臺灣博碩士論文加值系統

嗜鹽古細菌 (haloarchaea) 如何生活於高光照、高鹽的極端環境中，表現在膜上的視紫質扮演了重要角色。依照生理功能可以區分為兩大類—光驅動離子幫浦 (light-driven ion pump) 與光趨性受體 (phototaxis receptors)，前者如氫視紫質 (bacteriorhodopsin，BR) 與氯視紫質 (halorhodopsin，HR)，後者像是感覺型視紫質(sensory rhodopsin，SR)。本研究所探討的鹽方扁平古菌 (Haloquadratum walsbyi, Hw) 可存活於其他嗜鹽古菌無法生存的2.0 M MgCl2環境中且擁有三種視紫質，光驅動離子幫浦 BR、HR 以及第三種功能未知的Middle Rhodopsin (MR)，前人研究指出 MR 同時具有BR及SRII特有的保守關鍵胺基酸，且其生物化學特性介於兩者之間。比較先前對 Hw 上另外兩種視紫質的研究顯示，BR 具備高耐酸能力，HR 與氯離子的結合力強，那麼第三種 MR 是否也與某種離子具相關性?因H. walsbyi具有獨特的鎂離子高耐受性，故進行一系列特性分析探討其和鎂離子之關係。本研究在透過視紫質於不同離子濃度下，觀察其特徵吸收峰偏移程度，以評估是否對某一離子具專一性結合，並利用光週期驗證視紫質結構變化時，離子是否參與其中。實驗結果發現，Hw 的三種視紫質以及其他菌種的視紫質，只有 MR 在不同鎂、鈉離子濃度環境下，其特徵吸收峰顯著偏移；更進一步在光週期測定，發現只在不同鎂離子濃度下具有快慢的差異，此為突破性的發現，於是進一步挑選在MR上可能和鎂離子結合之胺基酸進行點突變，發現位於蛋白質胞外側的 D84 具有篩選可以進入蛋白質的離子種類，接近視黃醛口袋 (retinal binding pocket) 的 T216 以及位在蛋白質胞內側 D95則為可能的鎂離子專一性結合位，而鎂離子和MR結合之生理重要性則正在做更深入的研究。

In haloarchaeas, microbial rhodopsins (M-Rho) are vital proteins for solar energy capture, as well as seeking for optimal living environment. Based on different physiological functions and mechanisms, two types of M-Rho have been found: light-driven ion transporters (bacteriorhodopsin, BR and halorhodopsin, HR) and phototaxis receptors (sensory rhodopsin, SR). Haloquadratum walsbyi (Hw) survives a 2.0 M MgCl2 condition and three M-Rho were identified in its genome, namely light-driven ion transporters HwBR, HwHR, and the functionally unknown HwMR. Previous studies showed HwMR to have the conserved residues from both BR and SRII; it even possesses photochemical properties of both, too. Based on our previous findings that HwBR was acid-tolerant and HwHR had extremely high affinity to chloride. Since Hw cells were found to survive an otherwise harmful 2.0 M Mg2+ environment, the goal of this study was set to seek the specific ion species, including Mg2+, that associate with MR. Here, we present the maximum absorbance (Abs-max) variations of three Hw M-Rho proteins under different ion species and concentrations and ion-dependent photocycle kinetics were recorded. Our results showed only MR appeared to be Mg2+ and Na+ sensitive among three M-Rhos in Hw. Furthermore, only the photocycle kinetics of MR showed significant changes under Mg2+. In order to investigate the critical site(s) related to Mg2+ binding, we mutated several amino acids in MR, and concluded that D84 which locates on the extracellular half of protein contributed to ion selectivity; the T216 near the retinal binding pocket, as well as cytoplasmic side D95 might be the specific Mg2+ binding sites. Further physiological significance is under study.

