Title

透過光電化學測試揭露在不同氯視紫紅質間之氯離子-協助氫離子流動機制的差異

Translated Titles

Different Mechanisms of Chloride-associated Proton Flux in Various Halorhodopsins Unveiled by Photoelectrochemical Measurements

DOI

10.6342/NTU201601256

Authors

黃元祈

Key Words

古細菌 ; 氯視紫紅質 ; 光驅動氯離子幫浦 ; 光電流 ; 光週期 ; haloarchaea ; halorhodopsin ; light-driven chloride pump ; photocurrent ; photocycle

PublicationName

臺灣大學生化科技學系學位論文

Volume or Term/Year and Month of Publication

2016年

Academic Degree Category

碩士

Advisor

楊啟伸

Content Language

英文

Chinese Abstract

氯視紫紅質 (halorhodopsin, HR) 是存在於古細菌 (haloarchaea) 之中的一種七穿膜區且含視黃醛蛋白質,目前已知是一種光驅動氯離子幫浦。然而過去有研究發現,不同物種間的氯視紫紅質不完全相同,在結構和傳遞機制上存在著些微的差異。在此研究中我們使用氧化铟锡 (ITO, indium-tin oxide) 材料來測試由光驅動氫離子釋出所造成的光電流 (photocurrent) 產生之情形,發現了NpHR (halorhodopsin from Natronomonas pharaonis) 產生獨特的電流訊號,的確指出不同物種氯視紫紅質之間的不同。針對若干高度保留且和氯離子穩定相關的胺基酸進行點突變,發現特定帶電的胺基酸一旦突變後光電流訊號即消失。再者,針對光電流產生和光週期相對應的時間設計了一個可以同時測量光電流和光週期的系統。最後我們提出了三種可能的傳遞機制模型,並試圖解釋這個氯視紫紅質之間的差異。總而言之,此研究提供了一個高解析度的方法來探討氯離子-協助之氫離子傳輸的機制。

English Abstract

Halorhodopsin (HR) is a seven-transmembrane (7TM) retinylidene protein from haloarchaea that is commonly known to function as a light-driven inward chloride pump. However, previous studies have indicated that despite the general characteristics that most HRs share, HRs from distinct species differ in many aspects. We present indium-tin oxide (ITO)-based photocurrent measurements that demonstrate light-induced signal generated by proton release, which is observed solely in NpHR through purified protein-based assays, indeed showed HRs are not all identical. Mutagenesis studies were conducted on several conserved residues considered critical for chloride stability in HRs. Intriguingly, the photocurrent signals were eliminated after specific point mutation. Furthermore, the photocurrent and various photocycle intermediates were recorded simultaneously. We summarized three possible mechanisms in attempt to explain the differences among HRs. Overall, this approach provides a high-resolution method for further investigation of the chloride-associated proton-translocation mechanism.

