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Membrane proteins are of undeniable importance for cell physiology across all domains of life and a loss of their function, e.g., due to mutations in their coding sequence, is almost always linked to disease. In humans, mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR), an ATP-gated anion channel in epithelia, give rise to cystic fibrosis (CF). Over 2100 mutations of the CFTR gene are known, however, their disease liability remains mostly undetermined. Causal therapies, i.e., small-molecule drugs that target CFTR itself, have improved the lives of people with the most common mutations (e.g. ΔF508, G551D) over the last decade. In contrast, many rare CF-phenotypic mutations are not eligible for these novel treatments and would benefit from in vitro evaluation of their molecular consequences. In vitro studies of membrane proteins are often complicated by the intrinsic hydrophobicity and aggregation susceptibility of this protein group. However, this can be avoided by using short membrane protein fragments corresponding to the smallest in vivo folding unit of the respective protein at the ER membrane. These model proteins can be easily genetically modified, expressed and purified, making them a suitable tool to pinpoint the effects of mutations. This thesis demonstrates the utility of such a reductionist model system: TM3/4, the second helical hairpin of CFTR’s transmembrane domain 1, was used to study protein folding with a focus on disease-causing missense mutations of CFTR, which may cause CFTR misfolding in vivo. TM3/4 purification was first optimized by using a thioredoxin tag, which allowed heat purification of the fusion protein even after initial purification steps. Optimal heat treatment for maximal protein purity and recovery were determined for TM3/4 and another helical hairpin, ATP synthase subunit c. Moreover, tertiary folding of a CF-phenotypic loop mutation, E217G, introducing a non-native GXXXG interaction motif was analyzed by single-molecule Förster resonance energy transfer (smFRET) in different lipid bilayer conditions, showing unusually increased stability in comparison to wild type (WT) TM3/4. Furthermore, smFRET was used in tandem with circular dichroism and fluorescence spectroscopy to assess the effect of a specific membrane lipid, cholesterol, on TM3/4 variants showing significant changes on secondary but not tertiary structure. Lastly, a mutant library of 13 TM3/4 mutants was established to perform drug screenings with CFTR correctors – a class of small molecules rescuing or preventing misfolding of CFTR. This screening study demonstrated that (i) not all CF-phenotypic missense mutations are locally misfolded at a lipid bilayer comparable to the ER membrane; and (ii) in vitro restoration of a native WT-like conformation of locally misfolded TM3/4 mutants is not only possible but different drug-mutant pairings can be identified related to folding rescue efficiency of a given corrector on a respective mutant. The latter identified drug-mutant pairings may lead to drug repurposing if the effect can be confirmed in cell culture experiments. In conclusion, the TM3/4 minimal model of CFTR and biophysical methods, such as smFRET, proved as versatile tools not only for investigation of mutation and lipid effects on membrane protein folding but also for drug screenings in a disease context.:1 INTRODUCTION 2 THEORETICAL BACKGROUND 2.1 MEMBRANE PROTEINS AND THEIR NATIVE ENVIRONMENTS 2.1.1 Membrane protein families and their role in human health 2.1.2 Fundamental folding models of α-helical membrane proteins 2.1.3 Co-translational folding at the ER supported by the translocon 2.1.4 Folding-relevant interactions within membrane proteins 2.1.5 Biological membranes and lipid classes 2.1.6 Physical properties of lipid bilayers impacting membrane proteins 2.1.7 Membrane models for in vitro studies 2.2 CYSTIC FIBROSIS AND CFTR 2.2.1 Pathology of cystic fibrosis 2.2.2 Structure and function of the CFTR channel 2.2.3 A minimal model of CFTR to study rare CF