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Rubisco Folding and Oligomeric Assembly: Detailed Analysis of an Assembly Intermediate

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Rubisco Folding and Oligomeric Assembly: Detailed Analysis of an Assembly Intermediate Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München Rubisco folding and oligomeric assembly: Detailed analysis of an assembly intermediate Amanda Windhof (geb. Starling) aus Delaware, Ohio, USA 2011 Acknowledgments I especially want to thank Prof. Dr. F. Ulrich Hartl and Dr. Manajit Hayer-Hartl for giving me the opportunity to conduct my PhD research in the department. I have learned so much on this journey which would not have been possible without your intellectual support and scientific enthusiasm. I also want to thank PD Dr. Dr. Winklhofer for being my 2. Gutacherin and of course the other members of my PhD committee: Prof. Dr. Beckmann, Prof. Dr. Vothknecht, Prof. Dr. Gaul, and Prof. Dr. Hopfner. There are also many people I want to thank for within the department and institute. I am very grateful to Dr. Cuimin Liu for her help and guidance when I first came to this department. She was always open to answer any questions I had. Thank you Dr. Andreas Bracher for your support in many ways, in particular with your crystallographic knowledge and various ideas for developing enhanced methods. I also want to thank Dr. Oliver Müller- Cajar for your never ending passion for science, especially for Rubisco, and for your very insightful discussions. I am grateful for the lunch conversations with Dr. Yi-chin Tsai and your mass spectrometry expertise. I would also like to thank Thomas Hauser and Mathias Stotz for your valuable discussions and input. Thank you to Evelyn Frey-Royston, Silke Leuze-Bütün, and Andrea Obermayr-Rauter for their administrative support as well as Emmanuel Burghardt, Bernd Grampp, Romy Lange, Nadine Wischnewski, Verena Marcus, Elisabeth Schreil, Albert Ries, Andreas Scaia, and Kevin Gaughan for their invaluable technical support. Thank you to Dr. A. Young and Prof. Dr. R. Beckmann for your cryo-EM expertise and interpretation. I also want to thank Reinhard Mentele and Elisabeth Weyher-Stingl for technical support as well as the staff of the MPI Animal and Sequencing Facilities. Also, to the rest of the lab, I am very thankful for the help and assistance I have received while being here and all the fun times we have shared. Last but certainly not least I want to express my thankfulness for my family, particularly my husband, Tim Windhof, and my parents, Larry and Pam Starling. I cannot thank you enough for all the love and support you have given me over the years. I want to thank Tim for always being there for me, his practical guidance, his support, and for everything he has taught me. I CONTENTS Contents 1 Summary ______________________________________________________________ 1 2 Introduction ____________________________________________________________ 3 2.1 Protein folding _________________________________________________________ 3 2.2 Molecular chaperones ___________________________________________________ 6 2.2.1 De novo protein folding _______________________________________________ 6 2.2.1.1 Ribosome-associated chaperones ___________________________________ 7 2.2.1.2 Hsp70 system __________________________________________________ 9 2.2.1.3 Chaperonins ___________________________________________________ 11 2.2.1.3.1 E. coli chaperonin system ______________________________________ 11 2.2.1.3.2 Cyanobacterial chaperonin _____________________________________ 14 2.2.1.3.3 Chloroplast chaperonin ________________________________________ 17 2.3 Photosynthesis ________________________________________________________ 20 2.3.1 Light-dependent reactions ____________________________________________ 22 2.3.2 Light-independent reactions __________________________________________ 24 2.3.3 Carbon concentrating mechanisms _____________________________________ 25 2.3.3.1 C4 Plants _____________________________________________________ 26 2.3.3.2 Crassulacean acid metabolism (CAM) photosynthesis __________________ 28 2.3.3.3 Pyrenoid _____________________________________________________ 28 2.3.3.4 Carboxysome __________________________________________________ 29 2.3.4 Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) ________________ 30 2.3.4.1 Rubisco regulation ______________________________________________ 32 2.3.4.2 Rubisco structure _______________________________________________ 33 2.3.4.3 Rubisco folding and assembly_____________________________________ 36 2.3.4.4 RbcX ________________________________________________________ 38 2.4 Aim of study __________________________________________________________ 42 3 Materials and Methods __________________________________________________ 43 3.1 Materials ____________________________________________________________ 