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Emerging Roles for Natural and Artificial Lipids in Shaping the Catalytic Function, Stability and Oligomeric State of Membrane Proteins

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Emerging Roles for Natural and Artificial Lipids in Shaping the Catalytic Function, Stability and Oligomeric State of Membrane Proteins Emerging roles for natural and artificial lipids in shaping the catalytic function, stability and oligomeric state of membrane proteins. Batoul Srour To cite this version: Batoul Srour. Emerging roles for natural and artificial lipids in shaping the catalytic function, stability and oligomeric state of membrane proteins.. Other. Université de Strasbourg, 2015. English. NNT : 2015STRAF068. tel-01304805 HAL Id: tel-01304805 https://tel.archives-ouvertes.fr/tel-01304805 Submitted on 20 Apr 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. 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. UNIVERSITÉ DE STRASBOURG ÉCOLE DOCTORALE DES SCIENCE CHIMIQUES UMR 7140, Chimie de la matière complexe THÈSE présentée par : Batoul SROUR soutenue le : 24 Avril 2015 pour obtenir le grade de Docteur de l’université de Strasbourg Discipline/ Spécialité : Chimie Emerging roles for natural and artificial lipids in shaping the catalytic function, stability and oligomeric state of membrane proteins. Rôles émergents des lipides naturels et artificiels dans l'élaboration de la fonction catalytique, la stabilité et l'état d'oligomerisation des protéines membranaires. THÈSE dirigée par : Prof. Dr. HELLWIG Petra Université de Strasbourg RAPPORTEURS : Prof. Dr. SüSS Regine Albert-Ludwig université de Freiburg Prof. Dr. ANDRADE Susana Albert-Ludwig université de Freiburg AUTRES MEMBRES DU JURY: Prof. Dr. BECHINGER Burkhard Université de Strasbourg “If you can't fly then run if you can't run then walk if you can't walk then crawl but whatever you do you have to keep moving forward. Martin Luther King Jr. ACKNOWLEDGEMENTS Coming to the end of these three unforgettable years, lots of memories flash back into my mind where lots of people have played a major role and without their care and love, this thesis would not have been possible. I would like first to express my sincere appreciation and respect to my thesis director Prof. Petra Hellwig for her supervision, kindness and continuous help. It was a great pleasure for me to work within your team. I would also like to thank Prof. Regine Süss, Prof. Susane Andrade and Prof. Burkhard Bechinger for accepting the task of evaluating my thesis work and be my committee members. I want also to express my sincere thanks to the laboratory members: to Frederic, Thomas, Sinan, Sebastien, Michelle, Zahia, Alicia, Nathalia, Soumia, and especially for Mireille for the support and extra encouragement you gave. Thank you all for creating this lovely working ambiance. A special thanks to my Lebanese friends: To Rania Noureldeen, Joelle Magarian, Ola Srour, and Zeinab Wehbe for the nice moments we spent together and for being my support during the hard moments I passed especially in the last few months. Thanks to Nedal Taha, Joelle Mezher, Marianne Ibrahim, Fatima Ibrahim, Malak Abbas, Iman Abdallah, Nancy Watfa and Haifa Mokbel for their kindness. Thanks to Mohamad Rajab, Amal Mcheik, Dalia Fakih, Wassim Jaber and Ali Said for your continuous listening and motivation. Hussein Awada deserves extra thanks for being beside during this journey; your support is much appreciated. Special thanks to Hassan Hammoud, Ali and Hamed Wehbe for the good moments we shared together. Thanks to everyone who has participated either directly or indirectly in this accomplishment. My deepest thanks go to my family for being beside and for all the sacrifices you have made on my behalf. This work is dedicated to my father Ali, my mother Hala, my beloved sister Hawraa, and my two brothers Hassan and Hussein. List of abbreviations Å Angstrom ATR Attenuated total reflection Abs Absorbance ATP Adenonsine triphosphate Ag/AgCl Silver/silver chloride BCP Block copolymer CL Cardiolipin DTGS Deuterated triglycine sulfate DDM n-Dodecyl β-D-maltoside Da Dalton DSC Differential scanning calorimetry DQ Decylubiquinone E. coli Escherichia coli Em Midpoint potential EPR Electron paramagnetic resonance Fe-S Iron sulfur cluster FMN Flavin mononucleotide FTIR Fourier transform infrared IRE Internal reflection element IR Infrared MCT Mercury cadmium telluride MES 2-(N-morpholino) ethanesulfonic Acid MIR Mid infrared MD Molecular dynamic mV mili-volts NMR Nuclear magnetic resonance NADH Nicotinamide adenine dinucleotide NDF NADH dehydrogenase fragment Nuo NADH: ubiquinone oxidoreductase subunit Ox-red oxidized minus reduced PDB Protein data bank PC Phosphatidylcholine PE Phosphatidylethanolamine PG Phosphatidylglycerol PMOXA Polymethyloxazoline PDMS Polydimethylsiloxane Q Quinone (oxidized) QRF Quinone reductase fragment QH2 Reduced quinone QCM Quartz crystal microbalance RE Reference electrode ROS Reactive oxygen species RR Resonance Raman SHE Standard hydrogen electrode SERS Surface enhanced resonance Raman spectroscopy SQ Semiquinone UV Ultraviolet TM Transmembrane Tm Transition temperature vis Visible ν Stretching vibrations δ Bending vibrations ε Molar absorption coefficient The twenty amino acids I Table of contents 1 Introduction ...................................................................................................................... 