Microbial rhodopsin is a transmembrane protein that functions with a chromophore and is regulated by light. In nature, regulation through the retinal chromophore plays an important role in ...physiological phenomena. However, living organisms in soil, sea, and freshwater synthesize carotenoids preferentially over retinal in the biosynthetic pathway. Evolution has extended the energy conversion photosystem with additional pigments that act as antennae. Previously, Gloeobacter rhodopsin and xanthorhodopsin have been reported to form secondary chromophores with carotenoids. In this study, we report that a thermophilic rhodopsin (TR) and Tara76 rhodopsin, the latter of which is classified as a blue light‐absorbing proteorhodopsin, can form secondary chromophores with canthaxanthin (CAN). Tara76 rhodopsin and TR were found to exhibit high thermal stabilities and photophysical properties following their interaction with CAN. Isothermal titration calorimetry analysis, spectral shift measurements, and exciton analysis were used to examine the interactions of these rhodopsins with CAN. It was found that these interactions increased the stability toward temperature and pH through highly efficient chromophore formation, in addition to rapidly recruiting the retinal at a rate approximately twice as high as that obtained in the absence of CAN.
Microbial rhodopsins are distributed through many microorganisms. Heliorhodopsins are newly discovered but have an unclear function. They have seven transmembrane helices similar to type-I and ...type-II rhodopsins, but they are different in that the N-terminal region of heliorhodopsin is cytoplasmic. We chose 13 representative heliorhodopsins from various microorganisms, expressed and purified with an N-terminal His tag, and measured the absorption spectra. The 13 natural variants had an absorption maximum (λmax) in the range 530-556 nm similar to proteorhodopsin (λmax = 490-525 nm). We selected several candidate residues that influence rhodopsin color-tuning based on sequence alignment and constructed mutants via site-directed mutagenesis to confirm the spectral changes. We found two important residues located near retinal chromophore that influence λmax. We also predict the 3D structure via homology-modeling of Thermoplasmatales heliorhodopsin. The results indicate that the color-tuning mechanism of type-I rhodopsin can be applied to understand the color-tuning of heliorhodopsin.
Photoreceptors are light-sensitive proteins found in various organisms that respond to light and relay signals into the cells. Heliorhodopsin, a retinal-binding membrane protein, has been recently ...discovered, however its function remains unknown. Herein, we investigated the relationship between
Actinobacteria bacterium
IMCC26103 heliorhodopsin (AbHeR) and an adjacent glutamine synthetase (AbGS) in the same operon. We demonstrate that AbHeR binds to AbGS and regulates AbGS activity. More specifically, the dissociation constant (K
d
) value of the binding between AbHeR and AbGS is 6.06 μM. Moreover, the absence of positively charged residues within the intracellular loop of AbHeR impacted K
d
value as they serve as critical binding sites for AbGS. We also confirm that AbHeR up-regulates the biosynthetic enzyme activity of AbGS both in vitro and in vivo in the presence of light. GS is a key enzyme involved in nitrogen assimilation that catalyzes the conversion of glutamate and ammonia to glutamine. Hence, the interaction between AbHeR and AbGS may be critical for nitrogen assimilation in
Actinobacteria bacterium
IMCC26103 as it survives in low-nutrient environments. Overall, the findings of our study describe, for the first time, to the best of our knowledge, a novel function of heliorhodopsin as a regulatory rhodopsin with the capacity to bind and regulate enzyme activity required for nitrogen assimilation.
Microbial rhodopsins are light‐activated proteins that contain seven transmembrane alpha‐helices. Spectral tuning in microbial rhodopsins is a useful optogenetic tool. In this study, we report a new ...site that controls spectral tuning. In the proteorhodopsins ISR34 and ISR36, a single amino‐acid substitution at Cys189 caused an absorption maximum shift of 44 nm, indicating spectral tuning at a specific site. Comparison of single amino acid substitutions was conducted using photochemical and photobiological approaches. The maximum absorption for red‐shift was measured for mutations at positions 189 and 105 in ISR34, both residues being equally important. Structural changes resulting from amino acid substitutions are related to pKa values, pumping activity and spectral tuning.
