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Structural Analysis of Membrane Protein Biogenesis and Ribosome Stalling by Cryo-Electron Microscopy

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Structural Analysis of Membrane Protein Biogenesis and Ribosome Stalling by Cryo-Electron Microscopy Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München Structural Analysis of Membrane Protein Biogenesis and Ribosome Stalling by Cryo-Electron Microscopy Lukas Sebastian Bischoff aus Gräfelfing 2015 Erklärung Diese Dissertation wurde im Sinne von § 7 der Promotionsordnung vom 28.November 2011 von Herrn Prof. Dr. Roland Beckmann betreut. Eidesstattliche Versicherung Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet. München, den 05.02.2015 ………………………………… Lukas Bischoff Dissertation eingereicht am 05.02.2015 1. Gutachter: Prof. Dr. Roland Beckmann 2. Gutachter: PD. Dr. Dietmar Martin Mündliche Prüfung am 28.04.2015 Parts of this thesis have been published: Visualization of a polytopic membrane protein during SecY-mediated membrane insertion. Bischoff L, Wickles S, Berninghausen O, van der Sluis EO, Beckmann R. Nature Communications. 2014 Jun 10;5:4103. doi: 10.1038/ncomms5103. Molecular Basis for the Ribosome Functioning as an L-Tryptophan Sensor. Bischoff L, Berninghausen O, Beckmann R. Cell Reports. 2014 Oct 7. pii: S2211-1247(14)00779-7. doi: 10.1016/j.celrep.2014.09.011. A structural model of the active ribosome-bound membrane protein insertase YidC. Wickles S, Singharoy A, Andreani J, Seemayer S, Bischoff L, Berninghausen O, Soeding J, Schulten K, van der Sluis EO, Beckmann R. Elife. 2014 Jul 10;3:e03035. doi: 10.7554/eLife.03035. Dynamic behavior of Trigger Factor on the ribosome Deeng J, Chan KY, van der Sluis EO, Bischoff L, Berninghausen O, Han W, Gumbart J, Schulten K, Beatrix B. and Beckmann R. SUBMITTED Parts of this thesis have been presented at international conferences: Kuo Symposium on 3D cryo-EM Molecular Imaging, 26.07.14 – 30.07.14, Shanghai, China, poster presentation Gordon Research Conference: 'Three-Dimensional Electron Microscopy’, 22.06.14 – 27.06.14, Girona, Spain, poster presentation Gordon Research Conference: ‘Protein Transport Across Cell Membranes’, 09.03.14 – 14.03.14 Galveston, USA, oral and poster presentation EMBO Conference Series: ‘From Structure to Function of Translocation Machines’, 13.04.13 – 17.04.13, Dubrovnik, Croatia, poster presentation Gordon Research Conference: ‘Protein Transport Across Cell Membranes’, 11.03.12 – 16.03.12 Galveston, USA, oral and poster presentation I Table of Content 1. Introduction ................................................................................................................... 1 1.1 Protein translocation in bacteria ..................................................................................... 1 1.1.1 The SecYEG translocon ............................................................................................. 3 1.1.2 Co-translation protein targeting by SRP and FtsY .................................................... 4 1.1.4 Post-translation protein translocation by the SecA/B pathway ............................... 6 1.1.5 Translocation of secretory proteins through the SecYEG complex .......................... 8 1.1.6 Sealing of the channel ............................................................................................... 9 1.1.7 Membrane protein integration by SecY ................................................................... 9 1.1.8 Membrane protein topology and the positive inside rule ..................................... 12 1.1.9 The bacterial membrane insertase YidC ................................................................. 13 1.1.10 A thermodynamic view on membrane protein biogenesis .................................. 16 1.2 Translation regulation by arrest peptides ..................................................................... 18 1.2.1 General mechanisms of translation regulation by arrest peptides ........................ 18 1.2.2 Ribosomal stalling on the TnaC peptide in E.coli .................................................... 20 1.3 Cryo-EM and the resolution revolution ......................................................................... 24 2. Aims of this thesis ......................................................................................................... 27 3. Materials and Methods ................................................................................................ 28 3.1 General ........................................................................................................................... 28 3.2 Vectors ........................................................................................................................... 28 3.3 PCR Primers .................................................................................................................... 28 3.4 Bacteria and Media ........................................................................................................ 30 3.5 Molecular Cloning .......................................................................................................... 30 3.5.1 Polymerase chain reaction (PCR) ............................................................................ 30 3.5.2 Purification of PCR products ................................................................................... 32 3.5.3 Restriction ............................................................................................................... 32 3.5.4 Degradation of parental DNA with DpnI ................................................................. 32 3.5.5 Analysis of DNA fragments by agarose gel electrophoresis ................................... 32 3.5.6 Purification of DNA fragments from agarose gels .................................................. 33 3.5.7 Ligation .................................................................................................................... 33 3.5.8 Plasmid isolation ..................................................................................................... 34 3.5.9 DNA sequencing ...................................................................................................... 34 II 3.6 Analysis of proteins ........................................................................................................ 34 3.6.1 Protein concentration ............................................................................................. 34 3.6.2 Protein concentration determination ..................................................................... 34 3.6.3 Protein precipitation ............................................................................................... 35 3.6.4 SDS-polyacrylamid gel electrophoresis (PAGE) ...................................................... 35 3.6.5 Western blotting ..................................................................................................... 36 3.6.6 Antibody detection ................................................................................................. 36 3.7 Protein expression and purification from E.coli ............................................................ 37 3.7.1 Pre-culture, induction and expression in general ................................................... 37 3.7.2 Purification of E.coli SecY(His)EG ............................................................................ 37 3.7.3 Purification of E.coli SecA(His) ................................................................................ 38 3.7.4 Purification of E.coli FtsY(His) ................................................................................. 39 3.8 Purification of TnaC-stalled Ribosome nascent chain complexes (RNCs) ..................... 40 3.8.1 Purification of TnaC-stalled ribosome nascent chain complexes from the whole cell lysate .......................................................................................................................... 40 3.8.2 Purification of TnaC-stalled ribosome nascent chain-ligand complexes ................ 41 3.8.3 Purification of membrane bound Ribosome nascent chain complexes ................. 41 3.9 RNC-SecYEG complex purification for cryo-EM data collection .................................... 42 3.10 Site-directed cross-linking with an unnatural amino acid ........................................... 43 3.11 In vitro translocation assay .......................................................................................... 43 3.11.1 Preparation of inverted membrane vesicles ........................................................ 43 3.11.2 Preparation of SecYEG proteoliposomes .............................................................. 44 3.11.3 Translocation assay ............................................................................................... 44 3.12 Ribosome binding assay ............................................................................................... 44 3.13 Cryo-EM data collection ............................................................................................... 45 3.13.1 Data collection of the PR2Q-SecYE complex ........................................................ 45 3.13.2 Data collection of the TnaC-stalled ribosome ...................................................... 45 3.14 Cryo-EM data processing ............................................................................................. 46 3.14.1 Preprocessing of cryo-EM data ............................................................................. 46 3.14.2 Refinement and Sorting ........................................................................................ 46 3.14.3 Resolution determination
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