Topic Category 生命科學院 > 生化科技學系
生物農學 > 生物科學
Reference
  1. 1 Robertson, C. E., Harris, J. K., Spear, J. R. & Pace, N. R. Phylogenetic diversity and ecology of environmental Archaea. Current opinion in microbiology 8, 638-642 (2005).
    連結:
  2. 2 Pfeifer, F. Haloarchaea and the formation of gas vesicles. Life 5, 385-402 (2015).
    連結:
  3. 3 Govorunova, E. G., Sineshchekov, O. A., Janz, R., Liu, X. & Spudich, J. L. NEUROSCIENCE. Natural light-gated anion channels: A family of microbial rhodopsins for advanced optogenetics. Science 349, 647-650 (2015).
    連結:
  4. 5 Mevorat-Kaplan, K., Brumfeld, V., Engelhard, M. & Sheves, M. The protonated Schiff base of halorhodopsin from Natronobacterium pharaonis is hydrolyzed at elevated temperatures. Photochemistry and photobiology 82, 1414-1421 (2006).
    連結:
  5. 6 Spudich, J. L., Yang, C. S., Jung, K. H. & Spudich, E. N. Retinylidene proteins: structures and functions from archaea to humans. Annual review of cell and developmental biology 16, 365-392 (2000).
    連結:
  6. 9 Varo, G., Zimanyi, L., Fan, X., Sun, L., Needleman, R. & Lanyi, J. K. Photocycle of halorhodopsin from Halobacterium salinarium. Biophysical journal 68, 2062-2072 (1995).
    連結:
  7. 10 Facciotti, M. T., Rouhani, S. & Glaeser, R. M. Crystal structures of bR(D85S) favor a model of bacteriorhodopsin as a hydroxyl-ion pump. FEBS letters 564, 301-306 (2004).
    連結:
  8. 11 Lanyi, J. K. Halorhodopsin: a light-driven chloride ion pump. Annual review of biophysics and biophysical chemistry 15, 11-28 (1986).
    連結:
  9. 12 Spudich, J. L. & Bogomolni, R. A. Sensory rhodopsins of halobacteria. Annual review of biophysics and biophysical chemistry 17, 193-215 (1988).
    連結:
  10. 13 Fu, 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. Journal of bacteriology 192, 5866-5873 (2010).
    連結:
  11. 14 Stoeckenius, W., Lozier, R. H. & Bogomolni, R. A. Bacteriorhodopsin and the purple membrane of halobacteria. Biochimica et biophysica acta 505, 215-278 (1979).
    連結:
  12. 16 Oesterhelt, D. & Stoeckenius, W. Rhodopsin-like protein from the purple membrane of Halobacterium halobium. Nature: New biology 233, 149-152 (1971).
    連結:
  13. 17 Henderson, 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. Journal of molecular biology 213, 899-929 (1990).
    連結:
  14. 19 Sasaki, J. & Spudich, J. L. Signal transfer in haloarchaeal sensory rhodopsin- transducer complexes. Photochemistry and photobiology 84, 863-868 (2008).
    連結:
  15. 21 Sasaki, J., Takahashi, H., Furutani, Y., Kandori, H. & Spudich, J. L. Sensory rhodopsin-I as a bidirectional switch: opposite conformational changes from the same photoisomerization. Biophysical journal 100, 2178-2183 (2011).
    連結:
  16. 22 Pfisterer, C., Gruia, A. & Fischer, S. The mechanism of photo-energy storage in the Halorhodopsin chloride pump. The Journal of biological chemistry 284, 13562-13569 (2009).
    連結:
  17. 23 Matsuno-Yagi, A. & Mukohata, Y. Two possible roles of bacteriorhodopsin; a comparative study of strains of Halobacterium halobium differing in pigmentation. Biochemical and biophysical research communications 78, 237-243 (1977).
    連結:
  18. 24 Seki, A., Miyauchi, S., Hayashi, S., Kikukawa, T., Kubo, M., Demura, M., Ganapathy, V. & Kamo, N. Heterologous expression of Pharaonis halorhodopsin in Xenopus laevis oocytes and electrophysiological characterization of its light-driven Cl- pump activity. Biophysical journal 92, 2559-2569 (2007).
