서강대학교

초록

Microbial rhodopsins are photochemical reactive proteins that are composed of a 7-transmembrane alpha-helices and they contain retinal (vitamin A aldehyde) as a chromophore. This photoreceptive membrane protein consists of seven transmembrane α helices, with a conserved lysine residue in the seventh helix G bound to retinal via a Schiff Base (SB). Bacteriorhodopsin (BR) is a light-driven outward proton (H+) pump discovered in Halobacterium salinarum in 1971. Other forms of microbial rhodopsin, such as the inward chloride (Cl-) pump, outward sodium (Na+) pump, internal H+ pump, light-gated cation, and anion channels, and the enzyme rhodopsin, have since been discovered. The adaptive light absorption spectra shift of microbial rhodopsin depending on the chromophores in opsin and the wide range of the absorption spectrum of rhodopsin have been discovered and genetically engineered. The chromophore of microbial rhodopsin forms a photo-intermediate through light, thereby forming various photocycles related to the function of each rhodopsin. In addition, many studies are being reported as optogenetic tools through protein engineering for application. Here, this study provides insight photochemical and photophysical properties of microbial rhodopsin with chromophores and the interaction of these seven helical membrane proteins toward carotenoid forming secondary chromophore. And additionally reports a study on a new type of heliohodopsin with unknown functions, indicating that microbial rhodopsins have a variety of biological functions. This thesis will be divided into five sections. The chapter 2 is a study on a new site for spectral tuning through the substitution of main residues that have an influence on the absorption wavelength of the chromophore. The spectral tuned microbial rhodopsins can be applied as an optogenetic tool. This study reports a new site for spectral tuning. A single amino-acid substitution of Cys189 caused the absorption maximum shift of 44 nm indicating a specific site for spectral tuning. We performed a single substitution comparison through photochemical and photobiological approaches. We also measured the maximum absorption for red shift through mutations at positions 189 and 105 in ISR34. It suggests that structural changes by amino acids are related to pKa values, pumping activity, and spectral tuning. The chapter 3 is a study on the most stable microbial rhodopsin through photochemical and photophysical analysis of the stability of the chromophore. Tara76 rhodopsin is a typical proton pump rhodopsin with a λmax of 489 nm that was isolated from 76 stations of the Tara ocean expedition. This rhodopsin exhibits strong stability against extreme pH, detergent, temperature, salt stress, and dehydration stress. It also exhibits strong stability in dual and triple conditions and much stronger stability than other microbial rhodopsins. Tara76 rhodopsin has a thermal stability approximately 20-times 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 at high temperature 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. In chapter 4, mutation studies of GR to the carotenoid interaction revealed the strong carotenoid-rhodopsin binding motifs and provided a vivid example of structural-based insights into the evolution of rhodopsin, suggesting the diverged journey from carotenoid binding. 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 This study has provided another approach toward protein engineering beyond natural adaptation. In chapter 5, it is a study 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. In the last chapter, among the ion-pumping rhodopsins, the most abundant cluster of functions belong to a new type of heliohodopsin with unknown functions, indicating that microbial rhodopsins have a variety of biological functions. In this study, a helio-opsin gene found in Trichococcus flocculiformis, a gram-positive bacterium isolated from bulking sludge, we report the biological function of T. flocculiformis heliorhodopsin (TfHeR) relationship with CPDII Photolyase among several candidates located in the same operon where the helio-opsin gene is located. TfHeR is working as a regulatory helper rhodopsin that binding with CPDII Photolyase to broaden the spectrum and upregulating the DNA repair activity.