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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 楊?伸(Chii-Shen Yang) | |
| dc.contributor.author | Kuang-Ting Liu | en |
| dc.contributor.author | 劉冠廷 | zh_TW |
| dc.date.accessioned | 2021-06-16T06:42:33Z | - |
| dc.date.available | 2017-04-15 | |
| dc.date.copyright | 2014-08-13 | |
| dc.date.issued | 2014 | |
| dc.date.submitted | 2014-07-29 | |
| dc.identifier.citation | 參考文獻
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Jung, Bacteriorhodopsin-like proteins of eubacteria and fungi: the extent of conservation of the haloarchaeal proton-pumping mechanism. Photochemical & Photobiological Sciences, 2006. 5(6): p. 538-546. 8. Tsunoda, S.P., et al., H+-Pumping Rhodopsin from the Marine Alga Acetabularia. Biophysical Journal, 2006. 91(4): p. 1471-1479. 9. Fuhrman, J.A., M.S. Schwalbach, and U. Stingl, Proteorhodopsins: an array of physiological roles? Nature Reviews Microbiology, 2008. 6(6): p. 488-494. 10. Lanyi, J.K. and S.P. Balashov, Xanthorhodopsin: a bacteriorhodopsin-like proton pump with a carotenoid antenna. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 2008. 1777(7): p. 684-688. 11. Hoff, W.D., K.-H. Jung, and J.L. Spudich, Molecular mechanism of photosignaling by archaeal sensory rhodopsins. Annual Review of Biophysics and Biomolecular Structure, 1997. 26(1): p. 223-258. 12. Spudich, J.L., The multitalented microbial sensory rhodopsins. Trends in Microbiology, 2006. 14(11): p. 480-487. 13. 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Spudich, Identification of a third rhodopsin-like pigment in phototactic Halobacterium halobium. Proceedings of the National Academy of Sciences, 1982. 79(20): p. 6250-6254. 19. Oesterhelt, D. and W. Stoeckenius, Rhodopsin-like protein from the purple membrane of Halobacterium halobium. Nature, 1971. 233(39): p. 149-152. 20. Dunn, R., et al., Structure-function studies on bacteriorhodopsin. I. Expression of the bacterio-opsin gene in Escherichia coli. Journal of Biological Chemistry, 1987. 262(19): p. 9246-9254. 21. Luecke, H., et al., Structure of bacteriorhodopsin at 1.55 A resolution. Journal of Molecular Biology, 1999. 291(4): p. 899-911. 22. Sasaki, J., et al., Conversion of bacteriorhodopsin into a chloride ion pump. Science(Washington), 1995. 269(5220): p. 73-75. 23. Sudo, Y. and J.L. Spudich, Three strategically placed hydrogen-bonding residues convert a proton pump into a sensory receptor. Proceedings of the National Academy of Sciences, 2006. 103(44): p. 16129-16134. 24. Hampp, N., Bacteriorhodopsin as a photochromic retinal protein for optical memories. Chemical Reviews, 2000. 