Developement of strategies for the isotopic labeling of methyl groups for the NMR study of large protein assemblies Rime Kerfah To cite this version: Rime Kerfah. Developement of strategies for the isotopic labeling of methyl groups for the NMR study of large protein assemblies. Biomolecules [q-bio.BM]. Université de Grenoble, 2014. English. NNT : 2014GRENV043. tel-01343865 HAL Id: tel-01343865 https://tel.archives-ouvertes.fr/tel-01343865 Submitted on 10 Jul 2016 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. 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THÈSE Pour obtenir le grade de DOCTEUR DE L’UNIVERSITÉ DE GRENOBLE Spécialité : Biologie structurale et nanobiologie Présentée par Rime Kerfah Thèse dirigée par Jérôme Boisbouvier Préparée au sein du Groupe de Spéctrosopie RMN Biomoléculaire École Doctorale Chimie et Sciences du Vivant Développement de stratégies de marquage isotopique des groupements méthyles pour l'étude d'assemblages protéiques de grande taille par RMN Date de soutenance le 26 Septembre 2014, devant le jury composé de : Bruno Kieffer Président Carine Tisné Rapporteur Ewen Lescop Rapporteur Olivier Hamelin Membre Michael Plevin Membre Jérôme Boisbouvier Directeur de thèse ACKNOWLEDGMENTS I wish to thank my supervisor, Dr. Boisbouvier, for the guidance and generosity that he has provided me. I consider it a privilege to have had the opportunity in working within his research group. I would like to acknowledge with sincere gratitude to Elodie and Pavel for their assistance in my early life in science and also Guillaume for his ingenious IT advises. I am also really grateful for the scientific support of Pierre and Dominique and for their cultural discussions. I would like to thank the whole NMR, group, especially Adrien, Karine, Catherine, Lionel, Isabel, Paul, Hugo and Jia-ying for the collaboration and the excellent work environment that have proportioned to me. My thesis would not be possible without the presence of my parents, brothers and my best friend, Amal. For all of them I would like to express my gratitude for being always by my side. To conclude, I would like to thank Victor, who has been my energy source to pursue this step of my life and who has always managed to put a smile on my face in difficult circumstances. Content 1. INTRODUCTION……………….. .......................................................................................... 1 1.1 Overview of isotope labeling in solution NMR spectroscopy: ....................................................... 2 1.2 Selective protonation and isotopic labeling in a perdeuterated biological system ........................ 5 1.2.1 Production of labeled proteins .................................................................................................. 5 1.2.1.1 In vivo protein production ................................................................................................ 5 1.2.1.2 In vitro protein production ............................................................................................... 8 1.2.2 Optimal sites for selective protonation and isotopic labeling ................................................. 13 1.2.2.1 Selective protonation and isotopic labeling on the backbone: ..................................... 13 1.2.2.1.1 Selective protonation and labeling of amide groups ................................................ 13 1.2.2.1.2 Selective protonation and isotopic labeling at the C position ................................. 19 1.2.2.2 Selective protonation and isotopic labeling on the side chain ...................................... 20 1.2.2.2.1 Stereo-array Isotope Labeling (SAIL) ......................................................................... 21 1.2.2.2.2 Specific protonation position in aromatic amino acids ............................................. 23 1.2.2.2.3 Methyl groups labeling ............................................................................................. 25 1.3 Selective protonation and isotopic labeling of methyl groups in perdeuterated biological systems ………………………………………………………………………………………………………………………………………………25 1.3.1 Why methyl groups? ............................................................................................................... 25 1.3.2 Labeling strategy for each methyl-containing amino acid ...................................................... 29 1.3.2.1 Amino acids involved in irreversible metabolic pathways ............................................. 29 1.3.2.1.1 Isoleucine residue methyl groups labeling ................................................................ 29 1.3.2.1.2 Leucine and valine residues methyl groups labeling................................................. 37 1.3.2.1.3 Methionine residue methyl group labeling ............................................................... 44 1.3.2.2 Amino acids involved in reversible metabolic pathways ............................................... 45 1.3.2.2.1 Alanine residue methyl group labeling ..................................................................... 45 1.3.2.2.2 Threonine residue methyl group labeling ................................................................. 49 1.3.2.3 Grafted Methyl group .................................................................................................... 56 1.3.2.3.1 Grafted methyl group on cysteine ............................................................................ 57 1.3.2.3.2 Grafted methyl group on lysine: ............................................................................... 57 2. COMBINATORIAL LABELING OF METHYL GROUPS............................................. 59 2.1 Context ....................................................................................................................................... 60 2.2 Article I: scrambling-free combinatorial labeling of alanine-, isoleucine-1, leucine-pro-S and valine-pro-S groups for the detection of long-range NOEs ................................................................... 64 3. ASSIGNMENT OF METHYL GROUPS ......................................................................... 83 3.1 Context ....................................................................................................................................... 84 3.2 Methyl groups assignment in large proteins ............................................................................... 84 3.2.1 Methyl groups assignment based on bond-transfer NMR experiments: ................................ 85 3.2.2 Methyl group assignment using structure-based methods .................................................... 85 3.2.3 Stereospecific assignment of methyl groups .......................................................................... 88 3.2.4 Article II: A single step regio- and stereo-specific NMR assignment of isoleucine, leucine and valine residues in large proteins ................................................................................................... 90 3.3 Methyl groups assignment in supramolecular assemblies ........................................................ 110 3.3.1 Historical overview ................................................................................................................ 110 3.3.2 Methyl groups assignment using the “divide and conquer” strategy ................................... 110 3.3.3 Methyl groups assignments using mutagenesis .................................................................... 111 13 3.3.4 Article III: A cost-effective protocol for the parallel production of libraries of CH3- specifically labeled mutants for NMR studies of high molecular weight proteins ........................... 113 4. APPLICATIONS OF METHYL GROUPS LABELING STRATEGY ......................... 134 4.1 Labeling of methyl groups for real-time NMR studies of protein oligomerization process ........ 135 4.1.1 Context .................................................................................................................................. 135 4.1.2 Article IV: Probing protein self-assembly by real-time NMR spectroscopy ........................ 136 4.2 Combinatorial methyl groups labeling for long-range nOes detection ..................................... 156 4.2.1 Context .................................................................................................................................. 156 4.2.2 Combinatorial labeling of methyl groups for the detection of long-range nOes in perdeuterated large systems ............................................................................................................... 158 4.2.3 Detection of long-range nOes in MSG labeled simultaneously at AI1(LV)proS ....................... 162 4.2.4 Generalization to a supramolecular protein (PhTET-2) ......................................................... 164 4.3 Combinatorial methyl groups labeling for the filetering of inter-monomeric nOes in homo- oligomeric proteins ...........................................................................................................................