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| ???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
|---|---|---|
| dc.contributor.advisor | 楊?伸(Chii-Shen Yang) | |
| dc.contributor.author | Chun-Jie Cai | en |
| dc.contributor.author | 蔡存傑 | zh_TW |
| dc.date.accessioned | 2021-06-16T06:35:19Z | - |
| dc.date.available | 2017-09-03 | |
| dc.date.copyright | 2014-09-03 | |
| dc.date.issued | 2014 | |
| dc.date.submitted | 2014-08-01 | |
| dc.identifier.citation | 1 Pfluger, K. & Muller, V. Transport of compatible solutes in extremophiles. J. Bioenerg. Biomembr. 36, 17 (2004).
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Identification of a third rhodopsin-like pigment in phototactic Halobacterium halobium. Proc. Natl. Acad. Sci. U. S. A. 79, 6250-6254 (1982). 29 Mironovaa, O. S. et al. Functional characterization of sensory rhodopsin II from Halobacterium salinarum expressed in Escherichia coli. FEBS Lett. 579, 3147-3151 (2005). 30 Spudich, J. L. & A., B. R. Mechanism of colour discrimination by a bacterial sensory rhodopsin. Nature 312, 509-513 (1984). 31 Takahashi, T., Mochizuki, Y., Kamo, N. & Kobatake, Y. Evidence that the long-lifetime photointermediate of s-rhodopsin is a receptor for negative phototaxis in halobacterium halobium. Biochem. Biophys. Res. Commun. 127, 99-105 (1985). 32 Fog, A. & Keran, W. R. Electronic semiconducting oxides as pH sensors. Sensor. Actuator. 5, 137-144 (1984). 33 Chang, Y.-N. 由新發現於 Haloacula marismortui 與 Haloquadratum walsbyi 之氯視紫質研究揭露異於其他微生物中離子運送蛋白質之高度保守性. 國立臺灣大學生命科學院生化科技學系 (2010). 34 Grzesiek, S. & Dencher, N. A. 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Sci. 269, 81-87 (2013). 39 Chu, L.-K., Yen, C.-W. & El-Sayed, M. A. Bacteriorhodopsin-based photo-electrochemical cell. Biosens. Bioelectron. 26, 620-626 (2010). 40 Walsby, A. E. A square bacterium. Nature 283, 69-71 (1980). 41 Oren, A., Duker, S. & Ritter, S. The polar lipid composition of Walsby's square bacterium. FEMS Microbiol. Lett. 138, 135-140 (1996). 42 Burns, D. G., Camakaris, H. M., Janssen, P. H. & Dyall-Smith, M. L. Cultivation of Walsby's square haloarchaeon. FEMS Microbiol. Lett. 238, 469-473 (2004). 43 Bolhuis, H., Poele, E. M. t. & Rodriguez-Valera, F. Isolation and cultivation of Walsby's square archaeon. Environ. Microbiol. 6, 1287-1291 (2004). 44 Fu, H.-Y., Chang, Y.-N., Jheng, M.-J. & Yang, C.-S. Ser262 determines the chloride-dependent colour tuning of a new halorhodopsin from Haloquadratum walsbyi. Biosci. Rep. 32, 501-509 (2012). 45 Bolhuis, H. et al. The genome of the square archaeon Haloquadratum walsbyi: life at the limits of water activity. BMC Genomics 7 (2006). 46 Lobasso, S. et al. The light-activated proton pump bop I of the archaeon Haloquadratum walsbyi. Photochem. Photobiol. 