mutations 2.2.4 Missense mutations within the CFTR segmental model TM3/4 2.2.5 Novel modulator therapies for the treatment of cystic fibrosis 2.3 IN VITRO ASSESSMENT OF MEMBRANE PROTEIN FOLDING 2.3.1 Expression and purification of membrane proteins 2.3.2 Single-molecule FRET in single- and multi-well mode for protein folding 3 HEAT PURIFICATION OF TRX MEMBRANE PROTEIN FUSIONS 3.1 PREAMBLE AND SUMMARY 3.2 RESULTS AND DISCUSSION 4 IMPACT OF A CFTR LOOP MUTATION WITH ATYPICAL STABILITY 4.1 PREAMBLE AND SUMMARY 4.2 RESULTS AND DISCUSSION 5 EFFECTS OF CHOLESTEROL ON LOCAL CFTR FOLDING 5.1 PREAMBLE AND SUMMARY 5.2 RESULTS 5.2.1 Folding of TM3/4 hairpins in the presence of cholesterol 5.2.2 Folding of TM3/4 hairpins in the presence of Lumacaftor 5.2.3 Impact of Lumacaftor on membrane fluidity 5.3 DISCUSSION 6 CFTR CORRECTOR SCREENINGS WITH SINGLE-MOLECULE FRET 6.1 PRESCREENING TO IDENTIFY MISFOLDED TM3/4 VARIANTS 6.2 SCREENING OF MISFOLDED TM3/4 VARIANTS WITH CFTR CORRECTORS 7 CONCLUSIONS 8 OUTLOOK 9 MATERIALS AND METHODS 9.1 CONSTRUCT DESIGN OF HELICAL TRANSMEMBRANE HAIRPINS 9.2 PROTEIN EXPRESSION AND PURIFICATION 9.3 HEAT TREATMENT OF HELICAL TRANSMEMBRANE CONSTRUCTS 9.4 SINGLE-MOLECULE FRET EXPERIMENTS 9.4.1 Labeling of TM3/4 constructs 9.4.2 Liposome preparation and reconstitution of labeled protein constructs 9.4.3 Single-molecule FRET measurements in manual mode 9.4.4 Single-molecule FRET measurements in multi-well screening mode 9.5 CIRCULAR DICHROISM SPECTROSCOPY 9.5.1 Circular dichroism to determine protein heat stability 9.5.2 Circular dichroism to study protein structure in different lipid bilayers 9.6 FLUORESCENCE SPECTROSCOPY 9.6.1 Vesicle leakage assay to test lipid bilayer stability 9.6.2 Examining lipid bilayer fluidity with fluorescent probes 10 APPENDIX 10.1 GENERATION OF A TM3/4 MUTANT LIBRARY 10.2 TM3/4 SCREENINGS WITH CFTR CORRECTORS 10.2.1 SmFRET control screenings and supporting data 10.2.2 Extracted closed state fractions from smFRET screenings 10.2.3 DLS to measure vesicle integrity after corrector addition 11 REFERENCES 12 ACKNOWLEDGEMENTS 13 ERKLÄRUNG GEMÄß §5 ABS. 1 S. 3 DER PROMOTIONSORDNUNG / Membranproteine sind für die Zellphysiologie aller biologischen Domänen von unbestreitbarer Bedeutung und ein Verlust ihrer Funktion, z.B. durch Mutationen in ihrer kodierenden Sequenz, ist fast immer Auslöser von Krankheiten. Beim Menschen führen Mutationen im Gen für den Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), einen ATP-abhängigen Anionenkanal in Epithelien, zu Mukoviszidose (CF). Über 2100 Mutationen des CFTR-Gens sind bekannt – ob jedoch alle Mutationen tatsächlich CF auslösen, ist weitgehend ungeklärt. Kausale Therapien, d.h. niedermolekulare Medikamente, die auf CFTR selbst abzielen, haben in den letzten zehn Jahren die Lebensqualität von Menschen mit den häufigsten Mutationen (z.B. ΔF508, G551D) verbessert. Demgegenüber stehen jedoch viele seltene CF-phänotypische Mutationen, für welche diese neuartigen Behandlungen nicht zugelassen sind, wodurch diese Mutationen von einer In-vitro-Analyse ihrer molekularen Konsequenzen profitieren würden. In-vitro-Untersuchungen von Membranproteinen werden oft durch die intrinsische Hydrophobizität und Aggregationsanfälligkeit dieser Proteine erschwert. Dies kann jedoch vermieden werden, indem kurze Membranproteinfragmente verwendet werden, die der kleinsten in vivo Faltungseinheit des jeweiligen Proteins an der ER-Membran entsprechen. Diese Modellproteine können routiniert genetisch verändert, exprimiert und aufgereinigt werden, was sie zu einem geeigneten Werkzeug macht, um die Auswirkungen von Mutationen zu genau festzustellen. Diese Dissertation demonstriert die Nützlichkeit eines solchen reduktionistischen Modellsystems: TM3/4, das zweite helikale Haarnadel-Motiv der Transmembrandomäne 1 von CFTR, wurde verwendet, um Proteinfaltung mit Schwerpunkt auf krankheitsverursachende Missense-Mutationen von CFTR zu untersuchen, welche eine CFTR-Fehlfaltung in vivo verursachen können. Die TM3/4-Aufreinigung wurde zunächst durch die Verwendung eines Thioredoxin-Tags