    連結:
  19. 25 Dutta, S., Weiner, L. & Sheves, M. Cation binding to halorhodopsin. Biochemistry 54, 3164-3172 (2015).
    連結:
  20. 26 Mukohata, Y. & Kaji, Y. Light-induced ATP synthesis dependent on halorhodopsin-pH regulation. Archives of biochemistry and biophysics 208, 615-617 (1981).
    連結:
  21. 28 Kunji, E. R., von Gronau, S., Oesterhelt, D. & Henderson, R. The three-dimensional structure of halorhodopsin to 5 A by electron crystallography: A new unbending procedure for two-dimensional crystals by using a global reference structure. Proceedings of the National Academy of Sciences of the United States of America 97, 4637-4642 (2000).
    連結:
  22. 29 Kouyama, T., Kanada, S., Takeguchi, Y., Narusawa, A., Murakami, M. & Ihara, K. Crystal structure of the light-driven chloride pump halorhodopsin from Natronomonas pharaonis. J Mol Biol 396, 564-579 (2010).
    連結:
  23. 30 Sasaki, T., Kubo, M., Kikukawa, T., Kamiya, M., Aizawa, T., Kawano, K., Kamo, N. & Demura, M. Halorhodopsin from natronomonas pharaonis forms a trimer even in the presence of a detergent, dodecyl-beta-D-maltoside. Photochemistry and photobiology 85, 130-136 (2009).
    連結:
  24. 31 Essen, L. O. Halorhodopsin: light-driven ion pumping made simple? Current opinion in structural biology 12, 516-522 (2002).
    連結:
  25. 32 Gruia, A. D., Bondar, A. N., Smith, J. C. & Fischer, S. Mechanism of a molecular valve in the halorhodopsin chloride pump. Structure 13, 617-627 (2005).
    連結:
  26. 33 Braiman, M. S., Walter, T. J. & Briercheck, D. M. Infrared spectroscopic detection of light-induced change in chloride-arginine interaction in halorhodopsin. Biochemistry 33, 1629-1635 (1994).
    連結:
  27. 34 Kolbe, M., Besir, H., Essen, L. O. & Oesterhelt, D. Structure of the light-driven chloride pump halorhodopsin at 1.8 A resolution. Science 288, 1390-1396 (2000).
    連結:
  28. 35 Jardon-Valadez, E., Bondar, A. N. & Tobias, D. J. Electrostatic interactions and hydrogen bond dynamics in chloride pumping by halorhodopsin. Biochimica et biophysica acta 1837, 1964-1972 (2014).
    連結:
  29. 36 Otomo, J. Anion selectivity and pumping mechanism of halorhodopsin. Biophysical chemistry 56, 137-141 (1995).
    連結:
  30. 37 Lanyi, J. K. & Vodyanoy, V. Flash spectroscopic studies of the kinetics of the halorhodopsin photocycle. Biochemistry 25, 1465-1470 (1986).
    連結:
  31. 38 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).
    連結:
  32. 39 Gerscher, S., Mylrajan, M., Hildebrandt, P., Baron, M. H., Muller, R. & Engelhard, M. Chromophore-anion interactions in halorhodopsin from Natronobacterium pharaonis probed by time-resolved resonance Raman spectroscopy. Biochemistry 36, 11012-11020 (1997).
    連結:
  33. 40 Sasaki, J., Brown, L. S., Chon, Y. S., Kandori, H., Maeda, A., Needleman, R. & Lanyi, J. K. Conversion of bacteriorhodopsin into a chloride ion pump. Science 269, 73-75 (1995).
    連結:
  34. 41 Kubo, M., Kikukawa, T., Miyauchi, S., Seki, A., Kamiya, M., Aizawa, T., Kawano, K., Kamo, N. & Demura, M. Role of Arg123 in light-driven anion pump mechanisms of pharaonis halorhodopsin. Photochemistry and photobiology 85, 547-555 (2009).
    連結:
