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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 楊啟伸 | |
| dc.contributor.author | Xiao-Ru Chen | en |
| dc.contributor.author | 陳筱儒 | zh_TW |
| dc.date.accessioned | 2021-06-16T02:34:44Z | - |
| dc.date.available | 2016-07-30 | |
| dc.date.copyright | 2015-07-30 | |
| dc.date.issued | 2015 | |
| dc.date.submitted | 2015-07-28 | |
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Sequencing of bacterial and archaeal 16S rRNA genes directly amplified from a hypersaline environment. Systematic and applied microbiology 18, 574-581 (1995). 61 Ghai, R. et al. New abundant microbial groups in aquatic hypersaline environments. Scientific reports 1 (2011). 62 Burns, D. G., Camakaris, H. M., Janssen, P. H. & Dyall-Smith, M. L. Cultivation of Walsby's square haloarchaeon. FEMS microbiology letters 238, 469-473 (2004). 63 Bolhuis, H., Poele, E. M. & Rodriguez-Valera, F. Isolation and cultivation of Walsby's square archaeon. Environmental microbiology 6, 1287-1291 (2004). 64 Bolhuis, H. et al. The genome of the square archaeon Haloquadratum walsbyi : life at the limits of water activity. BMC genomics 7, 169 (2006). 65 Oren, A. The order halobacteriales. The prokaryotes ,113-164 ( 2006). 66 Tamogami, J., Kikukawa, T., Miyauchi, S., Muneyuki, E. & Kamo, N. A tin oxide transparent electrode provides the means for rapid time-resolved pH measurements: application to photoinduced proton transfer of bacteriorhodopsin and proteorhodopsin. Photochemistry and photobiology 85, 578-589 (2009). 67 Lanyi, J. K. & Schobert, B. Effects of chloride and pH on the chromophore and photochemical cycling of halorhodopsin. Biochemistry 22, 2763-2769 (1983). 68 Kanada, S., Takeguchi, Y., Murakami, M., Ihara, K. & Kouyama, T. Crystal structures of an O-like blue form and an anion-free yellow form of pharaonis halorhodopsin. Journal of molecular biology 413, 162-176 (2011). 69 Yamashita, Y. et al. Expression of salinarum halorhodopsin in Escherichia coli cells: solubilization in the presence of retinal yields the natural state. Biochimica et biophysica acta 1808, 2905-2912 (2011). 70 Yoshizawa, S. et al. Functional characterization of flavobacteria rhodopsins reveals a unique class of light-driven chloride pump in bacteria. 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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/53960 | - |
| dc.description.abstract | 嗜鹽古細菌 (haloarchaea) 生存在高鹽的嚴苛環境中，菌體中存在許多蛋白質協助抵抗極端環境，其中一類為微生物視紫蛋白質(Microbial rhodopsin)，當受到光刺激後會有不同的功能，主要可分為兩大類，光驅動離子幫浦及光感受器。在光驅動離子幫浦中，其中一種為氯視紫蛋白質 (halorhodopsin, HR)，目前已知是一種光驅動氯離子幫浦，可以維持古生菌的細胞滲透壓。當受光刺激後，氯離子會由胞外送至胞內，氫離子被動運輸至細胞內。在本研究中，透過對於氫離子敏感的光電流測試，進一步測量光驅動氫離子釋出的活性。結果發現當 Halobacterium salinarum (Hs) 及 Haloarcula marismortui (Hm) 上的氯視紫蛋白質 (HR) 以 E.coli 表現時，質子的確被動進入膜內。但是若將蛋白質純化後，以蛋白質溶液進行光電流測試，照光前後並不會產生質子梯度。然而，由結果發現在 Haloquadratum walsbyi 中的氯視紫蛋白質 (HwHR) 有別於 HsHR 及 HmHR，無論是以表現於 E.coli 的菌液或是純化後的蛋白質溶液，進行光電流測試，皆有質子梯度的產生。為了找出在 HwHR 中，與細菌視紫蛋白質 (bacteriorhodopsin) 相似的質子幫浦活性所涉及的胺基酸，找出帶負電胺基酸進行點突變後，發現 D254N 質子釋出的活性消失。D254N-HwHR在特徵吸收波長上往短波長移動 15 nm，光週期則比 wild type 慢 6 倍。綜合上述結果， Asp254 對於光驅動質子釋出及視黃醛重新異構化的效率上扮演重要角色。 | zh_TW |