optimiert, der eine Hitzeaufreinigung des Fusionsproteins auch nach anfänglichen Reinigungsschritten ermöglichte. Die optimale Hitzebehandlung für maximale Proteinreinheit und -ausbeute wurde für TM3/4 und ein weiteres helikales Haarnadelprotein, die ATP-Synthase-Untereinheit c, bestimmt. Weiterhin wurde die tertiäre Faltung einer CF-phänotypischen Mutation, E217G, die ein nicht-natives GXXXG-Interaktionsmotiv einführt, mittels einzelmolekularem Förster-Resonanzenergietransfer (smFRET) in verschiedenen Lipiddoppelschichten analysiert, welche eine ungewöhnlich erhöhte Stabilität im Vergleich zum TM3/4-Wildtyp (WT) zeigte. Darüber hinaus wurde smFRET in Verbindung mit Circulardichroismus und Fluoreszenzspektroskopie verwendet, um die Wirkung eines spezifischen Membranlipids, Cholesterin, auf TM3/4-Varianten zu untersuchen, welches signifikante Auswirkungen auf die sekundäre, aber nicht auf die tertiäre Proteinstruktur hatte. Schließlich wurde eine Mutantenbibliothek von 13 TM3/4-Mutanten eingerichtet, um Wirkstoffscreenings mit CFTR-Korrektoren durchzuführen – einer Klasse kleiner Moleküle, die die Fehlfaltung von CFTR verhindern können. Diese Screening-Studie zeigte, dass (i) nicht alle CF-phänotypischen Missense-Mutationen lokal an einer Lipiddoppelschicht fehlgefaltet sind, die mit der ER-Membran vergleichbar ist; und (ii) die In-vitro-Wiederherstellung einer nativen WT-ähnlichen Konformation von lokal fehlgefalteten TM3/4-Mutanten ist nicht nur möglich, sondern es können auch verschiedene Wirkstoff-Mutanten-Paare identifiziert werden, die mit der Faltungsrettungseffizienz eines Korrektors auf eine bestimmte Mutante zusammenhängen. Die letztgenannten Wirkstoff-Mutanten-Paare können zu Drug-Repurposings führen, wenn die Wirkung in Zellkulturexperimenten bestätigt werden kann. Im Allgemeinen, haben sich das TM3/4-Minimalfaltungsmodell von CFTR sowie biophysikalische Methoden, wie z.B. smFRET, als vielseitige Werkzeuge nicht nur für die Untersuchung von Mutations- und Lipideffekten auf die Membranproteinfaltung, sondern auch für das Screening von Medikamenten im Krankheitskontext erwiesen.:1 INTRODUCTION 2 THEORETICAL BACKGROUND 2.1 MEMBRANE PROTEINS AND THEIR NATIVE ENVIRONMENTS 2.1.1 Membrane protein families and their role in human health 2.1.2 Fundamental folding models of α-helical membrane proteins 2.1.3 Co-translational folding at the ER supported by the translocon 2.1.4 Folding-relevant interactions within membrane proteins 2.1.5 Biological membranes and lipid classes 2.1.6 Physical properties of lipid bilayers impacting membrane proteins 2.1.7 Membrane models for in vitro studies 2.2 CYSTIC FIBROSIS AND CFTR 2.2.1 Pathology of cystic fibrosis 2.2.2 Structure and function of the CFTR channel 2.2.3 A minimal model of CFTR to study rare CF mutations 2.2.4 Missense mutations within the CFTR segmental model TM3/4 2.2.5 Novel modulator therapies for the treatment of cystic fibrosis 2.3 IN VITRO ASSESSMENT OF MEMBRANE PROTEIN FOLDING 2.3.1 Expression and purification of membrane proteins 2.3.2 Single-molecule FRET in single- and multi-well mode for protein folding 3 HEAT PURIFICATION OF TRX MEMBRANE PROTEIN FUSIONS 3.1 PREAMBLE AND SUMMARY 3.2 RESULTS AND DISCUSSION 4 IMPACT OF A CFTR LOOP MUTATION WITH ATYPICAL STABILITY 4.1 PREAMBLE AND SUMMARY 4.2 RESULTS AND DISCUSSION 5 EFFECTS OF CHOLESTEROL ON LOCAL CFTR FOLDING 5.1 PREAMBLE AND SUMMARY 5.2 RESULTS 5.2.1 Folding of TM3/4 hairpins in the presence of cholesterol 5.2.2 Folding of TM3/4 hairpins in the presence of Lumacaftor 5.2.3 Impact of Lumacaftor on membrane fluidity 5.3 DISCUSSION 6 CFTR CORRECTOR SCREENINGS WITH SINGLE-MOLECULE FRET 6.1 PRESCREENING TO IDENTIFY MISFOLDED TM3/4 VARIANTS 6.2 SCREENING OF MISFOLDED TM3/4 VARIANTS WITH CFTR CORRECTORS 7 CONCLUSIONS 8 OUTLOOK 9 MATERIALS AND METHODS 9.1 CONSTRUCT DESIGN OF HELICAL TRANSMEMBRANE HAIRPINS 9.2 PROTEIN EXPRESSION AND PURIFICATION 9.3 HEAT TREATMENT OF HELICAL TRANSMEMBRANE CONSTRUCTS 9.4 SINGLE-MOLECULE FRET EXPERIMENTS 9.4.1 Labeling of TM3/4 constructs 9.4.2 Liposome preparation and reconstitution of