BR) functions as a light-driven proton pump that transitions between different states during the photocycle, such as all-trans (AT; BR568) and 13-cis, 15-syn (CS; BR548) state and K, L, M₁, M₂, N, and O intermediates. In this study, we used in situ photoirradiation ¹³C solid-state NMR to observe a variety of photo-intermediates and photoreaction pathways in [20-¹³C]retinal-WT-BR and its mutant [20-¹³C, 14-¹³C]retinal-D96N-BR. In WT-BR, the CS state converted to the CS* intermediate under photoirradiation with green light at −20 °C and consequently converted to the AT state in the dark. The AT state converted to the N intermediate under irradiation with green light. In D96N-BR, the CS state was converted to the CS* intermediate at −30 °C and consequently converted to the AT state. Simultaneously, the AT state converted to the M and L intermediates under green light illumination at −30 °C and subsequently converted to the AT state in the dark. The M intermediate was directly excited to the AT state by UV light illumination. We demonstrated that short-lived photo-intermediates could be observed in a stationary state using in situ photoirradiation solid-state NMR spectroscopy for WT-BR and D96N-BR, enabling insight into the light-driven proton pump activity of BR. Full article

The light-driven proton pump bacteriorhodopsin (BR) from the extreme halophilic archaeon Halobacterium salinarum is a retinal-binding protein, which forms highly ordered and thermally stable 2D crystals in native membranes (termed purple membranes). BR and purple membranes (PMs) have been and are still being intensively studied by numerous researchers from different scientific disciplines. Furthermore, PMs are being successfully used in new, emerging technologies such as bioelectronics and bionanotechnology. Most published studies used the wild-type form of BR, because of the intrinsic difficulty to produce genetically modified versions in purple membranes homologously. However, modification and engineering is crucial for studies in basic research and, in particular, to tailor BR for specific applications in applied sciences. We present an extensive and detailed protocol ranging from the genetic modification and cultivation of H. salinarum to the isolation, and biochemical, biophysical and functional characterization of BR and purple membranes. Pitfalls and problems of the homologous expression of BR versions in H. salinarum are discussed and possible solutions presented. The protocol is intended to facilitate the access to genetically modified BR versions for researchers of different scientific disciplines, thus increasing the application of this versatile biomaterial. Full article

The light-driven sodium pump KR2, found in 2013 in the marine bacteria Krokinobacter eikastus, serves as a model protein for the studies of the sodium-pumping microbial rhodopsins (NaRs). KR2 possesses a unique NDQ (N112, D116, and Q123) set of the amino acid residues in the functionally relevant positions, named the NDQ motif. The N112 was shown to determine the Na⁺/H⁺ selectivity and pumping efficiency of the protein. Thus, N112A mutation converts KR2 into an outward proton pump. However, no structural data on the functional conversions of the light-driven sodium pumps are available at the moment. Here we present the crystal structure of the N112A mutant of KR2 in the ground state at the resolution of 2.4 Å. The structure revealed a minor deflection in the central part of the helix C and a double conformation of the L74 residue in the mutant. The organization of the retinal Schiff base and neighboring water molecules is preserved in the ground state of KR2-N112A. The presented data provide structural insights into the effects of the alterations of the characteristic NDQ motif of NaRs. Our findings also demonstrate that for the rational design of the KR2 variants with modified ion selectivity for optogenetic applications, the structures of the intermediate states of both the protein and its functional variants are required. Full article

The utilization of light energy to power organic-chemical transformations is a fundamental strategy of the terrestrial energy cycle. Inspired by the elegance of natural photosynthesis, much interdisciplinary research effort has been devoted to the construction of simplified cell mimics based on artificial vesicles to provide a novel tool for biocatalytic cascade reactions with energy-demanding steps. By inserting natural or even artificial photosynthetic systems into liposomes or polymersomes, the light-driven proton translocation and the resulting formation of electrochemical gradients have become possible. This is the basis for the conversion of photonic into chemical energy in form of energy-rich molecules such as adenosine triphosphate (ATP), which can be further utilized by energy-dependent biocatalytic reactions, e.g., carbon fixation. This review compares liposomes and polymersomes as artificial compartments and summarizes the types of light-driven proton pumps that have been employed in artificial photosynthesis so far. We give an overview over the methods affecting the orientation of the photosystems within the membranes to ensure a unidirectional transport of molecules and highlight recent examples of light-driven biocatalysis in artificial vesicles. Finally, we summarize the current achievements and discuss the next steps needed for the transition of this technology from the proof-of-concept status to preparative applications. Full article