In this study, we identify a single amino acid that can control the colour tuning of proteorhodopsin, which is widely distributed in nature. Single amino acid substitution of Cys189 caused an absorption maximum shift of 44 nm, indicating spectral tuning at a specific site. Comparison of single amino acid substitutions was conducted using photochemical and photobiological approaches.
We present a CuAAC (Copper-Catalyzed Azide-Alkyne Cycloaddition) reaction protocol designed for the visualization of mRNA. To achieve this, we synthesized stable mRNA molecules incorporating the ...modified nucleoside analog, EU, a crucial element for fluorophore attachment. Leveraging this modified mRNA, we successfully executed the CuAAC reaction, wherein the pro-fluorophore, coumarin, was conjugated to EU on the mRNA through our meticulously designed CuAAC process. This innovative approach resulted in the emission of fluorescence, enabling both precise quantification and visual observation of mRNA. Furthermore, we demonstrated the feasibility of concurrent mRNA synthesis and visualization by seamlessly integrating the CuAAC reaction mix into the mRNA transcription process. Additionally, our novel methodology opens avenues for prospective real-time monitoring of mRNA transcription within artificial cells. These advancements hold significant promise for expanding our comprehension of fundamental cellular processes and finding applications across diverse biological contexts in the future.
Heliorhodopsins (HeRs) have been hypothesized to have widespread functions. Recently, the functions for few HeRs have been revealed; however, the hypothetical functions remain largely unknown. ...Herein, we investigate light-modulation of heterodimeric multidrug resistance ATP-binding cassette transporters (OmrDE) mediated by Omithinimicrobium cerasi HeR. In this study, we classifiy genes flanking the HeR-encoding genes and identify highly conservative residues for protein-protein interactions. Our results reveal that the interaction between OcHeR and OmrDE shows positive cooperatively sequential binding through thermodynamic parameters. Moreover, light-induced OcHeR upregulates OmrDE drug transportation. Hence, the binding may be crucial to drug resistance in O. cerasi as it survives in a drug-containing habitat. Overall, we unveil a function of HeR as regulatory rhodopsin for multidrug resistance. Our findings suggest potential applications in optogenetic technology.
Microbial rhodopsin is a simple solar energy-capturing molecule compared to the complex photosynthesis apparatus. Light-driven proton pumping across the cell membrane is a crucial mechanism ...underlying microbial energy production.
is one of the highly abundant bacterial phyla in freshwater habitats, and members of this lineage are considered to boost heterotrophic growth
phototrophy, as indicated by the presence of actino-opsin (ActR) genes in their genome. However, it is difficult to validate their function under laboratory settings because
are not consistently cultivable. Based on the published genome sequence of
sp. strain IMCC13023, actinorhodopsin from the strain (ActR-13023) was isolated and characterized in this study. Notably, ActR-13023 assembled with natively synthesized carotenoid/retinal (used as a dual chromophore) and functioned as a light-driven outward proton pump. The ActR-13023 gene and putative genes involved in the chromophore (retinal/carotenoid) biosynthetic pathway were detected in the genome, indicating the functional expression ActR-13023 under natural conditions for the utilization of solar energy for proton translocation. Heterologous expressed ActR-13023 exhibited maximum absorption at 565 nm with practical proton pumping ability. Purified ActR-13023 could be reconstituted with actinobacterial carotenoids for additional light-harvesting. The existence of actinorhodopsin and its chromophore synthesis machinery in
indicates the inherent photo-energy conversion function of this microorganism. The assembly of ActR-13023 to its synthesized chromophores validated the microbial community's importance in the energy cycle.
•Gas removal process in HER under sonication was recorded by high-speed camera.•Bubbles experience fragmentation, collision and attraction under sonication.•Ultrasound reduces the critical size and ...residence time of bubbles in HER.•Ultrasound enables the faster removal of bubbles from the electrode surface.•Ultrasound improves water electrolysis efficiency.
