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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 楊啟伸 | |
dc.contributor.author | Kang-Cheng Liu | en |
dc.contributor.author | 劉康正 | zh_TW |
dc.date.accessioned | 2021-06-15T01:18:04Z | - |
dc.date.available | 2014-07-29 | |
dc.date.copyright | 2009-07-29 | |
dc.date.issued | 2009 | |
dc.date.submitted | 2009-07-27 | |
dc.identifier.citation | 1. Baliga, N.S. et al. Genome sequence of Haloarcula marismortui: a halophilic archaeon from the Dead Sea. Genome Res 14, 2221-34 (2004).
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Proceedings of the National Academy of Sciences of the United States of America 97, 12176-12181 (2000). 9. Spudich, J.L., Yang, C.S., Jung, K.H. & Spudich, E.N. Retinylidene proteins: structures and functions from archaea to humans. Annu Rev Cell Dev Biol 16, 365-92 (2000). 10. 傅煦媛. 表現 Haloarcula marismortui 之六個光感受體揭露其獨特的感光特性. 國立台灣大學. 碩士論文. (2008). 11. Prof. Dr. John L. Spudich & Kwang-Hwan Jung. in Handbook of Photosensory Receptors (ed. Prof. Dr. Winslow R. Briggs, P.D.J.L.S.) 1-23 (2005). 12. Oesterhelt, D. & Stoeckenius, W. Functions of a new photoreceptor membrane. Proc Natl Acad Sci U S A 70, 2853-7 (1973). 13. Schobert, B. & Lanyi, J. Halorhodopsin is a light-driven chloride pump. J. Biol. Chem. 257, 10306-10313 (1982). 14. Bogomolni, R.A. & Spudich, J.L. Identification of a third rhodopsin-like pigment in phototactic Halobacterium halobium. Proceedings of the National Academy of Sciences of the United States of America 79, 6250-6254 (1982). 15. Tetsuo Takahashi, Hiroaki Tomioka, Naoki Kamo & Yonosuke Kobatake. A photosystem other than PS370 also mediates the negative phototaxis of Halobacterium halobium. FEMS Microbiology Letters 28, 161-164 (1985). 16. Hoff, W.D., Jung, K.-H. & Spudich, J.L. Molecular mechanism of photosignaling by archaeal sensory rhodopsins. Annual Review of Biophysics and Biomolecular Structure 26, 223-258 (1997). 17. Oesterhelt, D. The structure and mechanism of the family of retinal proteins from halophilic archaea. Current Opinion in Structural Biology 8, 489-500 (1998). 18. Sharma, A. et al. Evolution of rhodopsin ion pumps in haloarchaea. BMC Evolutionary Biology 7, 79 (2007). 19. Mukohata, Y. Comparative studies on ion pumps of the bacterial rhodopsin family. Biophys Chem 50, 191-201 (1994). 20. Lanyi, J.K. Bacteriorhodopsin as a model for proton pumps. Nature 375, 461-3 (1995). 21. Stoeckenius, W. & Rowen, R. A morphological study of Halobacterium halobium and its lysis in media of low salt concentration. J Cell Biol 34, 365-93 (1967). 22. Oesterhelt, D. & Stoeckenius, W. Rhodopsin-like protein from the purple membrane of Halobacterium halobium. Nat New Biol 233, 149-52 (1971). 23. Neutze, R. et al. Bacteriorhodopsin: a high-resolution structural view of vectorial proton transport. Biochimica et Biophysica Acta (BBA) - Biomembranes 1565, 144-167 (2002). 24. Henderson, R. & Unwin, P.N.T. Three-dimensional model of purple membrane obtained by electron microscopy. Nature 257, 28-32 (1975). 25. Rummel, G. et al. Lipidic Cubic Phases: New Matrices for the Three-Dimensional Crystallization of Membrane Proteins. J Struct Biol 121, 82-91 (1998). 26. Henderson, R. et al. Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy. J Mol Biol 213, 899-929 (1990). 27. Essen, L.-O., Siegert, R., Lehmann, W.D. & Oesterhelt, D. Lipid patches in membrane protein oligomers: Crystal structure of the bacteriorhodopsin-lipid complex. Proceedings of the National Academy of Sciences of the United States of America 95, 11673-11678 (1998). 28. Belrhali, H. et al. Protein, lipid and water organization in bacteriorhodopsin crystals: a molecular view of the purple membrane at 1.9 A resolution. Structure 7, 909-17 (1999). 