88, 690-700 (2012). 47 Sudo, Y. et al. A microbial rhodopsin with a unique retinal composition shows both sensory rhodopsin II and bacteriorhodopsin-like properties. J. Biol. Chem. 286, 5967-5976 (2011). 48 Fu, H.-Y. 視紫紅質在嗜鹽古細菌中的各式面貌. 國立臺灣大學生命科學院生化科技學系 (2013). 49 Yi, H.-P. 建立氧化銦錫 (ITO) 電化裝置偵測帶電分子與微生物視紫紅質之交互作用. 國立臺灣大學生命科學院生化科技學系 (2013). 50 Fu, H.-Y., Yi, H.-P., Lu, Y.-H. & Yang, C.-S. Insight into a single halobacterium using a dual-bacteriorhodopsin system with different functionally optimized pH ranges to cope with periplasmic pH changes associated with continuous light illumination. Mol. Microbiol. 88, 551-561 (2013). 51 Liu, H.-Y. 鹽方扁平古菌中兩種被預測為細菌視紫紅質之特性研究. 國立臺灣大學生命科學院生化科技學系 (2012). 52 Robertson, B. & Lukashev, E. P. Rapid pH change due to bacteriorhodopsin measured with a tin-oxide electrode. Biophys. J. 68, 1507-1517 (1995). 53 Good, N. E. et al. Hydrogen ion buffers for bifological research. Biochemistry 5, 467-477 (1966). 54 Schrodinger, LLC. The PyMOL Molecular Graphics System, Version 1.3r1 (2010). 55 Oren, A., Pri-El, N., Shapiro, O. & Siboni, N. Buoyancy studies in natural communities of square gas-vacuolate archaea in saltern crystallizer ponds. Saline Systems 2 (2006). 56 Bolhuis, H. Walsby's square archaeon; it's hip to be square, but even more hip to be culturable. N. Gunde-Cimerman et al. (eds.), Adaptation to Life at High Salt Concentrations in Archaea,Bacteria, and Eukarya, Netherlands. 185-199 (2005). 57 Yamaguchi, S. & Tahara, T. Two-photon absorption spectrum of all-trans retinal. Chem. Phys. Lett. 376, 237-243 (2003). 58 Wu, J. et al. Efficient approach to determine the pKa of the proton release complex in the photocycle of retinal proteins. J. Phys. Chem. B 113, 4482-4491 (2009). 59 Turner, G. J. et al. Bacteriorhodopsin D85N: Three spectroscopic species in equilibrium. Biochemistry 32, 1332-1337 (1993). 60 Sasaki, J. & Spudich, J. L. Proton transport by sensory rhodopsins and its modulation by transducer-binding. Biochim. Biophys. Acta 1460, 230-239 (2000). | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/57115 | - |
| dc.description.abstract | 水是生命不可或缺之物，而水中的離子對生物分子功能的行使扮演至關重要的角色。細胞透過離子的交換與輸送來維持生命，對於嗜鹽古生菌更是如此；其細胞膜上具有微生物視紫紅質，在光驅動之下，產生離子運送作用。其中氯視紫紅質協助氯離子輸送至胞內維持滲透壓，細菌視紫紅質則是將質子向外運送，使其累積在細胞膜外，促進 ATP 合成酶產生細胞能量 ATP。量測電位訊號為探討離子輸送的常見方法，因此本研究第一部份使用氧化銦錫玻片為電極所構築的光電流偵測系統，探討其離子敏感性以及生物分子的適用性。結果顯示本裝置對於質子具有高度專一性並可在廣泛的 pH 範圍內維持信號之高度線性關係，且可辨別光照下細胞膜上目標蛋白質運送質子的流向。藉此方法已可篩選到潛在質子傳輸蛋白質，並可以訊號波形協助研究菌種差異。第二部份使用第一部份之光電流偵測系統，嘗試分析生物中未解之謎。因鹽方扁平古菌 (Haloquadratum walsbyi) 被認為同時具有兩個不同的質子泵細菌視紫紅質，分別為 HwBR 和HwMR，但未能被證實。此部分研究以 E. coli 表達這兩個蛋白質，配合質子傳輸路徑關鍵氨基酸的點突變，在全細胞與蛋白質層次，探討光照下其二者質子運輸所造成之外界 pH 值變化、紫外光/可見光吸收光譜以及光電流訊號差異。以兩個質子泵最重要的保守胺基酸突變來研究，結果顯示 HwBR 為細菌視紫紅質，其 D93負責質子向外輸送，D104 主導質子回收。但 HwMR 僅具有微弱的質子傳輸能力，其 D84N 突變對質子釋出無顯著影響，D95N 突變則會阻撓質子向外傳輸。HwBR 與 HwMR 的特徵吸收光波段相差 55 nm，且後者微弱的質子傳輸能力與非典型的質子傳輸路徑，推測其可能扮演調控訊號傳遞之角色。總結兩大部份研究，以 ITO 為基材之光電流偵測裝置，適合篩選具質子傳輸能力的蛋白質，並可用於分析質子傳輸路徑。 | zh_TW |
