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Ancestral Β-Subunit Protein’ (Anbu) from Hyphomicrobium Sp

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Ancestral Β-Subunit Protein’ (Anbu) from Hyphomicrobium Sp Technische Universität München Fakultät für Chemie Lehrstuhl für Biochemie X-ray crystallographic analysis of the ‘ancestral β-subunit protein’ (Anbu) from Hyphomicrobium sp. and the hetero-oligomeric protease ClpP1/2 from Listeria monocytogenes Marie-Theres Vielberg Vollständiger Abdruck der von der Fakultät für Chemie der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigten Dissertation. Vorsitzender: Prof. Dr. Aymelt Itzen Prüfende der Dissertation: 1. Prof. Dr. Michael Groll 2. Prof. Dr. Ville Kaila Die Dissertation wurde am 07.11.2017 bei der Technischen Universität München eingereicht und durch die Fakultät für Chemie am 01.12.2017 angenommen. Für Mischa und meine Familie Table of Contents Table of Contents 1 Summary ....................................................................................................................... 1 2 Zusammenfassung ........................................................................................................ 3 3 Introduction .................................................................................................................. 5 4 Objective ....................................................................................................................... 8 5 Materials and Methods ............................................................................................... 10 5.1 Materials .............................................................................................................. 10 5.1.1 Chemicals ..................................................................................................... 10 5.1.2 Antibiotics .................................................................................................... 10 5.1.3 Bacterial strains ............................................................................................ 10 5.1.4 Buffer and Media ......................................................................................... 11 5.1.5 DNA and protein standards .......................................................................... 13 5.1.6 Enzymes ....................................................................................................... 13 5.1.7 Kits and pre-made solutions......................................................................... 13 5.1.8 Plasmids ....................................................................................................... 14 5.1.9 Primers ......................................................................................................... 14 5.1.10 Synthetic gene sequences ............................................................................. 15 5.1.11 Laboratory equipment .................................................................................. 15 5.1.12 Bioinformatic tools, software and databases ............................................... 17 5.2 Methods ............................................................................................................... 18 5.2.1 Molecular biology ........................................................................................ 18 5.2.1.1 Cultivation and storage of Escherichia coli ......................................... 18 5.2.1.2 Preparation of plasmid DNA from E. coli ............................................ 19 5.2.1.3 PCR Amplification ............................................................................... 19 5.2.1.4 Site-directed mutagenesis ..................................................................... 20 5.2.1.5 Agarose gel electrophoresis .................................................................. 20 5.2.1.6 DNA purification .................................................................................. 21 5.2.1.7 DNA concentration measurement ........................................................ 21 5.2.1.8 DNA sequencing................................................................................... 21 5.2.1.9 Restriction digest of DNA .................................................................... 21 5.2.1.10 Ligation of DNA fragments .................................................................. 22 5.2.1.11 Transformation of electro-competent E. coli cells ............................... 22 5.2.2 Protein chemistry ......................................................................................... 23 i Table of Contents 5.2.2.1 Heterologous gene expression in E. coli .............................................. 23 5.2.2.2 Production of ʟ-selenomethionine-labelled protein .............................. 23 5.2.2.3 Cell disruption by sonication ................................................................ 24 5.2.2.4 Chromatographic purification of HyAnbu ............................................ 24 5.2.2.5 Buffer exchange by dialysis ................................................................. 25 5.2.2.6 Concentration of a protein sample ........................................................ 25 5.2.2.7 Measurement of protein concentration ................................................. 26 5.2.2.8 Protein storage ...................................................................................... 26 5.2.2.9 SDS-PAGE ........................................................................................... 26 5.2.3 Activity assays ............................................................................................. 27 5.2.4 Protein crystallization and structure determination ..................................... 27 5.2.4.1 Crystallization ....................................................................................... 27 5.2.4.2 Data collection and processing ............................................................. 28 5.2.4.3 Experimental phase determination ....................................................... 29 5.2.4.4 Patterson search techniques .................................................................. 29 5.2.4.5 Model building and refinement ............................................................ 29 5.2.5 Graphic representation and biocomputational analyses ............................... 30 6 Structural and functional analysis of Anbu from Hyphomicrobium sp. strain MC1 .. 31 6.1 Introduction ......................................................................................................... 31 6.1.1 The 20S proteasome in eukaryotes and archaea .......................................... 31 6.1.2 Proteasomes and proteasome-like particles in bacteria ............................... 37 6.1.2.1 Actinobacteria and the 20S proteasome ............................................... 37 6.1.2.2 HslV – a bacterial homologue of the CP .............................................. 39 6.1.3 Anbu - the missing link in proteasomal evolution? ..................................... 40 6.2 Results ................................................................................................................. 42 6.2.1 Cloning and purification of HyAnbu ........................................................... 42 6.2.2 Activity measurements................................................................................. 43 6.2.3 Crystallization and structure determination ................................................. 44 6.2.4 The structure of HyAnbu ............................................................................. 47 6.2.4.1 Anbu’s subunits adopt the fold of Ntn-hydrolases ............................... 48 6.2.4.2 The protomer of Anbu is a dimer ......................................................... 51 6.2.4.3 The helical superstructure of HyAnbu .................................................. 53 6.2.4.4 The putative active site of Anbu ........................................................... 55 ii Table of Contents 6.3 Discussion ........................................................................................................... 57 6.3.1 Anbu proteins in various species – different or similar? ............................. 57 6.3.2 The relation between Anbu and its proteasomal homologues ..................... 59 6.3.3 The missing catalytic function ..................................................................... 62 6.3.4 Anbu - the ancestral -subunit protein? ....................................................... 64 7 Structural analysis of the ClpP1/2 heterocomplex from Listeria monocytogenes ...... 65 7.1 Introduction ......................................................................................................... 65 7.1.1 Antibiotic resistances threaten public health care ........................................ 65 7.1.2 Disarming bacteria instead of killing them .................................................. 66 7.1.3 Caseinolytic protease P is essential for the virulence of several pathogens 67 7.1.4 The bacterial Clp-degradation machinery .................................................... 67 7.1.5 The significance of ClpP for the virulence of Listeria monocytogenes ....... 71 7.1.6 The LmClpP1/2 heterocomplex ................................................................... 72 7.2 Results ................................................................................................................. 75 7.2.1 Solving the structure of ClpP1/2 from L. monocytogenes ........................... 75 7.2.2 Structural characteristics of LmClpP1/2 ...................................................... 76 7.2.2.1 General features of the
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