  35. 42 Sato, M., Kubo, M., Aizawa, T., Kamo, N., Kikukawa, T., Nitta, K. & Demura, M. Role of putative anion-binding sites in cytoplasmic and extracellular channels of Natronomonas pharaonis halorhodopsin. Biochemistry 44, 4775-4784 (2005).
    連結:
  36. 43 Sato, M., Kikukawa, T., Araiso, T., Okita, H., Shimono, K., Kamo, N., Demura, M. & Nitta, K. Roles of Ser130 and Thr126 in chloride binding and photocycle of pharaonis halorhodopsin. Journal of biochemistry 134, 151-158 (2003).
    連結:
  37. 44 Mukohata, Y., Ihara, K., Tamura, T. & Sugiyama, Y. Halobacterial rhodopsins. Journal of biochemistry 125, 649-657 (1999).
    連結:
  38. 45 Ernst, O. P., Lodowski, D. T., Elstner, M., Hegemann, P., Brown, L. S. & Kandori, H. Microbial and animal rhodopsins: structures, functions, and molecular mechanisms. Chemical reviews 114, 126-163 (2014).
    連結:
  39. 46 Varo, G. Analogies between halorhodopsin and bacteriorhodopsin. Biochimica et biophysica acta 1460, 220-229 (2000).
    連結:
  40. 48 Lakatos, M., Groma, G. I., Ganea, C., Lanyi, J. K. & Varo, G. Characterization of the azide-dependent bacteriorhodopsin-like photocycle of salinarum halorhodopsin. Biophysical journal 82, 1687-1695 (2002).
    連結:
  41. 49 Soliman, G. S. & Trüper, H. G. 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 3, 318-329 (1982).
    連結:
  42. 50 Robinson, J. L., Pyzyna, B., Atrasz, R. G., Henderson, C. A., Morrill, K. L., Burd, A. M., Desoucy, E., Fogleman, R. E., 3rd, Naylor, J. B., Steele, S. M., Elliott, D. R., Leyva, K. J. & Shand, R. F. Growth kinetics of extremely halophilic archaea (family halobacteriaceae) as revealed by arrhenius plots. Journal of bacteriology 187, 923-929 (2005).
    連結:
  43. 52 Aivaliotis, M., Gevaert, K., Falb, M., Tebbe, A., Konstantinidis, K., Bisle, B., Klein, C., Martens, L., Staes, A., Timmerman, E., Van Damme, J., Siedler, F., Pfeiffer, F., Vandekerckhove, J. & Oesterhelt, D. Large-scale identification of N-terminal peptides in the halophilic archaea Halobacterium salinarum and Natronomonas pharaonis. Journal of proteome research 6, 2195-2204 (2007).
    連結:
  44. 53 Kamekura, M., Dyall-Smith, M. L., Upasani, V., Ventosa, A. & Kates, M. Diversity of alkaliphilic halobacteria: proposals for transfer of Natronobacterium vacuolatum, Natronobacterium magadii, and Natronobacterium pharaonis to Halorubrum, Natrialba, and Natronomonas gen. nov., respectively, as Halorubrum vacuolatum comb. nov., Natrialba magadii comb. nov., and Natronomonas pharaonis comb. nov., respectively. International journal of systematic bacteriology 47, 853-857 (1997).
    連結:
  45. 54 Falb, M., Pfeiffer, F., Palm, P., Rodewald, K., Hickmann, V., Tittor, J. & Oesterhelt, D. Living with two extremes: conclusions from the genome sequence of Natronomonas pharaonis. Genome research 15, 1336-1343 (2005).
    連結:
  46. 55 Gonzalez, O., Oberwinkler, T., Mansueto, L., Pfeiffer, F., Mendoza, E., Zimmer, R. & Oesterhelt, D. Characterization of growth and metabolism of the haloalkaliphile Natronomonas pharaonis. PLoS computational biology 6, e1000799 (2010).
    連結:
  47. 56 Nakanishi, T., Kanada, S., Murakami, M., Ihara, K. & Kouyama, T. Large deformation of helix F during the photoreaction cycle of Pharaonis halorhodopsin in complex with azide. Biophysical journal 104, 377-385 (2013).
    連結:
  48. 57 Gmelin, W., Zeth, K., Efremov, R., Heberle, J., Tittor, J. & Oesterhelt, D. The crystal structure of the L1 intermediate of halorhodopsin at 1.9 angstroms resolution. Photochemistry and photobiology 83, 369-377 (2007).