| dc.description.abstract | Water is a necessarity to life, and it contains various ions known to be important for biological functions. Cells maintain their vitality through ion exchange and transportation, and it is particularly true for haloarchaea as they possess microbial rhodopsins embedded in cell membrane to serve such need. Among known microbial rhodopsins, halorhodopsin is a light-driven inward chloride pump that maintains osmobalance, while bacteriorhodopsin is a light-driven outward proton pump that subsequently accumulates protons that further drive ATP synthase to produce biological energy, ATP. To study those light-driven ion pumps, electrochemical and other methods are commonly adopted. In first stage of this study, we improved a previously reported ITO-based photocurrent analysis method via investigating its ion selectivity and application in biological molecules. The results showed our modified device to have high proton selectivity and it appeared to have highly linear relation to a wide pH range. Further, signal orientations of transported protons under illumination can be determined. Finally, we found both purified protein, native cells and E. coli cells expressing target proteins can all be applied in this system. The second stage of this study involved in applying this measurement system to resolve a biological system, Haluquadratum walsbyi (H. walsbyi), with two bacteriorhodopsin-like proteins, named HwBR and HwMR. We expressed wild type and mutated proteins in E.coli (C43) to investigate their functional pH change, UV/Vis spectrum, and signals in photocurrent under illumination at both whole cells and purified protein samples. The results showed HwBR is indeed a light-driven outward proton pump with residue D93 in charge of proton releasing, while D104 accerlate proton re-uptake from the cytoplasmic side. HwMR, on the other hand, possesses only weak proton pumping activity. When compared to HwBR, λmax of HwMR was a blue-shift 55 nm in UV/Vis spectrum without significant light-driven proton pumping activity, and possibly non-typical light-driven poroton transportation pathway inside the protein. We therefore proposed it might play signal-modulating role in H. walsbyi. In summary, ITO-based photocurrent method is appropriate for both screening potential light-driven proton-pumping protein and analysis of light-driven proton transportation pathway in any protein. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T06:35:19Z (GMT). No. of bitstreams: 1 ntu-103-R01b22050-1.pdf: 3304462 bytes, checksum: da1e411e1498cd9d709881c7ecabe772 (MD5) Previous issue date: 2014 | en |
