서강대학교

초록/요약

Microbial rhodopsin is a natural toolkit activated by light to perform different functions as a proton, ion translocation, photo-sensory, photo-enzymatic activity, and more to be explored with the recent discovery of heliorhodopsin. The photon is a vital energy source, and the photosystem of the plant is the well-known phototrophy apparatus on the earth. A much simpler photo-harvesting by all-trans-retinal as a single chromophore grabbed much attention for the microbial rhodopsin research and application. The first report of bacteriorhodopsin (BR) from halophilic Bacterium salinarum showed light-outward proton pumping, then follow by anion (chloride) pumping Halorhodopsin (HR) and Sensory rhodopsin (SRI and SRII) from the same bacteria. Rhodopsin is encoded by the opsin gene, which is found in all domains of life. Functional divergence of microbial was discovered, and sodium (Na+) pumping rhodopsin is fascinated for optogenetic application. Despite many discoveries of a new type of microbial rhodopsin, most of the cases were elucidated the protein function with a single all-trans-retinal as a chromophore. Xanthorhodopsin (XR) is named for dual chromophore rhodopsin, which assembles with carotenoid (salinixanthin) as a light-harvesting antenna was studied in Salinarum ruber. The confirmation of energy transfer from the carotenoid’s excited state to the retinal was studied. Xanthorhodopsin is a highly expressed gene in marine microbial community, yet the study of dual chromophore microbial rhodopsin is limited due to inexpressible of XR and salinixanthin. The reconstitution of salinixanthin and echinenone further studies the mystery of the carotenoid role in the retinylidene protein to homolog rhodopsin Gloeobacteria rhodopsin (GR) from cyanobacteria gloeobacter violaceus PCC 7421. Carotenoid binding characteristics and energy transfer from secondary chromophores have been studied. However, the input energy from carotenoids has not been verified experimentally in terms of functional improvement. Carotenoid is a well-known light excited molecule, the study of electron transfer and its role in the photosystem is well described. At the same time, carotenoid’s role in microbial rhodopsin is minimal. Here, this study provides insight into carotenoid’s role in microbial rhodopsin and the interaction of these seven helical membrane proteins toward carotenoid binding and selection. This thesis will be divided into four sections. First is discovering the natural existence of dual chromophore actinorhodopsin, which have a completed functional feature along with both chromophore retinal/carotenoid synthesized machinery in the same genome of actinobacteria: “Candidatus Aquiluna” sp. Strain IMCC13023 (ActRIMCC13023 actinorhodopsin). Exogenously added retinal and endogenously co-synthesized retinal and ActRIMCC13023 gene produce the same functional actinorhodopsin with maximum absorption at 565nm. Assembly of the dual chromophore to the ActRIMCC13023 is confirmed both In vivo and in vitro experiments, and dual chromophore rhodopsin showed significant improvement in light-outward proton pumping. In part two of this study, we introduce a new microbial rhodopsin expression system with secondary chromophore synthesized machinery. Canthaxanthin is successfully synthesized and bind to GR during protein expression and allows GR's purification with both chromophore retinal/canthaxanthin in short GRC. For the last 15 years, this is the first time we can prove that energy from canthaxanthin is transferred to retinal and improved proton pumping capacity up to 5 folds, and 3 times faster compared to GR in weak light condition. The binding of canthaxanthin also showed 126-folds heat tolerance and recovery better in drought stress. In part three, we describe the carotenoid binding pocket of GR: a case study of canthaxanthin bind to GR. We found that the carotenoid binding site is related to the retinal binding pocket with an interesting color tuning serines (CTS) with a compensated blue-red optical shifts. More importantly, our results proposed two important carotenoid binding motifs in microbial rhodopsin: (1) carotenoid locked motif (CLM) constructed by Gly178, Thr179, Thr182, and Phe185 residues on helix E that control the binding affinity of CAN, and (2) carotenoid aligned motif (CAM) consisted of Ser221, Pro226, and Ile227 residues on helix F, that preserve chiral retinal-carotenoid interaction to determine carotenoid interactive environment and energy transfer to the protein complex. The biological binding of CAN-GR showed a specific carotenoid-microbial rhodopsin characteristic, suggesting a precise binding partner is necessary. The carotenoid binding pocket of GR is like reported S. ruber XR, 3D simulation analysis of show docking spot near retinal beta-ring. Even with a similar binding pocket, we propose that microbial rhodopsin have carotenoid selectivity. In part four, we described biophysical study of GR binds to five different carotenoids suggested the perfect partner of one rhodopsin one carotenoid theory. The distinct structure of the carotenoid is recognized differently by the GR backbone, which yields different binding energy and associated constants. The five carotenoids are beta-carotene, canthaxanthin, echinenone, salinixanthin, and zeaxanthin. A 4-keto-ring of carotenoid is reported for an essential role in rhodopsin binding and energy transfer. We found a similar result where canthaxanthin, echinenone, and salinixanthin established a strong binding constant, while the weak and fragile binding is found in carotenoid without the 4-keto ring; beta-carotenoid and zeaxanthin. Among potent binding carotenoids, echinenone shows the fastest and highest flexibility fitted to the dynamic conformational change of the protein, while the other carotenoid lost its function during intense light illumination. So, GR's natural selection toward the carotenoid is based on binding constant, binding speed, and binding stability. Echinenone fastens light response by +119%, and proton yield by 250.8% compared to GR with single retinal. The full potential of this natural proton pump toolkit should be recognized for the energy cycle and further application. In the last part of this thesis, we discuss lessons to learn from the dual chromophore study and introduce three take-home ideas: (1). the possibility of other microbial rhodopsin that might employ carotenoid as a secondary chromophore, so their contribution toward the energy cycle would be a major solar energy capture on earth. Understanding their potential might to better policy and solutions in climate change response. (2). the carotenoid binding site allows the engineering of aromatic residues to maximize photo-harvesting and photo-damage tolerance. (3) the efficiency of light-driven ion translocation by dual chromophore microbial rhodopsin should further be studied for biotechnological application. In the last chapter, we discussed and proposed the significant microbial engineering method. One stone three birds engineered GR, we successfully engineer energy-efficient GR for better proton pumping and significantly improve fluorescence intensity from both spontaneous emission (non-reactive fluorescence) and reactive fluorescence. This engineering approach suggested a superior optogenetic tool to sense the voltage and visualize neurons. The interaction of carotenoid in microbial rhodopsin (wild-type and engineered variant) are also found and successfully co-expression across xanthorhodopsin clade with diverse function as a light-driven proton pump, anion (chloride pump), and cation (sodium pump). Finally, microbial rhodopsin, the simplest solar activated protein, is still holding many unique features, and unveiling those is a potential for protein-based tool in bio applications. We hope that introducing knowledge about carotenoid to the retinylidene protein and their natural selective fitness brings to light another feature in this protein family and benefits research and development shortly.