29. Luecke, H., Schobert, B., Richter, H.T., Cartailler, J.P. & Lanyi, J.K. Structure of bacteriorhodopsin at 1.55 A resolution. J Mol Biol 291, 899-911 (1999). 30. Subramaniam, S. & Henderson, R. Molecular mechanism of vectorial proton translocation by bacteriorhodopsin. Nature 406, 653-7 (2000). 31. Cao, Y. et al. Relationship of proton release at the extracellular surface to deprotonation of the schiff base in the bacteriorhodopsin photocycle. 68, 1518-1530 (1995). 32. Hirai, T. & Subramaniam, S. Protein Conformational Changes in the Bacteriorhodopsin Photocycle: Comparison of Findings from Electron and X-Ray Crystallographic Analyses. PLoS ONE 4, e5769 (2009). 33. Mogi, T., Stern, L.J., Marti, T., Chao, B.H. & Khorana, H.G. Aspartic acid substitutions affect proton translocation by bacteriorhodopsin. Proc Natl Acad Sci U S A 85, 4148-52 (1988). 34. Luecke, H., Schobert, B., Richter, H.-T., Cartailler, J.-P. & Lanyi, J.K. Structural Changes in Bacteriorhodopsin During Ion Transport at 2 Angstrom Resolution. Science 286, 255-260 (1999). 35. Moltke, S., Krebs, M.P., Mollaaghababa, R., Khorana, H.G. & Heyn, M.P. Intramolecular charge transfer in the bacteriorhodopsin mutants Asp85-->Asn and Asp212-->Asn: effects of pH and anions. Biophys J 69, 2074-83 (1995). 36. Shibata, M., Yoshitsugu, M., Mizuide, N., Ihara, K. & Kandori, H. Halide binding by the D212N mutant of Bacteriorhodopsin affects hydrogen bonding of water in the active site. Biochemistry 46, 7525-35 (2007). 37. QuikChange Primer Design Program. http://www.stratagene.com/tradeshows/feature.aspx?fpId=118 38. Lam, W.L. & Doolittle, W.F. Shuttle vectors for the archaebacterium Halobacterium volcanii. Proc Natl Acad Sci U S A 86, 5478-82 (1989). 39. Sass, H.J. et al. Structural alterations for proton translocation in the M state of wild-type bacteriorhodopsin. Nature 406, 649-653 (2000). 40. Enami, N. et al. Crystal Structures of Archaerhodopsin-1 and -2: Common Structural Motif in Archaeal Light-driven Proton Pumps. Journal of Molecular Biology 358, 675-685 (2006). 41. John L. Spudich. Variations on a molecular switch: transport and sensory signalling by archaeal rhodopsins. Molecular Microbiology 28, 1051-1058 (1998). | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/42626 | - |
dc.description.abstract | 光對於生物體是重要的能量來源,在過去於嗜鹽古生菌的研究中,鑑定出四種的光受體 (photoreceptor) 能夠使用外界光源調控生理現象。這類受體都屬於視紫紅質 (rhodopsin) 的構形,主要能分成兩種離子幫浦及兩種調控光趨性的感光受體,其中細菌視紫紅質( Bacteriorhodopsin, BR ) 是第一個所被發現也徹底研究的古生菌視紫紅質,其被鑑定為一個光趨動的氫離子幫浦,具有重要的生理功能。
在2004年完成的Haloarcula marismortui 基因體解序中1,總共預測出六個視紫質的基因,其中兩個基因序列bop和xop1,被預測為是接近細菌視紫質的蛋白質。在本實驗室先前的研究中,將其命名為HmBRI和HmBRII。本研究將其選殖入E. coli中,進行表達重組蛋白質,試圖了解在H. marismotrui中,為何同時存在兩個細菌視紫質基因的生理意義,因為這是從未在任何古生菌發現的 首先,在嘗試進行蛋白質表現及純化後,得到最適化條件,並且利用熱處理的方式,改進原本的純化方式。其次,將所得蛋白質經由吸收光譜、光週期 (photocycle) 測定和氫離子幫浦 (proton pump) 能力測試後,確定其光譜特性和過去研究的結果類似,並且同樣具有氫離子幫浦功能,初步證實兩者皆具有細菌視紫質的特性和功能;接著利用特定胺基酸位置突變,得到與過去研究的結果具有相同的性質;並進行蛋白質結晶實驗,提供結構上的瞭解。再其次,本研究以序列分析、吸收光譜、光週期、幫浦能力和結構上的各種觀點,分析BRI和BRII兩者在蛋白質特性的異同,探討其生理功能,雖然未能完全解釋其可能的差異,但藉由了解其蛋白質性質,提供深入研究和工業發展上特性的基礎背景之建立。 | zh_TW |
dc.description.abstract | Light represents one of the most important energy sources for life. Previous studies had identified photoreceptors from halophilic archaea that mediate four different physiological functions when responding to light. These photoreceptors belong to the rhodopsin family and can be classified as ion transporter or sensory receptor for phototaxis responses. Bacteriorhodopsin (BR) is the first well studied archaeal rhodopsin, which was identified as a light-driven proton pump responsible for energy generation under high intensity light illumination or under low oxygen condition.
