Heliorhodopsin-mediated light-modulation of ABC transporter.

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.

Carotenoid binding in Gloeobacteria rhodopsin provides insights into divergent evolution of xanthorhodopsin types.

The position of carotenoid in xanthorhodopsin has been elucidated. However, a challenging expression of this opsin and a complex biosynthesis carotenoid in the laboratory hold back the insightful study of this rhodopsin. Here, we demonstrated co-expression of the xanthorhodopsin type isolated from Gloeobacter violaceus PCC 7421-Gloeobacter rhodopsin (GR) with a biosynthesized keto-carotenoid (canthaxanthin) targeting the carotenoid binding site. Direct mutation-induced changes in carotenoid-rhodopsin interaction revealed three crucial features: (1) carotenoid locked motif (CLM), (2) carotenoid aligned motif (CAM), and color tuning serines (CTS). Our single mutation results at 178 position (G178W) confirmed inhibition of carotenoid binding; however, the mutants showed better stability and proton pumping, which was also observed in the case of carotenoid binding characteristics. These effects demonstrated an adaptation of microbial rhodopsin that diverges from carotenoid harboring, along with expression in the dinoflagellate Pyrocystis lunula rhodopsin and the evolutionary substitution model. The study highlights a critical position of the carotenoid binding site, which significantly allows another protein engineering approach in the microbial rhodopsin family.

Discovery of a new light-driven Li+/Na+-pumping rhodopsin with DTG motif.

Microbial pumping rhodopsin is a seven-transmembrane retinal binding protein, which is light-driven ion pump with a functional key motif. Ion-pumping with the key motif and charged amino acids in the rhodopsin is biochemically important. The rhodopsins with DTG motif have been discovered in various eubacteria, and they function as H+ pump. Especially, the DTG motif rhodopsins transported H+ despite the replacement of a proton donor by Gly. We investigated Methylobacterium populi rhodopsin (MpR) in one of the DTG motif rhodopsin clades. To determine which ions the MpR transport, we tested with various monovalent ion solutions and determined that MpR transports Li+/Na+. By replacing the three negatively charged residues residues which are located in helix B, Glu32, Glu33, and Asp35, we concluded that the residues play a critical role in the transport of Li+/Na+. The MpR E33Q transported H+ in place of Li+/Na+, suggesting that Glu33 is a Li+/Na+ binding site on the cytoplasmic side. Gly93 in MpR was replaced by Asp to convert from the Li+/Na+ pump to the H+ pump, resulting in MpR G93D transporting H+. Dissociation constant (Kd) values of Na+ for MpR WT and E33Q were determined to be 4.0 and 72.5 mM, respectively. These results indicated the mechanism by which MpR E33Q transports H+. Up to now, various ion-pumping rhodopsins have been discovered, and Li+/Na+-pumping rhodopsins were only found in the NDQ motif in NaR. Here, we report a new light-driven Na+ pump MpR and have determined the important residues required for Li+/Na+-pumping different from previously known NaR.

The role of carotenoids in proton-pumping rhodopsin as a primitive solar energy conversion system.

Rhodopsin and carotenoids are two molecules that certain bacteria use to absorb and utilize light. Type I rhodopsin, the simplest active proton transporter, converts light energy into an electrochemical potential. Light produces a proton gradient, which is known as the proton motive force across the cell membrane. Some carotenoids are involved in light absorbance and transfer of absorbed energy to chlorophyll during photosynthesis. A previous study in Salinibacter ruber has shown that carotenoids act as antennae to harvest light and transfer energy to retinal in xanthorhodopsin (XR). Here, we describe the role of canthaxanthin (CAN), a carotenoid, as an antenna for Gloeobacter rhodopsin (GR). The non-covalent complex formed by the interaction between CAN and GR doubled the proton pumping speed and improved the pumping capacity by 1.5-fold. The complex also tripled the proton pumping speed and improved the pumping capacity by 5-fold in the presence of strong and weak light, respectively. Interestingly, when canthaxanthin was bound to Gloeobacter rhodopsin, it showed a 126-fold increase in heat resistance, and it survived better under drought conditions than Gloeobacter rhodopsin. The results suggest direct complementation of Gloeobacter rhodopsin with a carotenoid for primitive solar energy harvesting in cyanobacteria.

Discovery of a microbial rhodopsin that is the most stable in extreme environments.

Microbial rhodopsin is a retinal protein that functions as an ion pump, channel, and sensory transducer, as well as a light sensor, as in biosensors and biochips. Tara76 rhodopsin is a typical proton-pumping rhodopsin that exhibits strong stability against extreme pH, detergent, temperature, salt stress, and dehydration stress and even under dual and triple conditions. Tara76 rhodopsin has a thermal stability approximately 20 times higher than that of thermal rhodopsin at 80°C and is even stable at 85°C. Tara76 rhodopsin is also stable at pH 0.02 to 13 and exhibits strong resistance in detergent, including Triton X-100 and SDS. We tested the current flow that electrical current flow across dried proteins on the paper at high temperatures using an electrode device, which was measured stably from 25°C up to 120°C. These properties suggest that this Tara76 rhodopsin is suitable for many applications in the fields of bioengineering and biotechnology.

Assembly of Natively Synthesized Dual Chromophores Into Functional Actinorhodopsin.

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. Actinobacteria is one of the highly abundant bacterial phyla in freshwater habitats, and members of this lineage are considered to boost heterotrophic growth via 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 Actinobacteria are not consistently cultivable. Based on the published genome sequence of Candidatus aquiluna 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 Actinobacteria 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.