2 1.1 The cell membrane ......................................................................................................... 2 1.1.1 Lipids ..................................................................................................................... 3 1.1.2 Properties of lipids ................................................................................................. 6 1.1.3 Experimental studies on lipids ............................................................................... 9 1.1.4 Computational studies .......................................................................................... 11 1.1.5 Lipids with cholesterol (experimental and simulation studies) ........................... 12 1.2 Liposomes .................................................................................................................... 15 1.2.1 Liposomes stability on solid surfaces .................................................................. 17 1.2.2 pH sensitive liposomes ........................................................................................ 18 1.3 Role of lipids in proteins .............................................................................................. 20 1.4 The respiratory chain ................................................................................................... 21 1.4.1 The NADH: ubiquinone oxidoreductase (complex I) .......................................... 22 1.4.2 NuoL mutants....................................................................................................... 32 1.4.3 Complex I and zinc .............................................................................................. 32 1.4.4 Complex I and synthetic lipids (polymer) ........................................................... 33 1.5 Aims of the thesis ......................................................................................................... 35 2 Characterization techniques and sample preparation ............................................... 37 2.1 Introduction to spectroscopy ........................................................................................ 37 2.2 UV-visible spectroscopy............................................................................................... 38 2.3 IR spectroscopy ............................................................................................................ 39 2.3.1 Fourier transform infrared spectroscopy (FTIR) ................................................. 43 2.3.2 IR spectroscopy of proteins ................................................................................. 45 2.3.3 Transmission mode .............................................................................................. 48 2.3.4 Difference spectroscopy....................................................................................... 48 2.3.5 Attenuated total reflection (ATR) spectroscopy .................................................. 50 2.4 Raman spectroscopy .................................................................................................... 52 2.4.1 Resonance Raman spectroscopy (RR) ................................................................. 53 II 2.4.2 Surface enhanced resonance Raman spectroscopy (SERS) ................................. 54 2.5 Sample Preparation ..................................................................................................... 54 2.5.1 Preparation of complex I protein and its variants ................................................ 54 2.5.2 Preparation of lipids and liposomes ..................................................................... 57 2.6 Experimental conditions .............................................................................................. 61 2.6.1 Infrared absorbance spectra of lipids ................................................................... 61 2.6.2 Study of liposomes ..............................................................................................
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