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Proteomanalysen an Halobacterium Salinarum

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Proteomanalysen an Halobacterium Salinarum Proteomanalysen an Halobacterium salinarum Christian Klein München 2005 Proteomanalysen an Halobacterium salinarum Christian Klein Dissertation an der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München vorgelegt von Christian Klein aus Gelsenkirchen München, den 27.01.2005 Erklärung Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung vom 29. Januar 1998 von Prof. Dr. D. Oesterhelt betreut. Ehrenwörtliche Versicherung: Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe angefertigt. München, am 26.01.2005 Christian Klein Dissertation eingereicht am 11. September 2004 1. Berichterstatter: Prof. Dr. D. Oesterhelt 2. Berichterstatter: Prof. Dr. N. A. Dencher Tag der mündlichen Prüfung: 14. Januar 2005 Publikationsliste Kokoeva, M.V., Storch, K.-F., Klein, C., Oesterhelt, D. (2002) A novel mode of sensory transduction in archaea: binding protein-mediated chemotaxis towards osmoprotectants and amino acids. EMBO, 21 (10), 2312-2322 Zischka, H., Gloeckner, C. J., Klein, C., Willmann, S., Swiatek de Lange, M., Ueffing, M. (2004) Improved mass spectrometric identification of gel-separated hydrophobic membrane proteins after sodium dodecyl sulphate removal by ion-pair extraction. Proteomics, 4 (12), 3776-3782 Tebbe, A.*, Klein, C.*, Bisle, B., Siedler, F., Scheffer, B., Garcia-Rizo, C., Wolfertz, J., Hickmann, V., Pfeiffer, F., Oesterhelt, D. (2005) Analysis of the Cytosolic Proteome of Halobacterium salinarum and its implication for genome annotation. Proteomics, 5 (1), 168- 179 (* contributed equally) Klein, C., Garcia-Rizo, C., Bisle, B., Scheffer, B., Zischka, H., Pfeiffer, F., Siedler, F, Oesterhelt, D. (2005) The Membrane Proteome of Halobacterium salinarum. Proteomics, 5 (1), 180-197 Garcia-Rizo, C., Pfeiffer, F., Klein, C., Siedler, F., Oesterhelt, D. (in preparation) Improvement of proteomic data analysis and its application to Halobacterium salinarum. Inhaltsverzeichnis 1. Einleitung .............................................................................................................................. 1 1.1 Vorkommen und Einordnung von halophilen Archaea .......................................................... 1 1.2 Halophilie..................................................................................................................................... 2 1.3 Halobakterielle Retinalproteine................................................................................................. 4 1.4 Aufbau der Zellhülle von halophilen Archaea ......................................................................... 6 1.4.1 Struktur des S-Layers von halophilen Archaea .................................................................................... 7 1.4.2 Struktur des Bilayers von halophilen Archaea...................................................................................... 8 1.4.2.1 Phospholipide ............................................................................................................................... 8 1.4.2.2 Glykolipide ................................................................................................................................... 9 1.4.3 Die Purpurmembran ........................................................................................................................... 10 1.5 Flagellen und Flagellarmotor................................................................................................... 11 1.6 Theorie der Elektrophorese ..................................................................................................... 14 1.6.1 Theorie der elektrischen Doppelschicht eines geladenen Teilchens................................................... 14 1.6.2 Bewegung von Polyionen im elektrischen Feld.................................................................................. 15 1.6.3 Theorie der isoelektrischen Fokussierung .......................................................................................... 16 1.6.4 Entwicklung der isoelektrischen Fokusierung.................................................................................... 18 1.6.5 SDS-Elektrophorese ........................................................................................................................... 19 1.6.6 Die zweidimensionale Elektrophorese................................................................................................ 19 1.7 Proteomics und Membranproteine.......................................................................................... 20 1.8 Zielsetzung................................................................................................................................. 22 2. Material und Methoden....................................................................................................... 23 2.1 Material, Geräte, Programme und Abkürzungen ................................................................. 23 2.1.1 Material .............................................................................................................................................. 23 2.1.2 Geräte ................................................................................................................................................. 24 2.1.3 Programme ......................................................................................................................................... 25 2.1.4 Abkürzungen ...................................................................................................................................... 25 2.2 Mikrobiologische Methoden..................................................................................................... 27 2.2.1 Anzucht von Halobacterium salinarum ............................................................................................. 27 2.2.1.1 Einzelkolonien................................................................................................................................. 27 2.2.1.2 Vorkultur (35 ml)............................................................................................................................. 27 2.2.1.3 Kultur (700 ml)................................................................................................................................ 27 2.2.1.4 Fermentor (10 Liter) ........................................................................................................................ 27 2.2.1.5 Fermentor (100 Liter) ...................................................................................................................... 27 I 2.3 Biochemische Methoden........................................................................................................... 28 2.3.1 Zellaufschlüsse ................................................................................................................................... 28 2.3.1.1 Zellernte...................................................................................................................................... 28 2.3.1.2 Zellaufschluss mittels Dialyse .................................................................................................... 28 2. 3.1.3 Zellaufschluss mittels French-Press........................................................................................... 28 2.3.1.4 Zellaufschluss mittels Sonifizieren ............................................................................................. 29 2.3.1.5 Zellaufschluss mittels Freeze/Thaw............................................................................................ 29 2.3.1.6 Zellaufschluss mittels Taurodeoxycholat.................................................................................... 29 2.3.1.7 Zellaufschluss mittels Gefriertrocknung..................................................................................... 30 2.3.1.8 Zellaufschluss mittels isopyknischer Aufarbeitung .................................................................... 30 2.3.1.9 Übersichtsdiagramm der Zellaufschlüsse und Aufarbeitungen................................................... 31 2.3.2 Membranreinigung ............................................................................................................................. 32 2.3.2.1 Diskontinuierlicher Dichtegradient............................................................................................. 32 2.3.2.2 Kontinuierlicher Dichtegradient ................................................................................................. 32 2.3.2.3 Partielle Solubilisierung mit Triton X-100 ................................................................................. 32 2.3.2.4 Phasenseparation mittels Triton X-114....................................................................................... 32 2.3.2.5 Carbonat-Extraktion.................................................................................................................... 33 2.3.2.6 Chloroform/Methanol-Extraktion ............................................................................................... 33 2.3.3 Cytosolgewinnung.............................................................................................................................. 33 2.3.3.1 Cytosolfraktionierung mittels Gelchromatographie.................................................................... 33 2.3.3.2 Cytosolfraktionierung mittels Ammoniumsulfat-Fällung........................................................... 34 2.3.4 Motorisolierung
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