labeled protein constructs 9.4.3 Single-molecule FRET measurements in manual mode 9.4.4 Single-molecule FRET measurements in multi-well screening mode 9.5 CIRCULAR DICHROISM SPECTROSCOPY 9.5.1 Circular dichroism to determine protein heat stability 9.5.2 Circular dichroism to study protein structure in different lipid bilayers 9.6 FLUORESCENCE SPECTROSCOPY 9.6.1 Vesicle leakage assay to test lipid bilayer stability 9.6.2 Examining lipid bilayer fluidity with fluorescent probes 10 APPENDIX 10.1 GENERATION OF A TM3/4 MUTANT LIBRARY 10.2 TM3/4 SCREENINGS WITH CFTR CORRECTORS 10.2.1 SmFRET control screenings and supporting data 10.2.2 Extracted closed state fractions from smFRET screenings 10.2.3 DLS to measure vesicle integrity after corrector addition 11 REFERENCES 12 ACKNOWLEDGEMENTS 13 ERKLÄRUNG GEMÄß §5 ABS. 1 S. 3 DER PROMOTIONSORDNUNG

<p dir="ltr">CaaX proteins, comprising approximately 300 members in the human protein database, represent a diverse group implicated in fundamental cellular processes, including proliferation, differentiation, trafficking, and gene expression. To carry out such vital cellular functions, CaaX proteins need to undergo three sequential post-translational modifications (PTM) through the CaaX pathway, which consists of isoprenylation (farnesylated or geranylgeranylated), endoproteolysis, and methylation. Among the CaaX family of protein, the Ras superfamily, plays a pivotal role in cell growth and survival. Mutations in <i>Ras proteins</i> are associated with a spectrum of cancers, presenting a significant challenge for therapeutic intervention. This thesis explores the intricate landscape of PTMs of CaaX proteins, with a focus on methylation, which is carried out by membrane protein isoprenylcysteine carboxyl methyltransferase (Icmt), and its potential as a therapeutic target, particularly for Ras-driven cancers.</p><p><br></p><p dir="ltr">Icmt is unique as it is the sole methyltransferase which carries out the third PTM of methyl esterification of CaaX proteins with the aid of co-substrate SAM, which serves as the methyl donor. Additionally, how Icmt, a membrane protein localized in the endoplasmic reticulum (ER), brings these two chemically diverse molecules in close enough proximity to promote catalysis, is very intriguing and is not yet fully understood. This thesis focuses on studying the structural and functional properties of Ste14, the yeast homolog of Icmt, in order to better understand the Icmt family of proteins. Ste14 is a functional homolog of human Icmt, sharing 41% sequence identity and 62% sequence similarity. Furthermore, Ste14 can be functionally purified unlike human Icmt. Together, these attributes make Ste14 an ideal system to study.</p><p dir="ltr"><br>The first project explores Ste14 and substrate binding, focusing on residues that determine farnesylated vs geranylgeranylated substrate specificity. It is essential to note that wild-type Ste14 recognizes farnesylated and geranylgeranylated substrate equally, with no preference to one over the other. Conserved residues found in Loop 2 and Transmembrane 3 of Ste14 were mutated to alanine and assessed for their activity with AGGC, the minimal geranylgeranylated CaaX substrate. Mutants which showed nearly zero percent activity with AGGC in comparison to wild type were further analyzed to understand if this loss of mutant activity with AGGC was potentially due to the mutant's inability to bind with AGGC. A photoreactive AGGC analog was used to carry out the photolabeling experiments and residues were analyzed for their binding ability with geranylgeranylated substrate. Mutants were further analyzed to understand the effect of mutation on structural integrity, to gauge which residues are essential for catalysis and for maintaining structural integrity of Ste14. Results demonstrated that residues F80 and E98 are essential for structural stability while L81 and L82 are essential for catalysis. This project would overall help better understand the lesser studied Ste14-substrate binding.