No description available.

No description available.

Understanding the molecular basis of protein function is a challenging task that lays the foundation for the pharmacological intervention in many diseases originating in altered structural states of the involved proteins. Dissecting a complex functional machinery into modules is a promising approach to protein function. The motivation for this work was to identify minimal requirements for “local” switching processes in the function of multidomain proteins that can adopt a variety of structural substates of different biological activity or representing intermediates of a complex reaction path. For example, modular switches are involved in signal transduction, where receptors respond to ligand-activation by specific conformational changes that are allosterically transmitted to “effector recognition sites” distant from the actual ligand-binding site. Heptahelical receptors have attracted particular attention due to their ubiquitous role in a large variety of pharmacologically relevant processes. Although constituting switches in their own right, it has become clear through mutagenesis and functional studies that receptors exhibit substates of partial active/inactive structure that can explain biological phenotypes of different levels of activity. Here, the notion that microdomains undergo individual switching processes that are integrated in the overall response of structurally regulated proteins is addressed by studies on the molecular basis of proton-dependent (chemical) and force-dependent (mechanical) conformational transitions. A combination of peptide synthesis, biochemical analysis, and secondary structure sensitive spectroscopy (Infrared, Circular dichroism, Fluorescence) was used to prove the switching capability of putative functional modules derived from three selected proteins, in which conformational transitions determine their function in transmembrane signaling (rhodopsin), transmembrane transport (bacteriorhodopsin) and chemical force generation (kinesin-1). The data are then related to the phenotypes of the corresponding full length-systems. In the first two systems the chemical potential of protons is crucial in linking proton exchange reactions to transmembrane protein conformation. This work addresses the hypothesized involvement of lipid protein interactions in this linkage (1). It is shown here that the lipidic phase is a key player in coupling proton uptake at a highly conserved carboxylic acid (DRY motif located at the C-terminus of helix 3) to conformation during activation of class-1 G protein coupled receptors (GPCRs) independently from ligand protein interactions and interhelical contacts. The data rationalize how evolutionary diversity underlying ligand-specifity can be reconciled with the conservation of a cytosolic ‘proton switch’, that is adapted to the general physical constraints of a lipidic bilayer described here for the prototypical class-1 GPCR rhodopsin (2). Whereas the exact sequence of modular switching events is of minor importance for rhodopsin as long as the final overall active conformation is reached, the related heptahelical light-transducing proton pump bacteriorhodopsin (bR), requires the precise relative timing in coupling protonation events to conformationtional switching at the cytosolic, transmembrane, and extracellular domains to guarantee vectorial proton transport. This study has focused on the cytosolic proton uptake site of this retinal protein whose proton exchange reactions at the cytosolic halfchannel resemble that of rhodopsin. It was a prime task in this work to monitor in real time the allosteric coupling between different protein regions. A novel powerful method based on the correlation of simultaneously recorded infrared absorption and fluorescence emission changes during bR function was established here (3), to study the switching kinetics in the cytosolic proton uptake domain relative to internal proton transfer reactions at the retinal and its counter ion. Using an uptake-impaired bR mutant the data proves the modular nature of domain couplings and shows that the energy barrier of the conformational transition in the cytosolic half but not its detailed structure is under the control of proton transfer reactions at the retinal Schiff base and its counter ion Asp85 (4). Despite the different functions of the two studied retinal proteins, the protonation is coupled to local switching mechanisms studied here at two levels of complexity, [a] a single carboxylic acid side chain acting as a lipid-dependent proton switch [b] a full-length system, where concerted modular regions orchestrate the functional coupling of proton translocation reactions. Switching on the level of an individual amino acid is shown to rely on localizable chemical properties (charge state, hydrophobicity, rotamer state). In contrast, switching processes involving longer stretches of amino acids are less understood, less generalizable, and can constitute switches of mechanical, rather than chemical nature. This applies particularly to molecular motors, where local structural switching processes are directly involved in force generation. A controversy exists with respect to the structural requirements for the cooperation of many molecular motors attached to a single cargo. The mechanical properties of the Hinge 1 domain of kinesin-1 linking the “neck” and motor domain to the “tail” were addressed here to complement single molecule data on torsional flexibility with secondary structure analysis and thermal stability of peptides derived from Hinge 1 (5). It is shown that the Hinge 1 exhibits an unexpected helix-forming propensity that resists thermal forces but unfolds under load. The data resolve the paradox that the hinge is required for motor cooperation, whereas it is dispensable for single motor processivity, clearly emphasizing the modular function of the holoprotein. However, the secondary-structural data reveal the functional importance of providing high compliance by force-dependent unfolding, i.e. in a fundamentally different way than disordered domains that are flexible but yet do not support cooperativity.