43 3.1.1 Chemicals ________________________________________________________ 43 3.1.2 Reagents and Purification kits _________________________________________ 43 3.1.3 Strains ___________________________________________________________ 43 3.1.4 Plasmids, DNA, and oligonucleotides ___________________________________ 44 3.1.5 Enzymes, proteins, peptides, and antibodies ______________________________ 47 3.1.6 Media ____________________________________________________________ 47 3.1.7 Buffers and Standard Solutions ________________________________________ 48 3.2 Instruments __________________________________________________________ 50 3.3 Molecular biological methods ___________________________________________ 51 3.3.1 DNA analytical methods _____________________________________________ 51 3.3.2 Competent E. coli cell preparation and transformation______________________ 52 3.3.2.1 Chemocompetent E. coli cells and chemical transformation _____________ 52 3.3.2.2 Electrocompetent E. coli cells and electroporation _____________________ 52 CONTENTS II 3.3.2.3 TSS transformation _____________________________________________ 53 3.3.3 Plasmid DNA and DNA fragment purification ____________________________ 53 3.3.4 PCR (polymerase chain reaction) ______________________________________ 53 3.3.5 Site-directed mutagenesis ____________________________________________ 54 3.3.6 Restriction digest and DNA ligation ____________________________________ 55 3.3.7 Cloning strategies __________________________________________________ 56 3.4 Protein biochemical and biophysical methods ______________________________ 57 3.4.1 Protein analytical methods ___________________________________________ 57 3.4.1.1 Protein quantification and sequence alignments _______________________ 57 3.4.1.2 SDS-PAGE ___________________________________________________ 57 3.4.1.3 Gradient SDS-PAGE ____________________________________________ 58 3.4.1.4 Native-PAGE _________________________________________________ 59 3.4.1.5 CN Bis-tris-PAGE ______________________________________________ 59 3.4.1.6 Coomassie blue staining of polyacrylamide gels ______________________ 60 3.4.1.7 Western blotting and immunodetection _____________________________ 60 3.4.1.8 Autoradiography _______________________________________________ 61 3.4.1.9 Generation of anti-serum _________________________________________ 61 3.4.1.10 TCA precipitation ______________________________________________ 62 3.4.1.11 SEC-MALS ___________________________________________________ 62 3.4.1.12 Proteolytic digestion and Edman degradation _________________________ 62 3.4.1.13 Mass spectrometry ______________________________________________ 63 3.4.1.14 CD (circular dichroism) spectroscopy _______________________________ 63 3.4.2 Protein crystallization _______________________________________________ 63 3.4.3 Protein expression and purification _____________________________________ 64 3.4.3.1 Syn6301-RbcL8 ________________________________________________ 64 3.4.3.2 Prokaryotic and Eukaryotic RbcL __________________________________ 65 3.4.3.3 Prokaryotic and Eukaryotic RbcS __________________________________ 65 3.4.3.4 Cyanobacterial-RbcX2 ___________________________________________ 66 3.4.3.5 Cyanobacterial-RbcX2(N-6His) ______________________________________ 66 3.4.3.6 At-RbcXI2 ____________________________________________________ 67 3.4.3.7 RbcL8/(RbcX2)8 complex ________________________________________ 67 3.4.3.8 Syn6301-RbcL8/(AnaCA-RbcX(TAG)2(N-6His))8 complexes ______________ 68 3.4.4 Functional analyses _________________________________________________ 69 3.4.4.1 Amber stop codon suppression ____________________________________ 69 3.4.4.2 Site-specific crosslinking ________________________________________ 69 3.4.4.3 Rubisco carboxylation activity assay of E. coli lysate __________________ 69 3.4.4.4 RTS: in vitro translation of Rubisco ________________________________ 70 3.4.4.5 PURE system __________________________________________________ 71 3.4.4.6 CABP synthesis from RuBP ______________________________________ 71 3.4.4.7 Disulfide crosslinking ___________________________________________ 72 3.4.4.8 In vivo expression of cyanobacterial GroEL and GroES in E. coli _________ 72 3.4.4.9 In vivo assembly of RbcL8/(RbcX2)8 complexes in E. coli _______________ 73 3.4.4.10 Analytical gel filtration of RbcL8/(RbcX2)8 complexes _________________ 73 3.4.4.11 Co-immunoprecipitation _________________________________________ 73 3.4.4.12 Rubisco in vitro refolding ________________________________________ 74 3.4.4.13 Peptide binding screen __________________________________________ 74 4 Results _______________________________________________________________ 76 4.1 Characterization of cyanobacterial chaperonin systems ______________________ 76 III CONTENTS 4.2 Role of GroEL/ES and RbcX2
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