100(5): p. 1755-1776. 25. Lanyi, J.K., Proton transfers in the bacteriorhodopsin photocycle. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 2006. 1757(8): p. 1012-1018. 26. Schobert, B., L.S. Brown, and J.K. Lanyi, Crystallographic structures of the M and N intermediates of bacteriorhodopsin: assembly of a hydrogen-bonded chain of water molecules between Asp-96 and the retinal Schiff base. Journal of Molecular Biology, 2003. 330(3): p. 553-570. 27. Rouhani, S., et al., Crystal structure of the D85S mutant of bacteriorhodopsin: model of an O-like photocycle intermediate. Journal of Molecular Biology, 2001. 313(3): p. 615-628. 28. Trivedi, S., O.P. Choudhary, and J. Gharu, Different proposed applications of bacteriorhodopsin. Recent Pat DNA Gene Seq, 2011. 5(1): p. 35-40. 29. Yi, H.-P., Establishment of an Indium tin oxide (ITO)-coated electrochemical device for monitoring the dynamic movements of ions and microbial rhodopsins Department of Biochemical Science and Technology College of Life Science National Taiwan University Master Thesis, 2013. 30. Liu, H.-Y., Characterization of two predicted bacteriorhodopsins in Haloquadratum walsbyi. Department of Biochemical Science and Technology College of Life Science National Taiwan University Master Thesis, 2012. 31. Javor, B., C. Requadt, and W. Stoeckenius, Box-shaped halophilic bacteria. Journal of Bacteriology, 1982. 151(3): p. 1532-1542. 32. Bolhuis, H., E.M.t. Poele, and F. Rodriguez‐Valera, Isolation and cultivation of Walsby's square archaeon. Environmental Microbiology, 2004. 6(12): p. 1287-1291. 33. Oren, A., S. Duker, and S. Ritter, The polar lipid composition of Walsby's square bacterium. FEMS Microbiology Letters, 1996. 138(2‐3): p. 135-140. 34. Ghai, R., et al., New abundant microbial groups in aquatic hypersaline environments. Scientific Reports, 2011. 1. 35. Bardavid, R.E. and A. Oren, Dihydroxyacetone metabolism in Salinibacter ruber and in Haloquadratum walsbyi. Extremophiles, 2008. 12(1): p. 125-131. 36. LoBasso, S., et al., Lipids of the ultra-thin square halophilic archaeon Haloquadratum walsbyi. Archaea, 1900. 2(3): p. 177-183. 37. Saponetti, M.S., et al., Morphological and structural aspects of the extremely halophilic archaeon Haloquadratum walsbyi. PLoS One, 2011. 6(4): p. e18653. 38. Zhou, P., et al., Genomic survey of sequence features for ultraviolet tolerance in Haloarchaea (family Halobacteriaceae). Genomics, 2007. 90(1): p. 103-109. 39. Chen, Y., et al., MUST: A system for identification of miniature inverted-repeat transposable elements and applications to Anabaena variabilis and Haloquadratum walsbyi. Gene, 2009. 