揭露法老嗜鹽鹼單胞菌氯視紫質中與氯離子相關之獨特光驅動質子運輸機制

Abstract

Natronomonas pharaonis 是一種嗜鹽古生菌，最早被分離於埃及鹼湖 （soda lake），生活於pH=11的極端鹼性環境中，擁有兩種菌式視紫質 （microbial rhodopsin）：氯視紫質 （halorhodopsin）與感受型視紫質第二型 （sensory rhodopsin II），其中氯視紫質已知是一種光驅動氯離子幫浦，可吸收光能並將氯離子運輸至胞內，以維持古生菌的細胞滲透壓。近年來在光遺傳學 （optogenetics）有廣泛的應用，可使神經細胞過極化抑制神經傳遞，亦可恢復受損的視覺。本實驗室先前發現，來自N. pharaonis的氯視紫質 （NpHR）可量測到獨特的氫離子訊號，因此進行了氯離子結合位附近重要胺基酸的點突變，發現無法傳遞氯離子的R123A、S130A、D252N等點突變蛋白質皆無法測得氫離子訊號，證明氫離子訊號與氯離子的運輸有緊密的關聯。本研究對此做更深入的分析，提出了胞內側氫離子循環的模型。氯視紫質受光激發之後會進行一連串的構型改變，稱之為光週期 （photocycle）：即HR→K→L→N→O→HR，先前的X光晶體繞射研究指出，在N state時穿膜區F與穿膜區C會構成胞內側水分子通道，由於該通道由非帶電胺基酸所組成，故氫離子可能協助帶負電的氯離子通過此疏水性通道，然而卻尚未有直接測量氫離子的實驗證實。本研究利用光化學電流裝置解開NpHR獨特的氫離子運輸機制，針對野生型WT-NpHR的實驗結果，證實胞內側氫離子循環的模型。接著更進一步針對氯視紫質的視黃醛結合袋 （retinal binding pocket）附近三個保守性色胺酸（tryptophan）設計點突變，發現靠近氯離子結合位的W127突變後無法測得氫離子訊號，且W229能協助視黃醛吸收紫外線波段，並加速光週期速率。研究的結論是NpHR中色胺酸在離子運輸與光能量傳遞上有關鍵的重要性，並提出氫離子結合位的可能模型。

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.