目錄 I
圖目錄 V
表目錄 VII
摘要 VII
Abstract IX
第一章 緒論 1
第一節 嗜鹽古生菌 1
第二節 微生物視紫質 (Microbial Rhodopsins) 2
第三節 Haloquadratum walsbyi 5
第四節 中間型態視紫質 7
I-4.1 蛋白質序列 7
I-4.2 紫外光/可見光譜 8
I-4.3 光週期 (Photocycle) 10
I-4.4 功能性測試—質子傳輸 11
I-4.5 演化探討 13
第五節 研究動機與目的 14
第二章 材料與方法 16
第一節 實驗材料與藥品 16
II-1.1 菌種 16
II-1.2 質體 16
II-1.3 藥品 16
第二節 實驗儀器與設備 18
II-2.1 核酸電泳 18
II-2.2 蛋白質電泳及轉印 18
II-2.3 離心機 18
II-2.4 酸鹼度計 18
II-2.5 光學設備 19
II-2.6 其他 19
第三節 實驗方法 20
II-3.1 生物資訊分析 20
II-3.2 質體轉型與建構 20
II-3.2.1 核酸萃取 20
II-3.2.2 聚合酶鏈鎖反應 (PCR) 20
II-3.2.3 DNA膠體純化 21
II-3.2.4 限制酶剪切 21
II-3.2.5 DNA黏合 21
II-3.2.6 大腸桿菌轉形 21
II-3.2.7 轉形株鑑定 22
II-3.2.8 突變株建構 22
II-3.3 重組蛋白質表現與純化 23
II-3.3.1 目標蛋白質表達 23
II-3.3.2 破菌與蛋白質萃取 23
II-3.3.3 膜分離與溶出膜蛋白質 24
II-3.3.4 親和層析法純化 24
II-3.3.5 膠體過濾法 24
II-3.3.6 超微膜過濾濃縮 24
II-3.3.7 透析 24
II-3.4 蛋白質定量 25
II-3.5 蛋白質定性分析 26
II-3.5.1 SDS-PAGE 變性膠體電泳 26
II-3.5.2 蛋白質轉印 27
II-3.5.3 免疫呈色 27
II-3.6 蛋白質光學性質分析 27
II-3.6.1 特徵吸收光譜測定 27
II-3.6.2 感光蛋白質光週期測定 27
II-3.6.3 光驅動離子幫浦活性測試 28
II-3.6.4 光電流訊號測試 29
II-3.7 蛋白質養晶 30
II-3.7.1 Pre crystallization test (PCT) 30
II-3.7.2 坐式液滴結晶條件篩選 (sitting drop) 30
第三章 實驗結果 31
第一節 生物資訊軟體分析 31
III-1.1 HwMR 結構模擬 31
III-1.2 序列比對 33
第二節 H.walsbyi 上三種視紫質吸收光譜特性分析 34
III-2.1 不同pH值之下 34
III-2.2 不同離子環境下 36
III-2.2.1 鎂離子與視紫質之相關性 36
III-2.2.2 鈉離子與視紫質之相關性 36
III-2.2.3 鈣離子與視紫質之相關性 37
第三節 H.walsbyi 上三種視紫質光週期特性分析 41
III-3.1 不同pH值之下 41
III-3.2 不同離子環境下 43
III-3.2.1 鎂離子與視紫質之相關性 43
III-3.2.2 鈉離子與視紫質之相關性 43
第四節HwMR 上與金屬離子作用的關鍵胺基酸分析 47
III-4.1 HwMR 點突變建構 47
III-4.2 蛋白質表現確認 47
III-4.3 HwMR 點突變蛋白質顏色調諧 (color-tuning) 50
III-4.4 不同離子之下吸收光譜測定 51
III-4.4.1 突變蛋白質與鎂離子之相關性 51
III-4.4.2 突變蛋白質與鈉離子之相關性 52
III-4.4.3 突變蛋白質與鈣離子之相關性 54
III-4.5 光週期量測 56
III-4.5.1 鎂離子環境下 56
III-4.5.2 鈉離子環境下 57
III-4.6 不同濃度鎂離子環境下對於特徵吸收波長的影響 59
第五節 其他菌種的視紫質與鎂離子之相關性 60
第六節 HwMR 內部質子傳遞情形之探討 61
III-6.1 蛋白質光電流訊號測試 61
III-6.2 全細胞氫離子幫浦活性與光電流測試 64
第七節 HwMR的養晶條件 66
第四章 結論與探討 68
第一節 HwMR 與鎂離子的交互作用 68
第二節 HwMR 在生理上的意義 70
IV-2.1 內部的質子循環 70
IV-2.2 特殊結構 70
第五章 未來展望 72
第六章 參考文獻 73

1Woese, C. R. & Fox, G. E. Phylogenetic Structure of the Prokaryotic Domain: The Primary Kingdoms. Proc Natl Acad Sci U S A 74, 5088-5090, (1977).