436(1): p. 1-7. 40. Bolhuis, H., et al., The genome of the square archaeon Haloquadratum walsbyi: life at the limits of water activity. BMC Genomics, 2006. 7(1): p. 169. 41. Sudo, Y., et al., A microbial rhodopsin with a unique retinal composition shows both sensory rhodopsin II and bacteriorhodopsin-like properties. Journal of Biological Chemistry, 2011. 286(8): p. 5967-5976. 42. Lobasso, S., et al., Isolation of Squarebop I bacteriorhodopsin from biomass of coastal salterns. Protein Expression and Purification, 2012. 84(1): p. 73-79. 43. Hsieh, S.-Y., Development of a Protein-assisted Protein Overexpression System for Membrane Proteins. Department of Biochemical Science and Technology College of Life Science National Taiwan University Master Thesis, 2011. 44. The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrodinger, LLC. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/57347 | - |
| dc.description.abstract | 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。 | zh_TW |
| dc.description.abstract | 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. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T06:42:33Z (GMT). No. of bitstreams: 1 ntu-103-R01b22051-1.pdf: 3841095 bytes, checksum: 886b0dd5541f5822a411f9b4b2bb529a (MD5) Previous issue date: 2014 | en |
| dc.description.tableofcontents | 目錄
目錄 2 圖目錄 4 表目錄 6 摘要 7 Abstract 8 第一章 緒論 9 第一節 微生物視紫紅質 9 第二節 Hw上發現新型細菌視紫紅質HwMR 15 第三節 研究動機與目的 16 第二章 材料與方法 19 第一節 實驗材料與藥品 19 1.1 菌種 19 1.2 質體 19 1.3 藥品 20 第二節 實驗儀器與設備 21 2.1 核酸電泳設備 21 2.2 蛋白質電泳與轉印設備 21 2.3 離心機 21 2.4 氫離子幫浦實驗用儀器 21 2.5光電流量測用儀器 21 2.6光週期實驗用量測儀器 21 2.7 其他 22 第三節 實驗方法 23 3.1 HwMR系列突變之質體建構（構築質體來自本實驗室傅煦媛博士） 23 3.2 HwMR與其系列突變蛋白質表達與純化 24 3.2.1 目標膜蛋白質表達 24 3.2.2破菌與蛋白質粗萃取 25 3.2.3膜分離與回溶膜蛋白質 25 3.2.4 親和層析法純化目標蛋白質 25 3.3 蛋白質基本分析 26 3.3.1 氫離子幫浦活性測試 26 3.3.2 特徵吸收峰光譜鑑定 27 3.3.3 光電流訊號測試 28 3.3.4 可見光光譜光週期測試 28 第三章 結果與討論 30 第一節 以生物資訊軟體分析HwMR 30 3.1.1 將HwMR轉變為光趨動氫離子幫浦的策略 30 3.1.2 以生物資訊軟體預測基因HwMR穿膜區 31 第二節 用Escherichia coli C43(DE3)大量表達系列突變蛋白質 33 3.2.1 以大腸桿菌Escherichia coli C43(DE3)表達目標蛋白質 33 3.2.2 HwBR、HwMR與其系列突變蛋白質可見光與紫外光光譜分析 35 第三節 HwMR系列突變蛋白質功能性分析 37 3.3.1 光驅動全細胞氫離子幫浦活性測試的條件最佳化 37 3.3.2 全細胞氫離子幫浦活性測試 38 3.3.3 全細胞光電流訊號測試 45 3.3.4 蛋白質光電流訊號測試 47 3.3.5可見光光譜光週期測試 49 第四節 實驗結果討論 53 3.4.1 使用SWISS-MODEL預測結果，配合PyMOL分析突變位置的影響 53 第四章 結論 56 第五章 未來展望 57 參考文獻 58 圖目錄 圖 1：四種Halobacterium salinarum中發現的細菌視紫紅質以及配對的傳訊蛋白質 10 圖 2：Haloquadratum walsbyi在顯微鏡之下的型態 14 圖 3：在BR與SRII中被預測會形成氫鍵的兩胺基酸 16 圖 4：本研究之實驗流程圖 18 圖 5：電子顯微鏡下拍攝Escherichia coli C43(DE3) 19 圖 6：二階段聚合酶連鎖反應 (Two-step PCR) 進行突變位建構 23 圖 7：大腸桿菌突變株C43(DE3)勝任細胞轉型與擴大培養步驟 24 圖 8：目標蛋白質與Ni-NTA親和性樹脂層析管柱結合，流洗並收集目標膜蛋白質。 