Elucidate the molecular mechanism of light driven chloride pumping-related unique proton signal in halorhodopsin from Natronomonas pharaonis

• Main Authors: Cheng-Hong Tu, 杜承宏
• Other Authors: Chii-Shen Yang
• Format: Others
• Language: zh-TW
• Published: 2019
• Online Access: http://ndltd.ncl.edu.tw/handle/37cdh5

碩士 === 國立臺灣大學 === 生化科技學系 === 107 === Natronomonas pharaonis belongs to the order of Halobacteriales, and was first isolated from soda lakes. To survive, N. pharaonis has to cope with extreme conditions of high salt and an alkaline pH of 11. In its two microbial rhodopsins, halorhodopsin (NpHR) functions as light-driven inward chloride pump, and NpSRII serves as light-sensing photophobic responsor. Halorhodopsin is believed to maintain osmolarity and generate PMF and it is also widely used in the field of optogenetics to silence nerve activity. Previous mutagenesis study showed a unique proton releasing signal closely related to chloride transport. In that study, a model of intracellular-side proton circulation was proposed. Among the photocycle of NpHR: HR→K→L→N→O→HR, the N state forms an intracellular water channel by its transmembrane helix F and C to facilitate chloride release. Since the water channel was composed of non-charged residues, it is postulated that a proton should facilitate the release of chloride ion in the form of HCl. However it lacks direct experimental proof. In this study, we first demonstrate detailed analysis about the unique proton signal of wild type NpHR under different environment, verifying the intracellular proton circulation model. On the other hand, we are also interested in how tryptophans in the retinal binding pocket help retinal re-isomerize. By mutagenesis study, we distinguished W127 in NpHR is one of the critical residues for proton signal, and thus propose a proton binding site model in the water cluster next to chloride binding site.