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標題: | 轉變鹽方扁平古菌中一未知功能感光蛋白質為似氫離子幫浦視紫紅質 Convert an unknown function microbial rhodopsin from Haloquadratum walsbyi into a light-driven proton pump |
作者: | Kuang-Ting Liu 劉冠廷 |
指導教授: | 楊?伸(Chii-Shen Yang) |
關鍵字: | 細菌視紫紅質,Middle Rhodopsin,嗜鹽方扁平古菌,氫離子幫浦, Bacteriorhodopsin,Middle Rhodopsin,Haloquadratum walsbyi,Proton pump, |
出版年 : | 2014 |
學位: | 碩士 |
摘要: | 視紫紅質在生物感光能力中扮演了重要的角色,其相關研究由1973年至今,在許多不同種嗜鹽古生菌中發現多種功能的視紫紅質,大約可分為光驅性離子幫浦和光感受器兩大類,細菌視紫紅質BR (Bacteriorhodopsin)即為前者,可將氫離子逆濃度梯度往細胞外運輸,造成氫離子濃度梯度,配合三磷酸線苷生成酶產生能量對抗嚴苛環境;後者SR (Sensory Rhodopsin)則控制細菌的光趨性行為,配合鞭毛運動使細菌在適合的光波長之下生長,並遠離有傷害的紫外線。嗜鹽古生菌Haloquadratum walsbyi在2006年基因體解碼後,被分析出有三種不同性質的視紫紅質HwBR、HwMR和HwHR,其中MR (Middle Rhodopsin) 其功能介於微生物視紫紅質BR (Bacteriorhodopsin) 與SRII (Sensory Rhodopsin II) 之間,雖然具有氫離子幫浦蛋白質的保守性關鍵胺基酸D85、D96,但是卻不具有光驅性氫離子幫浦活性,另一方面HwMR也具有光感受器蛋白質SRII的保守性關鍵胺基酸Y174和T204,但是該菌種卻沒有鞭毛。 本研究主旨在探討HwMR與微生物視紫紅質BR功能之間的轉換機制,經由胺基酸序列分析HwMR和其他物種的BR進行比較,透過點突變技術獲得系列突變蛋白。一雙點突變蛋白質HwMR-D84N-T216A的特徵吸收峰由原本的488 nm往長波長區域偏到550 nm,幾乎與BR的特徵吸收峰重疊且外觀同為紫色,再經光週期實驗、光電流測試與氫離子幫浦活性測試之後,發現該突變蛋白質的光週期加速一千倍而達到光驅動氫離子幫浦等級的速度,以高效率將氫離子運送進出蛋白質,但不能將氫離子送出細胞外,此突變使HwMR轉變成一高效率單邊氫離子循環幫浦蛋白質 ( high efficiency H+ single-sided circulation rhodopsin)。透過3D蛋白質結構模擬圖,得知HwMR-T216A的位置位於蛋白質中心視黃醛的旁邊,推測與該位置易形成氫鍵立體障礙有關,如果該阻礙消失,則有助於氫離子的釋出。本論文之實驗結果,可供往後感光蛋白質設計工程之參考。 The first microbial rhodopsins was found in Haloarchaea salinarum in 1973 and further studies have identified four kinds of retinal-binding proteins that use light as energy source to mediate different physiological functions. The two main functions are identified as light-driven ion pump and light-sensor for phototaxis system. The former include bacteriorhodopsin, which is a light-driven proton pump for proton gradient generation and later cooperates with ATP synthase to convert solar energy to biological consumable form. Three different microbial rhodopsins were proposed in Haloquadratum walsbyi. Among them, HwBR is proton pump, HwHR is halorhodopsin-like, but HwMR remained unclassified. After amino-acids alignments with HwBR, HwMR has many conserved residues suggesting it as a proton pump, but measurements showed it was merely a weak pump. The goal of this study is focusing on finding residues that can rebuild proton-pump activity on HwMR. After sequence analysis and mutagenesis, a D84N-T216A-HwMR double mutant appeared to significant speed up the photocycle. The maximan absorbance of HwMR-D84N-T216A was red-shifted to 550 nm; it is closer to bacteriorhodopsins. Based on photocurrent measurements and proton pump activity measurements, this mutant protein has converted into a high efficiency H+ single-sided circulation rhodopsin. According to 3D structure prediction, T216 locates right next to retinal-binding pocket, and it is a position known to form a hydrogen bond in SRII but not in BR. This study concluded a conversion of HwMR to either sensory- or ion-type is possible. Conclusions from this thesis can serve as technological refernces for further protein engineering in microbial rhodopsins. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/57347 |
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顯示於系所單位: | 生化科技學系 |
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