| dc.description.abstract | Microbial rhodopsins response to light and function as light-driven ion transportation or light sensor. Halorhodopsin (HR), one of these microbial rhodopsins, is known to be a light-driven inward chloride pump for osmolarity maintenance at least in haloarchaea. During inward transportation, chloride ions are proposed to passively carry protons across the membrane. In this study, we used a proton sensitive assay, photocurrent measurement, to measure light-driven proton releasing activity. Here we found halorhodopsin in Halobacterium salinarum (HsHR) and Haloarcula marismortui (HmHR), the protons indeed passively transported across the membrane when they were expressed in E. coli cells, but in the purified protein level, no light-driven photocurrent was recorded, indicating no proton gradients were formed inside and outside protein upon illumination. However, halorhodopsin in Haloquadratum walsbyi (HwHR) generated positive in both cell-based and protein photocurrent measurement. In order to find the residue(s) in HwHR mediating such bacteriorhodopsin-like proton activity, we mutated several negative residues and found D254N eliminate such activity in both cell-based and protein experiments. D254N-HwHR underwent a 15-nm blue-shifted in maximum absorbance and the recovery time of photocycle was ~6 time slower when compared to wild type. According to these results, we conclude Asp254 as the critical residue for light-driven proton releasing and the efficiency of retinal reisomerlization. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T02:34:44Z (GMT). No. of bitstreams: 1 ntu-104-R02b22052-1.pdf: 3618018 bytes, checksum: df769912076ea36ace8c7bf085662500 (MD5) Previous issue date: 2015 | en |
| dc.description.tableofcontents | 目錄 i
圖目錄 iv 表目錄 vii 摘要 viii Abstract ix 第一章 緒論 1 第一節 嗜鹽古生菌 1 第二節 微生物視紫蛋白質(Microbial Rhodopsins) 2 第三節 氯視紫蛋白質 (Halorhodopsin) 5 3.1 蛋白質結構 5 3.2 光週期 (Photocycle) 及傳遞機制 7 3.3 HR 的功能性測試 8 3.4 HR與 BR 傳遞機制的差異 9 3.5 氯離子結合與否對於氯視紫蛋白質的影響 10 3.6 Halorhodopsin 的應用 10 第四節 Haloquadratum walsbyi 12 第五節 研究動機與目的 13 第二章 材料與方法 15 第一節 實驗材料與藥品 15 1.1 菌種 15 1.2 質體 15 1.3 藥品 15 第二節 實驗儀器與設備 17 2.1 核酸電泳設備 17 2.2 蛋白質電泳與轉印設備 17 2.3 離心機 17 2.4 氫離子幫浦實驗用儀器 17 2.5 光電流量測用儀器 17 2.6 光週期實驗用量測儀器 17 2.7 其他 18 第三節 實驗方法 19 3.1 生物資訊分析 19 3.2 HwHR 突變株之質體建構 19 3.3 HwHR及其突變蛋白質表達與純化 21 3.4 蛋白質電泳與西方墨點法分析 23 3.5 蛋白質功能性分析 24 第三章 結果與討論 26 第一節 氯視紫蛋白質是光驅動氫離子幫浦 26 1.1 HwHR 蛋白質溶液光電流測試 26 1.2 HwHR 將氫離子由胞外主動運輸至胞內 26 第二節 氯視紫蛋白質在不同 pH 之下的特性分析 28 2.1 HwHR 釋出氫離子的效率會受外界環境 pH 值影響 28 2.2 HwHR 在不同 pH 值下光週期的變化 29 2.3 HwHR 在不同 pH 值之下特徵吸收波長的改變 30 第三節 氯視紫蛋白質氫離子與氯離子運送的關聯性 31 3.1 HwHR在不含氯離子下全細胞光誘導氫離子釋出活性測試 31 3.2 HwHR蛋白質溶液在不含氯離子下光電流測試 31 第四節 HwHR 在氯離子去除下的特徵吸收波長變化 33 第五節 HwHR 離子傳遞的關鍵胺基酸 34 5.1 HwHR 點突變建構 34 5.2 HwHR 點突變全細胞氫離子幫浦活性測試 36 5.3 HwHR 點突變全細胞光電流測試 38 5.4 HwHR 點突變蛋白質溶液光電流測試 40 5.5 HwHR 點突變蛋白質溶液在不同 pH 下的光電流測試 41 第六節 Asp254 對氯視紫蛋白質光譜性質之影響 43 6.1 特徵吸收波長 43 6.2 不同 pH 的環境對於特徵吸收波長的影響 45 6.3 不同氯離子濃度對於特徵吸收波長的影響 47 第七節 氯視紫蛋白質的光週期分析 49 7.1 HwHR 點突變後的光週期 49 7.2 HwHR 點突變在不同 pH 下的光週期 50 第八節 HwHR 生物資訊分析 51 8.1 HwHR 序列分析 51 8.2 HwHR 結構預測 52 第四章 總結 54 第五章 未來展望 55 參考文獻 58 | |
| dc.language.iso | zh-TW | |
| dc.subject | 嗜鹽古細菌 | zh_TW |
| dc.subject | 光週期 | zh_TW |
| dc.subject | 氯視紫蛋白質 | zh_TW |
| dc.subject | 微生物視紫蛋白質 | zh_TW |
| dc.subject | halorhodopsin | en |
| dc.subject | photocycle | en |
| dc.subject | microbial rhodopsin | en |
| dc.subject | haloarchaea | en |
| dc.title | 鹽方扁平古菌上氯視紫蛋白質光驅動離子傳遞能力之探討 | zh_TW |
| dc.title | Investigation on light-driven ion translocation capability of halorhodopsin from Haloquadratum walsbyi | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 103-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 梁博煌,許瑞祥,楊健志,林晉玄 | |
| dc.subject.keyword | 嗜鹽古細菌,微生物視紫蛋白質,氯視紫蛋白質,光週期, | zh_TW |
| dc.subject.keyword | haloarchaea,microbial rhodopsin,halorhodopsin,photocycle, | en |
| dc.relation.page | 63 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2015-07-28 | |
| 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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