    連結:
  49. 58 Bamberg, E., Tittor, J. & Oesterhelt, D. Light-driven proton or chloride pumping by halorhodopsin. Proceedings of the National Academy of Sciences of the United States of America 90, 639-643 (1993).
    連結:
  50. 59 Kouyama, T., Kawaguchi, H., Nakanishi, T., Kubo, H. & Murakami, M. Crystal structures of the L1, L2, N, and O states of pharaonis halorhodopsin. Biophysical journal 108, 2680-2690 (2015).
    連結:
  51. 60 陳筱儒. 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. 臺灣大學生化科技學系學位論文, 1-63 (2015).
    連結:
  52. 61 Chu, L. K., Yen, C. W. & El-Sayed, M. A. Bacteriorhodopsin-based photo-electrochemical cell. Biosensors & bioelectronics 26, 620-626 (2010).
    連結:
  53. 62 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. Molecular microbiology 88, 551-561 (2013).
    連結:
  54. 63 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. Bioscience reports 32, 501-509 (2012).
    連結:
  55. 64 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).
    連結:
  56. 65 Shibata, M., Saito, Y., Demura, M. & Kandori, H. Deprotonation of Glu234 during the photocycle of Natronomonas pharaonis halorhodopsin. Chemical physics letters 432, 545-547 (2006).
    連結:
  57. 68 Kulcsar, A., Groma, G. I., Lanyi, J. K. & Varo, G. Characterization of the proton-transporting photocycle of pharaonis halorhodopsin. Biophysical journal 79, 2705-2713 (2000).
    連結:
  58. 69 Muneyuki, E., Shibazaki, C., Ohtani, H., Okuno, D., Asaumi, M. & Mogi, T. Time-resolved measurements of photovoltage generation by bacteriorhodopsin and halorhodopsin adsorbed on a thin polymer film. Journal of biochemistry 125, 270-276 (1999).
    連結:
  59. 70 Chizhov, I. & Engelhard, M. Temperature and halide dependence of the photocycle of halorhodopsin from Natronobacterium pharaonis. Biophys J 81, 1600-1612 (2001).
    連結:
  60. 71 Hasegawa, C., Kikukawa, T., Miyauchi, S., Seki, A., Sudo, Y., Kubo, M., Demura, M. & Kamo, N. Interaction of the halobacterial transducer to a halorhodopsin mutant engineered so as to bind the transducer: Cl- circulation within the extracellular channel. Photochem Photobiol 83, 293-302 (2007).
    連結:
  61. 72 Varo, G., Brown, L. S., Sasaki, J., Kandori, H., Maeda, A., Needleman, R. & Lanyi, J. K. Light-driven chloride ion transport by halorhodopsin from Natronobacterium pharaonis. I. The photochemical cycle. Biochemistry 34, 14490-14499 (1995).
    連結:
  62. 74 Nakanishi, T., Kanada, S., Murakami, M., Ihara, K. & Kouyama, T. Large deformation of helix F during the photoreaction cycle of pharaonis halorhodopsin in complex with azide. Biophysical journal 104, 377-385 (2013).
    連結:
  63. 76 Tsai, F. K., Fu, H. Y., Yang, C. S. & Chu, L. K. Photochemistry of a dual-bacteriorhodopsin system in Haloarcula marismortui: HmbRI and HmbRII. The journal of physical chemistry. B 118, 7290-7301 (2014).
    連結:
  64. 77 Hsu, M.-F., Fu, H.-Y., Cai, C.-J., Yi, H.-P., Yang, C.-S. & Wang, A. H.-J. Structural and Functional Studies of a Newly Grouped Haloquadratum walsbyi Bacteriorhodopsin Reveal the Acid-resistant Light-driven Proton Pumping Activity. Journal of Biological Chemistry, jbc. M115. 685065, 29567–29577 (2015).
    連結:
  65. 78 Váró, G., Zimányi, L., Fan, X., Sun, L., Needleman, R. & Lanyi, J. K. Photocycle of halorhodopsin from Halobacterium salinarium. Biophysical journal 68, 2062-2072 (1995).