</p><p><br></p><p dir="ltr">In the second project, the focus shifts to study Ste14 and co-substrate SAM binding by using electron paramagnetic resonance spectroscopy (EPR) and site directed spin labeling (SDSL). The biophysical technique of EPR requires much less protein and serves as great tool to study conformational change Ste14 undergoes on SAM binding, 3 non conserved residues found in the SAM binding region of Ste14, were individually mutated to cysteine, and had a spin label MTSL attached to their purified active mutant forms. Through EPR the conformational changes of Ste14 during methylation specifically during SAM binding was analyzed by visualizing the movement of MTSL attached residue. Results showed of the three non-conserved residues, A223 and E227 were immobile during SAM binding while T164 residue displayed flexibility during SAM binding during SAM binding and release process. This study would help understand the protein dynamics that Icmt undergoes upon SAM binding.</p><p><br></p><p dir="ltr">The final section centers on inhibiting the third step of the CaaX pathway, which is methyl esterification, by targeting Icmt. The project involved testing a library of Icmt inhibitors and evaluating their ability to inhibit Icmt activity. Of this library of bi-substrate analog inhibitors, compounds YD 1-66, YD 1-67 and YD 1-77 emerge as promising inhibitors against human Icmt, laying the foundation for further studies to develop more potent inhibitors. This section accentuates the strategies employed to target Icmt and the potential of these inhibitors in combating Ras-driven cancers.</p><p><br></p><p dir="ltr">This thesis provides an extensive analysis of the structure and function of Ste14. The varied studies and their insights contribute to a comprehensive understanding of Icmt and pave the way for the rational design of potent chemotherapeutic inhibitors for Ras-driven cancers. The multifaceted research presented in this thesis reveals several new possibilities for targeted therapies in the field of oncology.</p>

Membrane protein mechanotransduction is the altered function of an integral membrane protein in response to mechanical force. Such mechanosensors are found in all kingdoms of life, and increasing numbers of membrane proteins have been found to exhibit mechanosensitivity. How they mechanotransduce is an active research area and the topic of this thesis. The methodology employed is classical molecular dynamics (MD) simulations. MD systems are complex, and two programs were developed to reduce this apparent complexity in terms of both visual abstraction and statistical analysis. Bendix detects and visualises helices as cylinders that follow the helix axis, and quantifies helix distortion. The functionality of Bendix is demonstrated on the symporter Mhp1, where a state is identified that had hitherto only been proposed. InterQuant tracks, categorises and orders proximity between parts of an MD system. Results from multiple systems are statistically interrogated for reproducibility and significant differences at the resolution of protein chains, residues or atoms. Using these tools, the interaction between membrane and the Escherichia coli mechanosensitive channel of small conductance, MscS, is investigated. Results are presented for crystal structures captured in different states, one of which features electron density proposed to be lipid. MD results supports this hypothesis, and identify differential lipid interaction between closed and open states. It is concluded that propensity for lipid to leave for membrane bulk drives MscS state stability. In a subsequent study, MscS is opened by membrane surface tension for the first time in an MD setup. The gating mechanism of MscS is explored in terms of both membrane and protein deformation in response to membrane stretch. Using novel tension methodology and the longest MD simulations of MscS performed to date, a molecular basis for the Dashpot gating mechanism is proposed. Lipid emerges as an active structural element with the capacity to augment protein structure in the protein structure-function paradigm.