Fungi possess diverse photosensory proteins that allow them to perceive different light wavelengths and to adapt to changing light conditions in their environment. The biological and physiological roles of the green light-sensing rhodopsins in fungi are not yet resolved. The rice plant pathogen Fusarium fujikuroi exhibits two different rhodopsins, CarO and OpsA. CarO was previously characterized as a light-driven proton pump. We further analyzed the pumping behavior of CarO by patch-clamp experiments. Our data show that CarO pumping activity is strongly augmented in the presence of the plant hormone indole-3-acetic acid and in sodium acetate, in a dose-dependent manner under slightly acidic conditions. By contrast, under these and other tested conditions, the Neurospora rhodopsin (NR)-like rhodopsin OpsA did not exhibit any pump activity. Basic local alignment search tool (BLAST) searches in the genomes of ascomycetes revealed the occurrence of rhodopsin-encoding genes mainly in phyto-associated or phytopathogenic fungi, suggesting a possible correlation of the presence of rhodopsins with fungal ecology. In accordance, rice plants infected with a CarO-deficient F. fujikuroi strain showed more severe bakanae symptoms than the reference strain, indicating a potential role of the CarO rhodopsin in the regulation of plant infection by this fungus. Full article

We have explored the use of optical oxygen electrodes to study oxygenic photosynthesis and heterotrophic activities in crystallizer brines of the salterns in Eilat, Israel. Monitoring oxygen uptake rates in the dark enables the identification of organic substrates that are preferentially used by the community. Addition of glycerol (the osmotic solute synthesized by Dunaliella) or dihydroxyacetone (produced from glycerol by Salinibacter) enhanced respiration rates. Pyruvate, produced from glycerol or from some sugars by certain halophilic Archaea also stimulated community respiration. Fumarate had a sparing effect on respiration, possibly as many halophilic Archaea can use fumarate as a terminal electron acceptor in respiration. Calculating the photosynthetic activity of Dunaliella by monitoring oxygen concentration changes during light/dark incubations is not straightforward as light also affects respiration of some halophilic Archaea and Bacteria due to action of light-driven proton pumps. When illuminated, community respiration of brine samples in which oxygenic photosynthesis was inhibited by DCMU decreased by ~40%. This effect was interpreted as the result of competition between two energy yielding systems: the bacteriorhodopsin proton pump and the respiratory chain of the prokaryotes. These findings have important implications for the interpretation of other published data on photosynthetic and respiratory activities in hypersaline environments. Full article

An ionic polymer microactuator driven by an organic photoelectric proton pump transducer is described in this paper. The light responsive transducer is fabricated by using molecular self-assembly to immobilize oriented bacteriorhodopsin purple membrane (PM) patches on a bio-functionalized porous anodic alumina (PAA) substrate. When exposed to visible light, the PM proton pumps produce a unidirectional flow of ions through the structure’s nano-pores and alter the pH of the working solution in a microfluidic device. The change in pH is sufficient to generate an osmotic pressure difference across a hydroxyethyl methacrylate-acrylic acid (HEMA-AA) actuator shell and induce volume expansion or contraction. Experiments show that the transducer can generate an ionic gradient of 2.5 μM and ionic potential of 25 mV, producing a pH increase of 0.42 in the working solution. The ΔpH is sufficient to increase the volume of the HEMA-AA microactuator by 80%. The volumetric transformation of the hydrogel can be used as a valve to close a fluid transport micro-channel or apply minute force to a mechanically flexible microcantilever beam. Full article

A few extremely halophilic Archaea (Halobacterium salinarum, Haloquadratum walsbyi, Haloferax mediterranei, Halorubrum vacuolatum, Halogeometricum borinquense, Haloplanus spp.) possess gas vesicles that bestow buoyancy on the cells. Gas vesicles are also produced by the anaerobic endospore-forming halophilic Bacteria Sporohalobacter lortetii and Orenia sivashensis. We have extensive information on the properties of gas vesicles in Hbt. salinarum and Hfx. mediterranei and the regulation of their formation. Different functions were suggested for gas vesicle synthesis: buoying cells towards oxygen-rich surface layers in hypersaline water bodies to prevent oxygen limitation, reaching higher light intensities for the light-driven proton pump bacteriorhodopsin, positioning the cells optimally for light absorption, light shielding, reducing the cytoplasmic volume leading to a higher surface-area-to-volume ratio (for the Archaea) and dispersal of endospores (for the anaerobic spore-forming Bacteria). Except for Hqr. walsbyi which abounds in saltern crystallizer brines, gas-vacuolate halophiles are not among the dominant life forms in hypersaline environments. There only has been little research on gas vesicles in natural communities of halophilic microorganisms, and the few existing studies failed to provide clear evidence for their possible function. This paper summarizes the current status of the different theories why gas vesicles may provide a selective advantage to some halophilic microorganisms. Full article