    連結:
  66. 79 Kouyama, T., Kawaguchi, H., Nakanishi, T., Kubo, H. & Murakami, M. Crystal Structures of the L 1, L 2, N, and O States of pharaonis Halorhodopsin. Biophysical journal 108, 2680-2690 (2015).
    連結:
  67. 80 Pal, R., Sekharan, S. & Batista, V. S. Spectral tuning in halorhodopsin: the chloride pump photoreceptor. Journal of the American Chemical Society 135, 9624-9627 (2013).
    連結:
  68. 4 Briggs, W. R. & Spudich, J. L. Handbook of photosensory receptors. (Wiley-VCH, 2005).
  69. 7 Oesterhelt, D., Hegemann, P. & Tittor, J. The photocycle of the chloride pump halorhodopsin. II: Quantum yields and a kinetic model. The EMBO journal 4, 2351-2356 (1985).
  70. 8 Hegemann, P., Oesterbelt, D. & Steiner, M. The photocycle of the chloride pump halorhodopsin. I: Azide-catalyzed deprotonation of the chromophore is a side reaction of photocycle intermediates inactivating the pump. The EMBO journal 4, 2347-2350 (1985).
  71. 15 Seehra, J. S. & Khorana, H. G. Bacteriorhodopsin precursor. Characterization and its integration into the purple membrane. The Journal of biological chemistry 259, 4187-4193 (1984).
  72. 18 Kagawa, Y., Ohno, K., Yoshida, M., Takeuchi, Y. & Sone, N. Proton translocation by ATPase and bacteriorhodopsin. Federation proceedings 36, 1815-1818 (1977).
  73. 20 Gordeliy, V. I., Labahn, J., Moukhametzianov, R., Efremov, R., Granzin, J., Schlesinger, R., Buldt, G., Savopol, T., Scheidig, A. J., Klare, J. P. & Engelhard, M. Molecular basis of transmembrane signalling by sensory rhodopsin II-transducer complex. Nature 419, 484-487 (2002).
  74. 27 Sharma, A. K., Walsh, D. A., Bapteste, E., Rodriguez-Valera, F., Ford Doolittle, W. & Papke, R. T. Evolution of rhodopsin ion pumps in haloarchaea. BMC evolutionary biology 7, 79 (2007).
  75. 47 Der, A., Szaraz, S., Toth-Boconadi, R., Tokaji, Z., Keszthelyi, L. & Stoeckenius, W. Alternative translocation of protons and halide ions by bacteriorhodopsin. Proceedings of the National Academy of Sciences of the United States of America 88, 4751-4755 (1991).
  76. 51 Konstantinidis, K., Tebbe, A., Klein, C., Scheffer, B., Aivaliotis, M., Bisle, B., Falb, M., Pfeiffer, F., Siedler, F. & Oesterhelt, D. Genome-wide proteomics of Natronomonas pharaonis. Journal of proteome research 6, 185-193 (2007).
  77. 66 Tittor, J., Soell, C., Oesterhelt, D., Butt, H. J. & Bamberg, E. A defective proton pump, point-mutated bacteriorhodopsin Asp96----Asn is fully reactivated by azide. The EMBO journal 8, 3477-3482 (1989).
  78. 67 Sasaki, J., Shichida, Y., Lanyi, J. K. & Maeda, A. Protein changes associated with reprotonation of the Schiff base in the photocycle of Asp96-->Asn bacteriorhodopsin. The MN intermediate with unprotonated Schiff base but N-like protein structure. The Journal of biological chemistry 267, 20782-20786 (1992).
  79. 73 Duschl, A., Lanyi, J. K. & Zimanyi, L. Properties and photochemistry of a halorhodopsin from the haloalkalophile, Natronobacterium pharaonis. The Journal of biological chemistry 265, 1261-1267 (1990).
  80. 75 MacDonald, R. E., Greene, R. V., Clark, R. D. & Lindley, E. V. Characterization of the light-driven sodium pump of Halobacterium halobium. Consequences of sodium efflux as the primary light-driven event. J Biol Chem 254, 11831-11838 (1979).