| dc.description.tableofcontents | 目錄 I
圖目錄 V 表目錄 VII 摘要 VIII Abstract X 第一章 緒論 1 第一節 蛋白質與小分子和離子間之交互作用 1 第二節 古生菌的微生物視紫紅質藉由光能進行離子傳輸 2 第三節 氯視紫紅質 4 第四節 細菌視紫紅質 6 第五節 感受型視紫紅質 8 第六節 離子傳輸蛋白質之研究方式 9 第七節 鹽方扁平古菌 (Haloquadratum walsbyi, Hw) 中的類細菌視紫紅 質 11 第八節 研究目的、動機與流程 12 第二章 材料與方法 15 實驗材料與藥品 15 菌種 15 質體 15 藥品名稱 15 溶劑配製 17 其它 18 實驗儀器與設備 18 離心機 18 質子幫浦實驗用儀器 18 光電流量測用儀器 18 其他 19 方法 19 目標蛋白質表現 19 目標蛋白質純化 20 蛋白質光電流偵測 20 ITO 離子敏感性辨別 22 ITO 玻片酸鹼變化偵測 23 全細胞 pH 值變化偵測 23 全細胞光電流偵測 24 原生菌培養與光電流偵測 24 UV/Vis 光譜掃描 24 第三章 結果與討論-探究新式光電流離子偵測技術 25 第一節 ITO-based 光電流偵測技術偵測到質子釋放 25 第二節 以緩衝溶液探討 ITO-based 光電流偵測技術之質子緩衝效應 26 第三節 藉由不同酸鹼值之待測液探討 ITO-based 光電流偵測技術所造成訊號 強度與方向變化趨勢 27 第四節 ITO-based 光電流偵測技術能用於偵測菌體外質子濃度變化 30 第五節 以光電流偵測技術篩選潛在質子傳輸蛋白質 31 第六節 光電流偵測技術亦適用於原生菌種 33 討論 34 結論 37 第四章 結果與討論-應用新式光電流技術定性鹽方扁平古菌兩種預測的類細菌視紫 紅質 38 第一節 生物資訊軟體分析揭露出 Haloquadratum walsbyi具有兩種類細 菌視紫紅質蛋白質 38 第二節 以 pH 偵測計無法量測到 HwMR 照光時所造成之質子變化 40 第三節 以新式光電流偵測技術確認 HwMR 為質子幫浦 41 第四節 HwBR 與 HwMR 在 UV/Vis 光譜呈現不同特徵吸收峰 42 第五節 HwBR-WT 與 HwMR-WT 在不同 pH 值下所呈現的圖譜變化 43 第六節 HwBR-WT 與 HwMR-WT 在不同 pH 值下所偵測到的光電流訊號 44 第七節 HwMR 之 D84N 突變於全細胞實驗無法顯著降低質子釋放能力 46 第八節 HwMR 之 D95N 突變於全細胞實驗中顯著破壞其質子釋放能力 48 第九節 HwBR-D93N 與 HwMR-D84N 在不同 pH 值下所呈現的 UV/Vis 圖 譜變化 50 第十節 HwBR-D93N 與 HwMR-D84N 在不同 pH 值下所偵測到的光電流訊號 51 第十一節 HwBR-D104N 與 HwMR-D95N 在不同 pH 值下所呈現的 UV/Vis 圖譜變化 52 第十二節 HwBR-D104N 與 HwMR-D95N 在不同 pH 值下所偵測到的光電流訊 號 53 第十三節 以結構觀點探討質子傳輸路徑上氨基酸對應位置的影響 54 討論 58 結論 62 未來展望 63 參考文獻 64 | |
| dc.language.iso | zh-TW | |
| dc.subject | 細菌視紫紅質 | zh_TW |
| dc.subject | 鹽方扁平古菌 | zh_TW |
| dc.subject | 光電流 | zh_TW |
| dc.subject | 微生物視紫紅質 | zh_TW |
| dc.subject | 質子傳輸路徑 | zh_TW |
| dc.subject | 氧化銦錫 | zh_TW |
| dc.subject | 氯視紫紅質 | zh_TW |
| dc.subject | indium tin oxide (ITO) | en |
| dc.subject | proton transportation pathway | en |
| dc.subject | halorhodopsin (HR) | en |
| dc.subject | bacteriorhodopsin (BR) | en |
| dc.subject | Haloquadratum walsbyi | en |
| dc.subject | photocurrent | en |
| dc.subject | microbial rhodopsin | en |
| dc.title | 以新式光電流量測觀點剖析微生物視紫紅質之光驅動離子傳輸功能 | zh_TW |
| dc.title | New insights of a photocurrent method for functional measurement of light-driven ion transportation in microbial rhodopsins | 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 | 微生物視紫紅質,氧化銦錫,光電流,鹽方扁平古菌,細菌視紫紅質,氯視紫紅質,質子傳輸路徑, | zh_TW |
| dc.subject.keyword | microbial rhodopsin,indium tin oxide (ITO),photocurrent,Haloquadratum walsbyi,bacteriorhodopsin (BR),halorhodopsin (HR),proton transportation pathway, | en |
| dc.relation.page | 72 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2014-08-04 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| Appears in Collections: | 生化科技學系 | |
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| ntu-103-1.pdf Restricted Access | 3.23 MB | Adobe PDF |
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