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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 楊啓伸(Chii-Shen Yang) | |
| dc.contributor.author | Hsiang-Yu Wu | en |
| dc.contributor.author | 吳翔祐 | zh_TW |
| dc.date.accessioned | 2021-06-16T05:14:01Z | - |
| dc.date.available | 2021-01-01 | |
| dc.date.copyright | 2020-08-20 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-08-11 | |
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FEBS Lett, 2015. 589(10): p. 1067-1071. 31. Luecke, H., et al., Structural changes in bacteriorhodopsin during ion transport at 2 angstrom resolution. Science, 1999. 286(5438): p. 255-261. 32. Mak-Jurkauskas, M.L., et al., Energy transformations early in the bacteriorhodopsin photocycle revealed by DNP-enhanced solid-state NMR. Proc Natl Acad Sci U S A, 2008. 105(3): p. 883-888. 33. Matsuyama, T., et al., Covalent bond between ligand and receptor required for efficient activation in rhodopsin. J Biol Chem, 2010. 285(11): p. 8114-8121. 34. Mogi, T., T. Marti, and H.G. Khorana, Structure-function studies on bacteriorhodopsin. IX. Substitutions of tryptophan residues affect protein-retinal interactions in bacteriorhodopsin. J Biol Chem, 1989. 264(24): p. 14197-14201. 35. Mogi, T., et al., Aspartic acid substitutions affect proton translocation by bacteriorhodopsin. Proceedings of the National Academy of Sciences of the United States of America, 1988. 85(12): p. 4148-4152. 36. Mostafa, H.I., Effect of beta-particles on the retinal chromophore in bacteriorhodopsin of Halobacterium salinarium. Radiat Meas, 2004. 38(2): p. 217-225. 37. Oh, D., et al., Diversity of Haloquadratum and other haloarchaea in three, geographically distant, Australian saltern crystallizer ponds. Extremophiles, 2010. 14(2): p. 161-169. 38. Oren, A., et al., Haloarcula marismortui (Volcani) sp. nov., nom. rev., an extremely halophilic bacterium from the Dead Sea. Int J Syst Bacteriol, 1990. 40(2): p. 209-210. 39. Oren, A., et al., Haloarcula quadrata sp. nov., a square, motile archaeon isolated from a brine pool in Sinai (Egypt). Int J Syst Bacteriol, 1999. 49 Pt 3: p. 1149-1155. 40. Petkova, A.T., et al., Tryptophan interactions in bacteriorhodopsin: a heteronuclear solid-state NMR study. Biochemistry, 2002. 41(7): p. 2429-2437. 41. Pikuta, E.V., R.B. Hoover, and J. Tang, Microbial extremophiles at the limits of life. Crit Rev Microbiol, 2007. 33(3): p. 183-209. 42. Robertson, C.E., et al., Phylogenetic diversity and ecology of environmental Archaea. Curr Opin Microbiol, 2005. 8(6): p. 638-642. 43. Rothschild, K.J., et al., Vibrational spectroscopy of bacteriorhodopsin mutants: chromophore isomerization perturbs tryptophan-86. Biochemistry, 1989. 28(17): p. 7052-7059. 44. Schenkl, S., et al., Insights into excited-state and isomerization dynamics of bacteriorhodopsin from ultrafast transient UV absorption. Proc Natl Acad Sci U S A, 2006. 103(11): p. 4101-4106. 45. Shevchenko, V., et al., Crystal structure of Escherichia coli-expressed Haloarcula marismortui bacteriorhodopsin I in the trimeric form. PLoS One, 2014. 9(12): p. e112873. 46. Soliman, G.S.H. and H.G. Trüper, Halobacterium pharaonis sp. nov., a New, Extremely Haloalkaliphilic Archaebacterium with Low Magnesium Requirement. Zentralblatt für Bakteriologie Mikrobiologie und Hygiene: I. Abt. Originale C: Allgemeine, angewandte und ökologische Mikrobiologie, 1982. 