26 圖 9：重組質體轉型至大腸桿菌並小量液態培養 26 圖 10：氫離子幫浦活性測試系統配置示意圖 27 圖 11：光電流訊號測試 (Photocurrent) 設備架設示意圖 28 圖 12：可見光光譜光週期測試儀器示意圖 29 圖 13：七個MR與各BR的不同點進行點突變 31 圖 14：SOSUI (左) 與TMHMM (右) 分析HwMR皆為六個穿膜區的膜蛋白質 32 圖 15：SWISS-MODEL分析HwMR為七個穿膜區的膜蛋白質 32 圖 16：大腸桿菌Escherichia coli C43(DE3)的電子顯微鏡照片 33 圖 17：純化出的HwBR、HwMR與HwMR系列突變蛋白質 33 圖 18：HwMR系列突變蛋白質變性電泳圖 34 圖 19：HwBR、HwMR與HwMR系列突變蛋白質可見光光譜 35 圖 20：HwBR、HwMR、HwMR-T216A與HwMR-D84N-T216A突變蛋白質可見光光譜 36 圖 21：C43-pET21b-532 nm laser-1 W 圖22：C43-pET21b-532 nm laser-1 W (+CCCP) 37 圖 23: C43-pET21b-532 nm laser-0.5 W 圖24: C43-pET21b-532 nm laser-0.5 W (+CCCP) 38 圖 25: C43-HwBR-532 nm laser-0.5 W 圖26: C43-HwBR-532 nm laser-0.5 W (+CCCP) 38 圖 27：HwMR-WT氫離子幫浦活性測試 (加CCCP前後) 39 圖 28：HwMR-WT氫離子幫浦活性測試 (加CCCP前紅框) 39 圖 29：HwMR-WT氫離子幫浦活性測試 (加CCCP後紅框) 40 圖 30：HwMR-T216A氫離子幫浦活性測試 (加CCCP前後) 40 圖 31：HwMR-T216A氫離子幫浦活性測試 (加CCCP前紅框) 41 圖 32：HwMR-T216A氫離子幫浦活性測試 (加CCCP後紅框) 41 圖 33：全細胞氫離子幫浦活性測試 43 圖 34： HwMR-T216A、HwMR-D84N-T216A與HwMR光驅動氫離子幫浦活性 44 圖 35：全細胞光電流訊號測試 46 圖 36：蛋白質在不同波長的雷射光激發之下，所產生的光電流訊號 49 圖 37：蛋白質在飛梭雷射光激發之下，所產生的光週期訊號 51 圖 38：電腦軟體預測HwMR蛋白質的結構 53 圖 39：HwMR在突變前 (圖左) 及T216A突變後 (圖右) 的氫鍵結構示意圖 54 圖 40：HwMR、HwMR-T216A與HwMR-D84N-T216A蛋白質視覺顏色比較 55 圖 41：HwBR、HwMR-T216A與HwMR-D84N-T216A的氫離子運送示意圖 56 表目錄 表 1：存在於Haloquadratum walsbyi之三種視紫紅質。 15 表 2：本論文所使用之藥品清單 20 表 3：本研究進行定點突變所使用引子。 23 表 4：HwBR與HwMR相對於HsBR上D85N和D96N突變的性質測試 [30]。 30 表 5：不同的預測軟體所預測的HwMR穿膜區數目。 32 表 6：蛋白質純化產量。 34 表 7：各蛋白質的UV-Vis光譜特徵吸收峰峰值。 36 表 8：HwBR、HwMR與HwMR系列突變的酸鹼值改變量與比例 44 表 9：各蛋白質的光週期半生期比較 52 | |
| dc.language.iso | zh-TW | |
| dc.subject | 嗜鹽方扁平古菌 | zh_TW |
| dc.subject | Middle Rhodopsin | zh_TW |
| dc.subject | 細菌視紫紅質 | zh_TW |
| dc.subject | 氫離子幫浦 | zh_TW |
| dc.subject | Bacteriorhodopsin | en |
| dc.subject | Middle Rhodopsin | en |
| dc.subject | Haloquadratum walsbyi | en |
| dc.subject | Proton pump | en |
| dc.title | 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 | zh_TW |
| dc.title | Convert an unknown function microbial rhodopsin from Haloquadratum walsbyi into a light-driven proton pump | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 102-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 吳韋訥(Wailap Victor Ng),許瑞祥(Ruey-Shyang Hseu),楊健志(Chien-Chih Yang),林晉玄(Ching-Hsuan Lin) | |
| dc.subject.keyword | 細菌視紫紅質,Middle Rhodopsin,嗜鹽方扁平古菌,氫離子幫浦, | zh_TW |
| dc.subject.keyword | Bacteriorhodopsin,Middle Rhodopsin,Haloquadratum walsbyi,Proton pump, | en |
| dc.relation.page | 60 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2014-07-29 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| 顯示於系所單位： | 生化科技學系 | |
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