2Robertson, C. E., Harris, J. K., Spear, J. R. & Pace, N. R. Phylogenetic Diversity and Ecology of Environmental Archaea. Curr Opin Microbiol 8, 638-642, (2005).
3Albers, S. V., Szabo, Z. & Driessen, A. J. Protein Secretion in the Archaea: Multiple Paths Towards a Unique Cell Surface. Nat Rev Microbiol 4, 537-547, (2006).
4Engelhardt, H. & Peters, J. Structural Research on Surface Layers: A Focus on Stability, Surface Layer Homology Domains, and Surface Layer-Cell Wall Interactions. J Struct Biol 124, 276-302, (1998).
5Kandler, O. & Konig, H. Cell Wall Polymers in Archaea (Archaebacteria). Cell Mol Life Sci 54, 305-308, (1998).
6Howland, J. L. The Surprising Archaea : Discovering Another Domain of Life. 63-69 (Oxford University, 2000).
7Lanyi, J. K. Salt-Dependent Properties of Proteins from Extremely Halophilic Bacteria. Bacteriol Rev 38, 272-290, (1974).
8Larsen, H., Omang, S. & Steensland, H. On the Gas Vacuoles of the Halobacteria. Arch Mikrobiol 59, 197-203, (1967).
9Bang, C. & Schmitz, R. A. Archaea Associated with Human Surfaces: Not to Be Underestimated. FEMS Microbiol Rev 39, 631-648, (2015).
10Spudich, 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, 365-392, (2000).
11Rao, V. R. & Oprian, D. D. Activating Mutations of Rhodopsin and Other G Protein-Coupled Receptors. Annu Rev Biophys Biomol Struct 25, 287-314, (1996).
12Fu, H. Y., Lin, Y. C., Chang, Y. N., Tseng, H., Huang, C. C., Liu, K. C., Huang, C. S., Su, C. W., Weng, R. R., Lee, Y. Y., Ng, W. V. & Yang, C. S. A Novel Six-Rhodopsin System in a Single Archaeon. J Bacteriol 192, 5866-5873, (2010).
13Costanzi, S., Siegel, J., Tikhonova, I. G. & Jacobson, K. A. Rhodopsin and the Others: A Historical Perspective on Structural Studies of G Protein-Coupled Receptors. Curr Pharm Des 15, 3994-4002, (2009).
14Lawson, M. A., Zacks, D. N., Derguini, F., Nakanishi, K. & Spudich, J. L. Retinal Analog Restoration of Photophobic Responses in a Blind Chlamydomonas reinhardtii Mutant. Evidence for an Archaebacterial Like Chromophore in a Eukaryotic Rhodopsin. Biophys J 60, 1490-1498, (1991).
15Tsunoda, S. P., Ewers, D., Gazzarrini, S., Moroni, A., Gradmann, D. & Hegemann, P. H+ -Pumping Rhodopsin from the Marine Alga Acetabularia. Biophys J 91, 1471-1479, (2006).
16Wald, G., Brown, P. K. & Smith, P. H. Iodopsin. J Gen Physiol 38, 623-681, (1955).
17Spudich, J. L. & Jung, K. H. Microbial Rhodopsins. Protein Science Encyclopedia, 1-23, (2005).
18Inoue, K., Ono, H., Abe-Yoshizumi, R., Yoshizawa, S., Ito, H., Kogure, K. & Kandori, H. A Light-Driven Sodium Ion Pump in Marine Bacteria. Nat Commun 4, 1678, (2013).
