Rubisco folding and oligomeric assembly [Elektronische Ressource] : Detailed analysis of an assembly intermediate / Amanda Windhof. Betreuer: Franz-Ulrich Hartl

Amanda Windhof

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2011
• Nombre de lectures: 21
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

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-RbcL ________________ 64 8
3.4.3.2 Prokaryotic and Eukaryotic RbcL __ 65
3.4.3.3 Prokaryotic aryotic RbcS __ 65
3.4.3.4 Cyanobacterial-RbcX ___________ 66 2
3.4.3.5 Cyanobacterial-RbcX ______________________________________ 66 2(N-6His)
3.4.3.6 At-RbcXI ____________________ 67 2
3.4.3.7 RbcL /(RbcX ) complex ________ 67 8 2 8
3.4.3.8 Syn6301-RbcL /(AnaCA-RbcX(TAG) ) complexes ______________ 68 8 2(N-6His) 8
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 RbcL /(RbcX ) complexes in E. coli _______________ 73 8 2 8
3.4.4.10 Analytical gel filtration of RbcL /(RbcX ) complexes _________________ 73 8 2 8
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 RbcX in Rubisco folding and assembly _______________ 77 2
4.2.1 RbcX binding motif ________________________________________________ 79 2
4.3 Analysis of the RbcL /(RbcX ) interaction ________________________________ 84 8 2 8
4.3.1 Cryo-EM structure of the RbcL /(RbcX ) complex ________________________ 84 8 2 8
4.3.1.1 Examination of the cryo-EM structure ______________________________ 84
4.3.1.2 Crosslinking of RbcL and RbcX ___ 87
4.3.1.3 MechanismX during in vitro refolding of RbcL ________________ 90 2
4.3.1.4 Role of RbcX in RbcL C-terminal stabilization ______________________ 91 2 8
4.3.2 Crystal structure of RbcL /(RbcX ) complex ____________________________ 92 8 2 8
4.3.2.1 RbcL/RbcX interface ____________________________________________ 93
4.3.2.2 Mechanism of RbcX function in RbcL dimer assembly ________________ 97 2
4.3.2.3 The role of RbcS in RbcX displacement and Rubisco catalysis _________ 100 2
4.4 Analysis of eukaryotic Rubisco and refoldin