An aqueous droplet in a solution of lipids in oil acquires a lipid monolayer coat, and two such droplets adhere to form a bilayer at their interface. Networks of droplets have been constructed in this way that function as light sensors, batteries and electrical circuits by using membrane proteins incorporated into the bilayers. However, the droplets have been confined to a bulk oil phase, which precludes direct communication with physiological environments. Further, the networks typically have been assembled manually, which limits their scale and complexity. This thesis addresses these limitations, and thereby enables prospective medical and technological applications for droplet networks. In the first part of the work, defined droplet networks are encapsulated within mm-scale drops of oil in water to form structures called multisomes. The encapsulated droplets adhere to one another and to the surface of the oil drop to form interface bilayers that allow them to communicate with each other and with the surrounding aqueous environment through membrane pores. The contents of the droplets can be released by changing the pH or temperature of the surrounding solution. Multisomes have potential applications in synthetic biology and medicine. In the second part of the work, a three-dimensional printing technique is developed that allows the construction of complex networks of tens of thousands of heterologous droplets ~50 µm in diameter. The droplets form a self-supporting material in bulk oil or water analogous to biological tissue. The mechanical properties of the material are calculated to be similar to those of soft tissues. Membrane proteins can be printed in specific droplets, for example to establish a conductive pathway through an otherwise insulating network. Further, the networks can be programmed by osmolarity gradients to fold into designed shapes. Printed droplet networks can serve as platforms for soft devices, and might be interfaced with living tissues for medical applications.

Je utilise le AFM et le HS-AFM pour étudier les caractéristiques mécaniques du cellule du cristallin et aussi des protéines de membrane de la cellule, AQP0 et Connexon. L’énergie d'interaction de la AQP0 est -2.7 kBT, très nécessaire pour former les microdomaines de jonctions (junctional microdomain). Aussi c' est la première fois qu il est possible de voir des protéines individuel et son mouvement en cellules vivants. La formation de microdomaines est important pour la transparence du cristallin, et le AQP1 ne le peux faire. / I used the AFM and HS-AFM for characterise the eye lens and the eye lens membrane protein, AQP0 and connexon.A QP0-AQP0 interaction energy is -2.7kBT, it is important for the formation of junctional microdomains, which keep the distance between the cells lens and lens transparency. this is the first report which is present time the visualization of unlabelled membrane proteins on living cells under physiological conditions. AQP1 can not maintain the lens transparency because it does not form junctional microdomains.

Les protéines membranaires intégrales jouent un rôle essentiel dans le maintien de l’intégrité cellulaire (transports d’ions et de nutriments, transduction de signal, interaction cellule-cellule). Afin de les étudier, ces protéines doivent être produites in vitro. La production classique de ces protéines membranaires intégrales dans des microorganismes présente de nombreuses difficultés liées à leur structure complexe mais aussi à des problèmes de toxicité, empêchant la production de nombre d’entre elles. En outre, pour être produites efficacement, ces protéines ont besoin d’un environnement amphiphile. Dans cette thèse, afin de pallier à ces difficultés, nous avons d’une part utilisé un système d’expression protéique acellulaire, non affecté par la physiologie des cellules vivantes. En outre, nous avons choisi de les intégrer dans des bicouches lipidiques planes reconstituées artificiellement. Dans une première partie, nous avons mis au point l’intégration d’une protéine membranaire intégrale formant un pore, l’alpha hémolysine, dans une bicouche lipidique supportée. Certaines protéines nécessitant un espace plus important de part etd’autre de la membrane, nous avons, dans une seconde partie, développé une bicouche lipidique espacée et ancrée par fusion de liposomes sur des surfaces d’or. Nous démontrons qu’il est possible d’y incorporer des protéines membranaires de type Aquaporine Z sous certaines conditions. Dans une troisième partie, dédiée à la formation de membranes biomimétiques utilisant des molécules lipidiques provenant d’Escherichia coli, nous montrons que la modification de la composition membranaire ne semble pas avoir d’incidence sur l’incorporation de protéines. Enfin, dans une dernière partie, nous avons réalisé des premiers essais d’insertion de protéines membranaires, de type alpha hémolysine, dans des bicouches suspendues afin de montrer que ces protéines produites par le système d’expression acellulaire sont fonctionnelles. / Integral membrane proteins play an essential role in the cell integrity preservation (transport of nutrients and ions, signal transduction, cell-cell interaction). In order to study these proteins, they have to be produced in vitro. Classical production of integral membrane proteins in microorganisms present many difficulties associated with their complex structure and also toxicity problems, preventing production of many of them. Moreover, to be efficiently produced, these proteins require an amphiphilic environment. In order to overcome these difficulties, we used a cell-free protein expression system, unaffected by the physiology ofliving cells. In addition, we chose to integrate them into artificial planar lipid bilayers. In a first part, we have developed the integration of an integral membrane protein forming a pore, the alpha hemolysin, in a supported lipid bilayer. Some proteins require more space on each side of the membrane, therefore in a second part, we have developed a tethered lipid bilayer membrane by liposome fusion on gold surfaces. We demonstrate that it is possible to incorporate membrane protein Aquaporin Z under certain conditions. The third part is dedicated to the formation of biomimetic membranes using lipid molecules from Escherichiacoli, we show that the membrane composition do not affect the protein incorporation. Finally, we have tested alpha hemolysin membrane proteins insertion in suspended lipid bilayers membranes to show that these proteins produced by the cell-free expression system are functional.

