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標題: | 鎂離子對鹽方扁平古菌一功能未知視紫質 HwMR 光學特性之影響研究 Probing the Mg2+-dependent optical property changes of the functionally unknown Middle Rhodopsin from Haloquadratum walsbyi, HwMR |
作者: | Ling-Ning Ko 柯齡甯 |
指導教授: | 楊?伸(Chii-Shen Yang) |
關鍵字: | 鹽方扁平古菌 Haloquadratum walsbyi,Middle Rhodopsin,特徵吸收峰,光週期,鎂離子, Haloquadratum walsbyi,Middle Rhodopsin,maximum absorbance,photocycle kinetics,Mg2+, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | 嗜鹽古細菌 (haloarchaea) 如何生活於高光照、高鹽的極端環境中,表現在膜上的視紫質扮演了重要角色。依照生理功能可以區分為兩大類—光驅動離子幫浦 (light-driven ion pump) 與光趨性受體 (phototaxis receptors),前者如氫視紫質 (bacteriorhodopsin,BR) 與氯視紫質 (halorhodopsin,HR),後者像是感覺型視紫質(sensory rhodopsin,SR)。本研究所探討的鹽方扁平古菌 (Haloquadratum walsbyi, Hw) 可存活於其他嗜鹽古菌無法生存的2.0 M MgCl2環境中且擁有三種視紫質,光驅動離子幫浦 BR、HR 以及第三種功能未知的Middle Rhodopsin (MR),前人研究指出 MR 同時具有BR及SRII特有的保守關鍵胺基酸,且其生物化學特性介於兩者之間。比較先前對 Hw 上另外兩種視紫質的研究顯示,BR 具備高耐酸能力,HR 與氯離子的結合力強,那麼第三種 MR 是否也與某種離子具相關性?因H. walsbyi具有獨特的鎂離子高耐受性,故進行一系列特性分析探討其和鎂離子之關係。本研究在透過視紫質於不同離子濃度下,觀察其特徵吸收峰偏移程度,以評估是否對某一離子具專一性結合,並利用光週期驗證視紫質結構變化時,離子是否參與其中。實驗結果發現,Hw 的三種視紫質以及其他菌種的視紫質,只有 MR 在不同鎂、鈉離子濃度環境下,其特徵吸收峰顯著偏移;更進一步在光週期測定,發現只在不同鎂離子濃度下具有快慢的差異,此為突破性的發現,於是進一步挑選在MR上可能和鎂離子結合之胺基酸進行點突變,發現位於蛋白質胞外側的 D84 具有篩選可以進入蛋白質的離子種類,接近視黃醛口袋 (retinal binding pocket) 的 T216 以及位在蛋白質胞內側 D95則為可能的鎂離子專一性結合位,而鎂離子和MR結合之生理重要性則正在做更深入的研究。 In haloarchaeas, microbial rhodopsins (M-Rho) are vital proteins for solar energy capture, as well as seeking for optimal living environment. Based on different physiological functions and mechanisms, two types of M-Rho have been found: light-driven ion transporters (bacteriorhodopsin, BR and halorhodopsin, HR) and phototaxis receptors (sensory rhodopsin, SR). Haloquadratum walsbyi (Hw) survives a 2.0 M MgCl2 condition and three M-Rho were identified in its genome, namely light-driven ion transporters HwBR, HwHR, and the functionally unknown HwMR. Previous studies showed HwMR to have the conserved residues from both BR and SRII; it even possesses photochemical properties of both, too. Based on our previous findings that HwBR was acid-tolerant and HwHR had extremely high affinity to chloride. Since Hw cells were found to survive an otherwise harmful 2.0 M Mg2+ environment, the goal of this study was set to seek the specific ion species, including Mg2+, that associate with MR. Here, we present the maximum absorbance (Abs-max) variations of three Hw M-Rho proteins under different ion species and concentrations and ion-dependent photocycle kinetics were recorded. Our results showed only MR appeared to be Mg2+ and Na+ sensitive among three M-Rhos in Hw. Furthermore, only the photocycle kinetics of MR showed significant changes under Mg2+. In order to investigate the critical site(s) related to Mg2+ binding, we mutated several amino acids in MR, and concluded that D84 which locates on the extracellular half of protein contributed to ion selectivity; the T216 near the retinal binding pocket, as well as cytoplasmic side D95 might be the specific Mg2+ binding sites. Further physiological significance is under study. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/71252 |
DOI: | 10.6342/NTU201801864 |
全文授權: | 有償授權 |
顯示於系所單位: | 生化科技學系 |
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