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Structural Characterization of Bacterial Defense Complex Marko Nedeljković

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Structural Characterization of Bacterial Defense Complex Marko Nedeljković Structural characterization of bacterial defense complex Marko Nedeljković To cite this version: Marko Nedeljković. Structural characterization of bacterial defense complex. Biomolecules [q-bio.BM]. Université Grenoble Alpes, 2017. English. NNT : 2017GREAV067. tel-03085778 HAL Id: tel-03085778 https://tel.archives-ouvertes.fr/tel-03085778 Submitted on 22 Dec 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. THÈSE Pour obtenir le grade de DOCTEUR DE LA COMMUNAUTE UNIVERSITE GRENOBLE ALPES Spécialité : Biologie Structurale et Nanobiologie Arrêté ministériel : 25 mai 2016 Présentée par Marko NEDELJKOVIĆ Thèse dirigée par Andréa DESSEN préparée au sein du Laboratoire Institut de Biologie Structurale dans l'École Doctorale Chimie et Sciences du Vivant Caractérisation structurale d'un complexe de défense bactérienne Structural characterization of a bacterial defense complex Thèse soutenue publiquement le 21 décembre 2017, devant le jury composé de : Monsieur Herman VAN TILBEURGH Professeur, Université Paris Sud, Rapporteur Monsieur Laurent TERRADOT Directeur de Recherche, Institut de Biologie et Chimie des Protéines, Rapporteur Monsieur Patrice GOUET Professeur, Université Lyon 1, Président Madame Montserrat SOLER-LOPEZ Chargé de Recherche, European Synchrotron Radiation Facility, Examinateur Madame Andréa DESSEN Directeur de Recherche, Institut de Biologie Structurale , Directeur de These 2 Contents ABBREVIATIONS ............................................................................................................................... 5 Summary ................................................................................................................................................ 7 Résumé ................................................................................................................................................... 9 Acknowledgements ............................................................................................................................. 11 INTRODUCTION ............................................................................................................................... 13 1.1. Host-pathogen interaction and virulence factors ................................................................. 15 1.2. Proteases as virulence factors ................................................................................................ 16 1.3. Protease inhibitors .................................................................................................................. 18 1.4. The α2M subfamily ................................................................................................................. 19 1.5. Structure of human α-2-macroglobulin ................................................................................ 23 1.6. Bacterial α2Ms ........................................................................................................................ 24 1.7. The bacterial cell wall ............................................................................................................. 27 1.8. Peptidoglycan precursor synthesis ........................................................................................ 28 1.9. Penicillin-Binding Proteins..................................................................................................... 31 1.10. Glycan chain polymerization ............................................................................................. 32 1.11. Cross-linking ....................................................................................................................... 33 1.12. Class A PBPs ....................................................................................................................... 35 1.13. Penicillin-binding protein 1c .............................................................................................. 37 AIMS OF THE PROJECT ................................................................................................................ 38 MATERIALS AND METHODS ....................................................................................................... 41 2.1. PBP1c sequence analysis ........................................................................................................ 43 2.2. Cloning ..................................................................................................................................... 43 2.3. Protein expression and purification ...................................................................................... 44 2.4. Electrophoresis ........................................................................................................................ 47 2.5. Western blotting ...................................................................................................................... 49 2.6. Mass spectrometry .................................................................................................................. 49 2.7. Analytical ultracentrifugation (AUC) and SEC-MALS ...................................................... 50 3 2.8. Thermal shift assay ................................................................................................................. 53 2.9. The PBP1c activity test ........................................................................................................... 55 2.10. Small-Angle X-ray Scattering (SAXS) .............................................................................. 56 2.11. Electron microscopy ........................................................................................................... 57 2.12. Crystallization trials ........................................................................................................... 58 RESULTS ............................................................................................................................................ 59 3.1. Expression and purification of ECAM .................................................................................. 61 3.2. Purification and characterization of PBP1c ......................................................................... 63 3.2.1. Amino acid sequence analysis ........................................................................................ 63 3.2.2. Expression and solubilization ........................................................................................ 66 3.2.3. Purification of PBP1c ...................................................................................................... 69 3.2.4. SEC-MALS analysis of PBP1c ....................................................................................... 72 3.2.5. Cell-free expression and purification of PBP1c ............................................................ 74 3.2.6. Mass spectrometry of PBP1c ......................................................................................... 75 3.2.7. Thermal stability analysis .............................................................................................. 76 3.3. Complex reconstitution and characterization ...................................................................... 79 3.3.1. Complex formation ......................................................................................................... 79 3.3.2. AUC analysis ................................................................................................................... 80 3.3.3. Peptidoglycan synthesis assay ........................................................................................ 83 3.3.4. Crystallization trials of PBP1c in apo form and in the presence of moenomycin ..... 87 3.3.5. Negative staining electron microscopy .......................................................................... 89 3.3.6. SAXS ................................................................................................................................ 92 DISCUSSION AND PERSPECTIVES ........................................................................................... 103 References .......................................................................................................................................... 110 INDEX ................................................................................................................................................ 122 ANNEXES ......................................................................................................................................... 124 4 ABBREVIATIONS α2M α-2-macroglobulin AMP Adenosine monophosphate ATP Adenosine triphosphate AUC Analytical ultracentrifugation C55-PP Undecaprenyl pyrophosphate CMC Critical micellar concentration CTP Cytosine triphosphate DDM n-dodecyl-β-D-maltopyranoside DM n-decyl-β-D-maltopyranoside DNA Deoxyribonucleic acid DTT Dithiothreitol ECAM E. coli α-2-macroglobulin EM Electron microscopy GlcNAc N-acetylglucosamine GT, GTase Glucosyltransferase GTP Guanosine triphosphate HPLC High-performance
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