Le complexe NADPH oxydase est un élément essentiel de l’immunité inné. Présent dans les cellules phagocytaires (neutrophile), sa fonction est de produire massivement, dans le phagosome, des anions superoxyde et générer ainsi des espèces encore plus réactives de l’oxygène qui vont détruire acides nucléiques, lipides et protéines des bactéries phagocytées. Le cœur membranaire catalytique du complexe NADPH oxydase est constitué d’un hétérodimère membranaire, le cytochrome b₅₅₈ (Cyt b₅₅₈). Après activation de celui-ci par les partenaires protéiques cytosoliques p47phox, p67phox, p40phox et Rac, une succession de réactions de transferts d’électron de part et d’autre de la membrane a lieu au sein du Cyt b₅₅₈ pour aboutir à la réduction du dioxygène de manière très contrôlée. Afin de mieux comprendre cette régulation, nous nous sommes d’abord intéressés aux stéreoisomères trans de l’acide arachidonique, activateur naturel de cet enzyme (cis), sur le fonctionnement de la NADPH oxydase et avons abordé cette étude parallèlement sur du Cytb₅₅₈ d’origine bovine présent dans des membranes de neutrophiles et dans des membranes de levures exprimant le Cytb₅₅₈ de manière hétérologue. Nous avons montré que la géométrie joue un rôle important sur l’activation du complexe enzymatique. Dans un deuxième temps, afin d’étudier le rôle de l’environnement membranaire sur le fonctionnement de la NADPH oxydase, nous avons déterminé les propriétés cinétiques et thermodynamiques de l’activité NADPH oxydase du Cytb₅₅₈ recombinant exprimé en levures, purifié, puis reconstitué en liposomes de composition lipidique variée. Après comparaison avec ces mêmes propriétés obtenues pour le Cytb₅₅₈ dans les membranes plasmiques et du réticulum endoplasmique de levures, nous avons montré que l’activité NADPH oxydase très sensible à la température peut être modulée par la composition et l’état physique de la membrane. / NADPH oxidase complex is a major actor of both antimicrobial host defense and inflammation by generating highly regulated superoxide anion, rapidly converted into reactive oxygen species (ROS). The NADPH oxidase complex consists of a heterodimeric integral membrane flavocytochrome b₅₅₈ and three cytosolic components p67phox, p47phox and p40phox, and the small GTP binding protein Rac. In response to a cellular stimulus, cytosolic proteins are recruited to the phagosomal membrane where they are assembled with the Cytb₅₅₈ to form the active NADPH oxidase. The aim of the work was to better understand the modulation of superoxide anion production by this enzyme. For this purpose, we performed experiments with both bovine neutrophil membranes and yeast membranes expressing the bovine recombinant Cytb₅₅₈. We first investigated the effect of the trans-isomerization of the cis-arachidonic acid, the activator of NADPH oxidase in vitro and showed that specific geometry of the activator plays an important role in the activation of the complex. We also studied the role of the membrane environment on the functioning of NADPH oxidase and determined the kinetics and thermodynamics of NADPH oxidase activity depending on the lipid composition of Cytb₅₅₈ proteoliposomes. Comparison with these properties obtained with recombinant Cytb₅₅₈ embedded into endoplasmic reticulum and plasma membranes, we showed that the NADPH oxidase activity is highly temperature dependent and can be modulated by the lipid environment and the physic state of the membrane.