19Nagel, G., Szellas, T., Huhn, W., Kateriya, S., Adeishvili, N., Berthold, P., Ollig, D., Hegemann, P. & Bamberg, E. Channelrhodopsin-2, a Directly Light-Gated Cation-Selective Membrane Channel. Proc Natl Acad Sci U S A 100, 13940-13945, (2003).
20Hoff, W. D., Jung, K. H. & Spudich, J. L. Molecular Mechanism of Photosignaling by Archaeal Sensory Rhodopsins. Annu Rev Biophys Biomol Struct 26, 223-258, (1997).
21Vogeley, L., Sineshchekov, O. A., Trivedi, V. D., Sasaki, J., Spudich, J. L. & Luecke, H. Anabaena Sensory Rhodopsin: A Photochromic Color Sensor at 2.0 A. Science 306, 1390-1393, (2004).
22Oesterhelt, D. & Stoeckenius, W. Rhodopsin-Like Protein from the Purple Membrane of Halobacterium halobium. Nat New Biol 233, 149-152, (1971).
23Luecke, 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).
24Pebay-Peyroula, E., Rummel, G., Rosenbusch, J. P. & Landau, E. M. X-Ray Structure of Bacteriorhodopsin at 2.5 Angstroms from Microcrystals Grown in Lipidic Cubic Phases. Science 277, 1676-1681, (1997).
25Hartmann, R., Sickinger, H. D. & Oesterhelt, D. Quantitative Aspects of Energy Conversion in Halobacteria. FEBS Lett 82, 1-6, (1977).
26Pfisterer, C., Gruia, A. & Fischer, S. The Mechanism of Photo-Energy Storage in the Halorhodopsin Chloride Pump. J Biol Chem 284, 13562-13569, (2009).
27Chen, X. R., Huang, Y. C., Yi, H. P. & Yang, C. S. A Unique Light-Driven Proton Transportation Signal in Halorhodopsin from Natronomonas pharaonis. Biophys J 111, 2600-2607, (2016).
28Walsby, A. E. A Square Bacterium. Nature 283, 69, (1980).
29Kessel, M. & Cohen, Y. Ultrastructure of Square Bacteria from a Brine Pool in Southern Sinai. J Bacteriol 150, 851-860, (1982).
30Stoeckenius, W. Walsby''s Square Bacterium: Fine Structure of an Orthogonal Procaryote. J Bacteriol 148, 352-360, (1981).
31Sublimi Saponetti, M., Bobba, F., Salerno, G., Scarfato, A., Corcelli, A. & Cucolo, A. Morphological and Structural Aspects of the Extremely Halophilic Archaeon Haloquadratum walsbyi. PLoS One 6, e18653, (2011).
32Bolhuis, H., Poele, E. M. & Rodriguez-Valera, F. Isolation and Cultivation of Walsby''s Square Archaeon. Environ Microbiol 6, 1287-1291, (2004).
33Oren, A., Duker, S. & Ritter, S. The Polar Lipid Composition of Walsby''s Square Bacterium. FEMS microbiology letters 138, 135-140, (1996).
34Oren, A., Ventosa, A., Gutierrez, M. C. & Kamekura, M. Haloarcula quadrata sp. nov., a Square, Motile Archaeon Isolated from a Brine Pool in Sinai (Egypt). Int J Syst Bacteriol 49 Pt 3, 1149-1155, (1999).
35Benlloch, S., Rodríguez‐Valera, F. & Martinez‐Murcia, A. J. Bacterial Diversity in Two Coastal Lagoons Deduced from 16s rDNA PCR Amplification and Partial Sequencing. FEMS Microbiology Ecology 18, 267-279, (1995).
36Ghai, R., Pasic, L., Fernandez, A. B., Martin-Cuadrado, A. B., Mizuno, C. M., McMahon, K. D., Papke, R. T., Stepanauskas, R., Rodriguez-Brito, B., Rohwer, F., Sanchez-Porro, C., Ventosa, A. & Rodriguez-Valera, F. New Abundant Microbial Groups in Aquatic Hypersaline Environments. Sci Rep 1, 135-144, (2011).