3(2): p. 318-329. 47. Sublimi Saponetti, M., et al., Morphological and structural aspects of the extremely halophilic archaeon Haloquadratum walsbyi. PLoS One, 2011. 6(4): p. e18653. 48. Tamogami, J., et al., A tin oxide transparent electrode provides the means for rapid time-resolved pH measurements: application to photoinduced proton transfer of bacteriorhodopsin and proteorhodopsin. Photochem Photobiol, 2009. 85(2): p. 578-589. 49. Trivedi, S., O.P. Choudhary, and J. Gharu, Different proposed applications of bacteriorhodopsin. Recent Pat DNA Gene Seq, 2011. 5(1): p. 35-40. 50. Valentine, D.L., Adaptations to energy stress dictate the ecology and evolution of the Archaea. Nat Rev Microbiol, 2007. 5(4): p. 316-323. 51. van Stokkum, I.H.M. and R.H. Lozier, Target Analysis of the Bacteriorhodopsin Photocycle Using a Spectrotemporal Model. The Journal of Physical Chemistry B, 2002. 106(13): p. 3477-3485. 52. Zaremba-Niedzwiedzka, K., et al., Asgard archaea illuminate the origin of eukaryotic cellular complexity. Nature, 2017. 541(7637): p. 353-358 53. 陳筱儒 鹽方扁平古菌上氯視紫蛋白質光驅動離子傳遞之探討 台灣大學生化科技學系學位論文(2015) 54. 林宏軒 視黃醛結合袋中保守色胺酸點突變對兩種氫視紫質酸耐受性能力之不同影響 台灣大學生化科技學系學位論文 (2018) 55. 周蔚 死海嗜鹽方形古菌氫視紫質II上Arg80和Thr199胺基酸對酸耐受度重要性之研究 台灣大學生化科技學系學位論文 (2018) 56. 柯齡甯 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 台灣大學生化科技學系學位論文 (2018) | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/56050 | - |
| dc.description.abstract | 對嗜鹽古生菌而言，氫視紫質會受光激發並作為氫離子幫浦，製造胞內外質子濃度梯度，進而驅動ATP合成酶運轉，對生產ATP相當重要。因此，如何在幫浦打出質子後，維持其功能不受外界提升後的質子濃度回饋抑制，為其能否達到更高的能源效率的關鍵。在2015年本實驗室分類的一種新BR亞型qR，發現到此類BR可以承受較低的pH環境，而窩氏鹽方扁平古菌 (Haloquadratum walsbyi) 的BR HwBR為其一員，同研究中亦已解得結構。鑑於BR是製造胞內外質子梯度來間接協助合成ATP，因此，可以耐胞外酸也表示，製造胞內外質子濃度差的能力是增加的。在先前實驗結果，發現到HwBR有較高的酸耐受性 (acid-tolerance)，且透過與結構比較，初步發現到有兩個區域與此性質有關，分別是在胞外側的R82與T201形成的脫水元 (dehydron) 結構，及在視黃醛結合口袋 (retinal binding pocket) D2位置的W94。在本研究中藉由將此三位點突變為R82E、T201S、W94F組合為三、雙、單點突變，藉由測試其光化學性質並比較不同突變組合的差異，進而了解各點位對HwBR酸耐受性之重要性。在光譜的實驗結果中，發現到W94F之有無決定了最高吸收峰 (Ab-max) 的藍移與否。在光電流的測試中，組合三突變點位後，蛋白質所能耐受酸的能力會因而下降，在pH 5.8的環境下便無法對抗環境中質子濃度進而打出質子，了解到三位點之間有抗酸的協同作用產生；而降低pH後，發現W94F突變株的酸耐受性明顯降低。進一步測試光週期狀態，在基態 (G state) 的結果中，W94F突變會造成光週期後段拖尾的現象；M態 (M state) 測試中，得到未受改變的結果；O態 (O state) 亦有明顯的拖尾，顯示前述W94F對BR作用機制造成的影響主要發生於O態。最後，本研究總結W94能獨立調節HwBR酸耐受性，然而BR整體仍仰賴胞外側的R82-T201及視黃醛結合口袋的W94共同合作，才能達到其高酸耐受性。 | zh_TW |
| dc.description.abstract | Bacteriorhodopsin is an important membrane protein of halobacteria for its light-driven property, working as a proton pump and causing a proton gradient to make ATP by ATP synthase. Therefore, the ability for BR to make proton gradient is directly related to the property of BR maintaining its function in increased extracellular proton concentrate. The higher proton concentration it stands, the more efficiently ATPs are produced. However, only qR, a newly cluster of BR subtype classified in 2015, can tolerate higher proton concentration including HwBR, BR from Haloquadratum walsbyi. In the same study, the structure of HwBR have been resolved. In previous studies, it showed that HwBR have higher acid tolerance and two regions in HwBR structure might be related to this property. Respectively, two regions are R82 and T201 in extracellular side which forming dehydron, and W94 