Haloarcula marismortui genome sequencing was completed in 2004 and a total of six rhodopsin genes were predicted, the most numerous in a single archaeon. Two genes-bop and xop1, were identified and determined to encode proteins called HmBRI and HmBRII, respectively, which formed a unique two-proton pump system. The goal of this thesis was set to compare protein features and functions between HmBRI and II to further understand the possible explanation why there are two BRs in the same cell. First, the HmRBI and II genes were expressed in E. coli and the optimum conditions were obtained and a new heat treatment procedure was introduced. Secondly, several assays for protein function probing were performed, including UV-vis absorption spectrum scanning, duration of photocycle and direct proton pump measurements, both HmBRI and HmBRII were proton pump and response to the same wavelength were thus concluded. Thirdly, protein sequence analysis, absorption spectra, photocycle measurements, proton pump activity and protein structure prediction analysis were carried out to extend our understanding of these two proteins. Finally, protein crystallization was performed to study the protein structures and needle-shaped crystals were successfully obtained. Even no clear conclusion was obtained; this study established the basic characteristics of those two BR proteins for any further study or development of industry applications. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T01:18:04Z (GMT). No. of bitstreams: 1 ntu-98-R96b47407-1.pdf: 6614839 bytes, checksum: b83927844907c9f3f9fb6e8af635e8be (MD5) Previous issue date: 2009 | en |
dc.description.tableofcontents | 謝誌 I
Contents II List of Tables IV List of Figures V Abstract VI 中文摘要 VII Introduction 1 Haloarcula marismortui 1 Archaeal rhodopsins 4 Bacteriorhodopsin: Photobiology and Structure 8 The purpose of this study 12 Flow chart 13 Materials and Methods 14 Bacteria strain 14 Primer 15 Plasmid vector 16 Construction 16 Protein expression 17 Heat treatment purification 17 SDS-PAGE and western blotting 18 UV-vis wavelength scan spectrum 18 Photocycle measurement 19 Proton pump activity measurement 20 Crystallization screening and modification 20 Bioinformatics analysis 21 Results 22 Summary of all constructions and their features 22 Protein expression 22 Protein purification 24 Bioinformatics analysis 28 Transmembrane helix prediction 28 Sequence alignment 30 Protein structure modeling 33 Spectrum 35 UV-vis spectrum and maximum absorption 35 Intermediate state absorption 36 Photocycle measurement 39 Proton pump activity measurements 41 The compare between HmBRI and HmBRII 47 Conclusion 48 References 49 | |
dc.language.iso | en | |
dc.title | Haloarcula marismortui 中 HmBRI 及 HmBRII 蛋白質特性及功能研究 | zh_TW |
dc.title | Functional analysis of HmBRI and HmBRII from Haloarcula marismortui | en |
dc.type | Thesis | |
dc.date.schoolyear | 97-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 黃青真,黃慶璨,許瑞祥,賴美津 | |
dc.subject.keyword | 細菌視紫紅質,光週期, | zh_TW |
dc.subject.keyword | Haloarcula marismortui,Bacteriorhodopsin,photocycle, | en |
dc.relation.page | 52 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2009-07-27 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 微生物與生化學研究所 | zh_TW |
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