37Boetius, A. & Joye, S. Ecology. Thriving in Salt. Science 324, 1523-1525, (2009).
38Oren, A. Halobacterium sodomense sp. nov., a Dead Sea Halobacterium with an Extremely High Magnesium Requirement. International Journal of Systematic and Evolutionary Microbiology 33, 381-386, (1983).
39Oren, A., Weisburg, W. G., Kessel, M. & Woese, C. R. Halobacteroides halobius gen. nov., sp. nov., a Moderately Halophic Anaerobic Bacterium from the Bottom Sediments of the Dead Sea. Systematic and applied microbiology 5, 58-70, (1984).
40Oren, A., Gurevich, P., Gemmell, R. T. & Teske, A. Halobaculum gomorrense gen. nov., sp. nov., a Novel Extremely Halophilic Archaeon from the Dead Sea. Int J Syst Bacteriol 45, 747-754, (1995).
41Lobasso, S., Lopalco, P., Mascolo, G. & Corcelli, A. Lipids of the Ultra-Thin Square Halophilic Archaeon Haloquadratum walsbyi. Archaea 2, 177-183, (2008).
42Bolhuis, H., Palm, P., Wende, A., Falb, M., Rampp, M., Rodriguez-Valera, F., Pfeiffer, F. & Oesterhelt, D. The Genome of the Square Archaeon Haloquadratum walsbyi : Life at the Limits of Water Activity. BMC Genomics 7, 169, (2006).
43Zhou, P., Wen, J., Oren, A., Chen, M. & Wu, M. Genomic Survey of Sequence Features for Ultraviolet Tolerance in Haloarchaea (Family Halobacteriaceae). Genomics 90, 103-109, (2007).
44Sudo, Y., Ihara, K., Kobayashi, S., Suzuki, D., Irieda, H., Kikukawa, T., Kandori, H. & Homma, M. A Microbial Rhodopsin with a Unique Retinal Composition Shows Both Sensory Rhodopsin II and Bacteriorhodopsin-Like Properties. J Biol Chem 286, 5967-5976, (2011).
45Lanyi, J. K. Bacteriorhodopsin. Annu Rev Physiol 66, 665-688, (2004).
46Sudo, Y., Furutani, Y., Kandori, H. & Spudich, J. L. Functional Importance of the Interhelical Hydrogen Bond between Thr204 and Tyr174 of Sensory Rhodopsin II and Its Alteration During the Signaling Process. J Biol Chem 281, 34239-34245, (2006).
47Suzuki, D., Irieda, H., Homma, M., Kawagishi, I. & Sudo, Y. Phototactic and Chemotactic Signal Transduction by Transmembrane Receptors and Transducers in Microorganisms. Sensors (Basel) 10, 4010-4039, (2010).
48Kuschmitz, D. & Hess, B. Coupling of Proton and Cycle and Photocycle in Bacterio-rhodopsin. Hoppe-seylers Zeitschrift Für Physiologische Chemie 358, 1383-1384 (1977).
49Essen, L. O. Halorhodopsin: Light-Driven Ion Pumping Made Simple? Curr Opin Struct Biol 12, 516-522, (2002).
50Scharf, B., Pevec, B., Hess, B. & Engelhard, M. Biochemical and Photochemical Properties of the Photophobic Receptors from Halobacterium halobium and Natronobacterium pharaonis. Eur J Biochem 206, 359-366, (1992).
51Ohtani, H., Kobayashi, T. & Tsuda, M. Branching Photocycle of Sensory Rhodopsin in Halobacterium halobium. Biophys J 53, 493-496, (1988).
52Sasaki, J. & Spudich, J. L. The Transducer Protein HtrII Modulates the Lifetimes of Sensory Rhodopsin II Photointermediates. Biophys J 75, 2435-2440, (1998).
53Sasaki, J. & Spudich, J. L. Proton Transport by Sensory Rhodopsins and Its Modulation by Transducer-Binding. Biochim Biophys Acta 1460, 230-239, (2000).