at retinal binding pocket D2 site. This study investigates how rhodopsin structure affects the acid-tolerance especially in HwBR by mutating amino acids in two domains as R82E, T201S and W94F, to combine different mutant sites and test photochemical properties. In spectum and photocycle test, we found W94F is the main character makes red-shift and cycle delay. On the other side, we see signal revesed at pH 5.8 in photocurrent test with triple mutant, and acid tolerance of mutant W94F decrease when downgrading pH. Further, conducting photocurrent test, we find that W94F makes G state and O state extending but M state. This experiment explains that W94F influence O state mainly. In this study, we conclude that the two regions in BR actually have their own functions of avoiding extracellular protons affect BR function as a proton pump, and still the acid-tolerance property needs cooperation of .different structural regions. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T05:14:01Z (GMT). No. of bitstreams: 1 U0001-2807202010533200.pdf: 3385345 bytes, checksum: 41455ad0881ad47c2a6c8795331a1009 (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | 致謝 II 摘要 III ABSTRACT IV 目錄 V 圖目錄 VII 表目錄 VIII 第一章 緒論 1 第一節 嗜鹽古生菌 1 1.1.1窩氏鹽方扁平古菌 2 第二節 微生物視紫蛋白質 3 1.2.1 菌式視紫質 5 1.2.2 質子傳遞機制 6 1.2.3 視黃醛結合口袋與命名 8 第三節 QR視紫質抗酸性與結構 11 1.3.1 胞外側脫水元點突變研究 14 1.3.2 視黃醛結合口袋點突變研究 15 第四節 研究動機與目的 16 第二章 實驗材料與方法 17 第一節 實驗材料與藥品 17 2.1.1 菌種 17 2.1.2 質體 17 2.1.3 酵素藥品 17 2.1.4 化合物藥品 17 第二節 實驗儀器與設備 19 2.2.1 核酸電泳設備 19 2.2.2 蛋白質電泳與轉印設備 19 2.2.3 離心機 19 2.2.4 光電流量測用儀器 19 2.2.5 光週期實驗用量測儀器 19 2.2.6 其它 20 第三節 實驗方法 21 2.3.1 HwBR 突變株之質體建構 21 2.3.2 HwBR野生型及突變株蛋白質表達與純化 23 2.3.3 蛋白質電泳與西方墨點法分析 25 2.3.4 特徵吸收波長光譜掃描 26 2.3.5 光電流訊號測試6 27 2.3.6 單波長光週期測試 28 第三章 結果 29 第一節 三點突變株對HWBR造成的劇烈變化 29 3.1.1 三突變與野生株之吸收光譜 29 3.1.3 三突變與野生株之視紫質光週期 31 第二節 各突變株與野生型之吸收光譜比較 32 3.2.1 於pH 5.8之吸收光譜結果 32 3.2.2 pH 5.8到pH 5之吸收光譜變化 32 第三節 各突變株與野生型之質子幫浦功能性測試 34 3.3.1 各突變株於pH 5.8之光電流測試 34 3.3.2 各突變株於pH 5之光電流測試 35 第四節 各突變株與野生型之光週期比較 37 3.4.1 各突變株於基態之光週期 37 3.4.2 各突變株於M中間態之光週期 40 3.4.3 各突變株於O中間態之光週期 42 第四章 總結與討論 44 第一節 D2位點對抗酸之重要性 44 第二節 脫水元對抗酸的重要性 45 第三節 兩區域間對抗酸性質的合作 47 第五章 未來展望 48 第六章 參考文獻 49 | |
| dc.language.iso | zh-TW | |
| dc.subject | 酸耐受性 | zh_TW |
| dc.subject | 脫水元 | zh_TW |
| dc.subject | 視黃醛結合口袋 | zh_TW |
| dc.subject | 窩氏方扁平古菌 | zh_TW |
| dc.subject | 氫視紫質 | zh_TW |
| dc.subject | Haloquadratum walsbyi | en |
| dc.subject | retinal binding pocket | en |
| dc.subject | dehydron | en |
| dc.subject | acid-tolerance | en |
| dc.subject | bacteriorhodopsin | en |
| dc.title | 比較兩關鍵結構區域對窩氏鹽方扁平古菌之HwBR其高抗酸性質之重要性 | zh_TW |
| dc.title | Compare the importance of two key structural regions conferring the high acid-tolerance property in HwBR from Haloquadratum walsbyi
| en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 梁博煌(Po-Huang Liang),徐駿森(Chun-Hua Hsu),李昆達(Kung-Ta Lee),吳亘承(Hsuan-Chen Wu) | |
| dc.subject.keyword | 窩氏方扁平古菌,氫視紫質,酸耐受性,脫水元,視黃醛結合口袋, | zh_TW |
| dc.subject.keyword | Haloquadratum walsbyi,bacteriorhodopsin,acid-tolerance,dehydron,retinal binding pocket, | en |
| dc.relation.page | 54 | |
| dc.identifier.doi | 10.6342/NTU202001951 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2020-08-12 | |
| 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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