In electrochemical processes, gas bubbles on the electrode can cause an increase in both overpotential and ohmic voltage drop which leads to higher energy consumption. Applying power ultrasound during water electrolysis can help to reduce the overpotential, enhance mass transfer, and save energy. In this study, we investigated the effect of ultrasound (20 kHz) on the hydrogen evolution reaction (HER) on a stainless steel plate with varying concentrations of NaOH solutions at 298 K, using linear sweep voltammetry (LSV). We especially focused on understanding the bubble behavior on the stainless steel plate during HER using high-speed imaging in ultrasonic field. When ultrasound was applied to solutions with NaOH concentrations of 0.1, 0.5, 1 M, the current density increased by about 9.0, 5.9, 2.8 %, respectively. As the ultrasound irradiation began, the bubbles tended to hover around on the electrode surface, coalescing with other bubbles, rather than rising. When the size of the coalesced bubbles became too large to stay on the surface of the electrode, they were expelled from the ultrasonic field. The repeated collapse and coalescence of these bubbles was observed while they were rising. The velocity increased about 2 times when ultrasound irradiation began, and increased by more than 6 times in the ultrasonic field. More nucleation of bubbles was observed on the electrode in the ultrasonic field. Using ultrasound reduced the critical diameter of bubbles which detached from the electrode, from 58.0 to 15.9 μm, and the residence time of the bubbles, from 533 to 118 ms. Further, when the ultrasound was applied, the mean diameter of bubbles decreased from 71.8 to 17 μm. Hence, bubble coverage on the electrode surface decreased from 8.3 to 1 % despite an increase in the total number of bubbles. As a result, ultrasound was found to be effective for hydrogen production during water electrolysis, increasing current by the faster removal of gas from the stainless steel plate.
Light quality is a significant factor for living organisms that have photosensory systems, such as rhodopsin, a seven alpha-helical transmembrane protein with the retinal chromophore. Here, we ...report, for the first time, the function of new rhodopsin, which is an inverted 7-transmembrane protein, isolated from Trichococcus flocculiformis
heliorhodopsin (TfHeR) works as a regulatory helper rhodopsin that binds with class 2 cyclobutane pyrimidine dimer (CPDII) photolyase to broaden the spectrum and upregulate DNA repair activity. We have confirmed their interaction through isothermal titration calorimetry (dissociation constant of 21.7 μM) and identified the charged residues for the interaction. Based on
and
experiments, we showed that the binding of heliorhodopsin with photolyase improved photolyase activity by about 3-fold to repair UV-caused DNA damage. Also, the DNA repair activity of TfHeR/
photolyase (TfPHR) was observed in the presence of green light. Our results suggested that heliorhodopsin directly controls the activity of photolyase and coevolves to broaden the activity spectrum by protein-protein interaction.
This study reports a function for Heliorhodopsin working as a regulatory helper rhodopsin that with CPDII photolyase to broaden the spectrum and upregulating the DNA repair activity. Our results suggested that heliorhodopsin directly controls photolyase activity and coevolves to broaden the DNA repair capacity by protein-protein interaction.
Abstract
In this study, we specifically visualized DNA molecules at their AT base pairs after in vitro phage ejection. Our AT-specific visualization revealed that either end of the DNA molecule could ...be ejected first with a nearly 50% probability. This observation challenges the generally accepted theory of Last In First Out (LIFO), which states that the end of the phage λ DNA that enters the capsid last during phage packaging is the first to be ejected, and that both ends of the DNA are unable to move within the extremely condensed phage capsid. To support our observations, we conducted computer simulations that revealed that both ends of the DNA molecule are randomized, resulting in the observed near 50% probability. Additionally, we found that the length of the ejected DNA by LIFO was consistently longer than that by First In First Out (FIFO) during in vitro phage ejection. Our simulations attributed this difference in length to the stiffness difference of the remaining DNA within the phage capsid. In conclusion, this study demonstrates that a DNA molecule within an extremely dense phage capsid exhibits a degree of mobility, allowing it to switch ends during ejection.
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