54Bogomolni, R. A., Stoeckenius, W., Szundi, I., Perozo, E., Olson, K. D. & Spudich, J. L. Removal of Transducer Htri Allows Electrogenic Proton Translocation by Sensory Rhodopsin I. Proc Natl Acad Sci U S A 91, 10188-10192, (1994).
55Sasaki, J. & Spudich, J. L. Proton Circulation During the Photocycle of Sensory Rhodopsin II. Biophys J 77, 2145-2152, (1999).
56Inoue, K., Tsukamoto, T. & Sudo, Y. Molecular and Evolutionary Aspects of Microbial Sensory Rhodopsins. Biochim Biophys Acta 1837, 562-577, (2014).
57Royant, A., Nollert, P., Edman, K., Neutze, R., Landau, E. M., Pebay-Peyroula, E. & Navarro, J. X-Ray Structure of Sensory Rhodopsin II at 2.1-a Resolution. Proc Natl Acad Sci U S A 98, 10131-10136, (2001).
58Sudo, Y. & Spudich, J. L. Three Strategically Placed Hydrogen-Bonding Residues Convert a Proton Pump into a Sensory Receptor. Proc Natl Acad Sci U S A 103, 16129-16134, (2006).
59Spudich, J. L., Zacks, D. N. & Bogomolni, R. A. Microbial Sensory Rhodopsins: Photochemistry and Function. Israel journal of chemistry 35, 495-513, (1995).
60Shimono, K., Hayashi, T., Ikeura, Y., Sudo, Y., Iwamoto, M. & Kamo, N. Importance of the Broad Regional Interaction for Spectral Tuning in Natronobacterium pharaonis Phoborhodopsin (Sensory Rhodopsin II). J Biol Chem 278, 23882-23889, (2003).
61Hsu, M. F., Fu, H. Y., Cai, C. J., Yi, H. P., Yang, C. S. & Wang, A. H. Structural and Functional Studies of a Newly Grouped Haloquadratum walsbyi Bacteriorhodopsin Reveal the Acid-Resistant Light-Driven Proton Pumping Activity. J Biol Chem 290, 29567-29577, (2015).
62Fu, H. Y., Chang, Y. N., Jheng, M. J. & Yang, C. S. Ser(262) Determines the Chloride-Dependent Colour Tuning of a New Halorhodopsin from Haloquadratum walsbyi. Biosci Rep 32, 501-509, (2012).
63Tamogami, 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. Photochem Photobiol 85, 578-589, (2009).
64Henderson, R., Baldwin, J. M., Ceska, T. A., Zemlin, F., Beckmann, E. & Downing, K. H. Model for the Structure of Bacteriorhodopsin Based on High-Resolution Electron Cryo-Microscopy. J Mol Biol 213, 899-929, (1990).
65Zhang, J., Mizuno, K., Murata, Y., Koide, H., Murakami, M., Ihara, K. & Kouyama, T. Crystal Structure of Deltarhodopsin-3 from Haloterrigena thermotolerans. Proteins 81, 1585-1592, (2013).
66Fu, 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. Molecular microbiology 88, 551-561, (2013).
67Chen, P. C., Chen, T. W., Han, Y. A., Ng, W. V. & Yang, C. S. Complete Genome Sequence of a New Halophilic Archaeon, Haloarcula taiwanensis, Isolated from a Solar Saltern in Southern Taiwan. Genome Announc 6, e01529-17, (2018).
68Zimanyi, L., Varo, G., Chang, M., Ni, B., Needleman, R. & Lanyi, J. K. Pathways of Proton Release in the Bacteriorhodopsin Photocycle. Biochemistry 31, 8535-8543, (1992).
69Eisenhauer, K., Kuhne, J., Ritter, E., Berndt, A., Wolf, S., Freier, E., Bartl, F., Hegemann, P. & Gerwert, K. In Channelrhodopsin-2 Glu-90 Is Crucial for Ion Selectivity and Is Deprotonated During the Photocycle. J Biol Chem 287, 6904-6911, (2012).