The processes localized to biological membranes are of great interest, both from a scientific and pharmaceutical point of view. Understanding aspects such as the detailed mechanism and regulation of these processes requires investigation of the structure and function of the membrane-bound proteins in which they take place. The study of these processes is often complicated by the need to create in vitro systems that mimic the environment in which these proteins are normally found in vivo. This thesis describes some of the methods available for membrane-protein studies in membrane-mimetic systems, as well as our work aimed at developing such systems. Furthermore, results from studies using these systems are described. In the first two studies, described in Papers I &amp; II, we investigated the use of silica particle-supported lipid bilayers, both for membrane-protein studies and as possible drug-delivery vehicles. Successful reconstitution of a multisubunit proton-pump, cytochrome c oxidase is described and characterized. Initial attempts to develop drug-delivery systems with two different targeting peptides are also described in the thesis. The second part of this thesis revolves around our work with membraneprotein dependent pathways. Results from studies of systems where the proton- pump bo3 oxidase and ATP synthase work in concert are described. The results show a surprising lipid-composition dependence for the coupled bo3- ATP-synthase activity (Paper III). Finally, a new system utilizing synaptic vesicle-fusion proteins for coreconstitution of membrane proteins is described, showing successful coreconstitution of a small respiratory chain, delivery of soluble proteins to preformed liposomes and reconstitution of ATP synthase in native membranes (Paper IV). / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p>

In lipid membranes lacking activating lipids, the nicotinic acetylcholine receptor adopts an uncoupled conformation that binds ligand, but does not transition into an open conformation. Understanding the mechanisms of lipid-dependent uncoupling is essential to understanding lipid-nAChR interactions, which may be implicated in pathological conditions such as nicotine addition. Here, I tested two structural features of a proposed uncoupling method to elucidate the mechanism of lipid-dependent uncoupling. First, infrared measurements and electrophysiological characterization performed in prokaryotic homologues indicate that lipid sensitivity is largely controlled by the most peripheral α-helix in the transmembrane domain, M4. My data show that tighter association of M4 with the adjacent M1 and M3 transmembrane α-helices decreases a receptor’s propensity to adopt a lipid-dependent uncoupled conformation. Second, I indirectly tested the hypothesis that uncoupling results from a conformational change at the extracellular/transmembrane domain interface that leads to an increased separation between the two domains and ultimately to a constriction of the channel pore. Finally, biophysical studies presented in this dissertation shed light on the complex binding of a number of non-competitive channel blockers to the nicotinic acetylcholine receptor channel pore in both the resting and desensitized states. The data provide further insight into the structural rearrangements that occur upon uncoupling of ligand binding and gating in the nicotinic acetylcholine receptor.

Pentameric ligand-gated ion channels (pLGICs) are membrane bound receptors found in the nervous system. They are responsible for detecting neurotransmitters released from neurons and subsequently mediating responses of the cells on which they are found. Thus, pLGICs play an invaluable role in communication between cells of the nervous system and understanding their function is pivotal to understanding how the nervous system works in general. One factor which is known to mediate pLGIC function is lipids found in the membrane environment in which pLGICs are embedded. This dissertation explores the various ways in which lipids interact with and modulate the function of pLGIC. Potential mechanisms and biological consequences of this modulation will be presented and discussed within the context of our current state of knowledge of pLGIC and nervous system function.

Pentameric ligand-gated ion channels (pLGICs) are membrane bound receptors found in the nervous system. They are responsible for detecting neurotransmitters released from neurons and subsequently mediating responses of the cells on which they are found. Thus, pLGICs play an invaluable role in communication between cells of the nervous system and understanding their function is pivotal to understanding how the nervous system works in general. One factor which is known to mediate pLGIC function is lipids found in the membrane environment in which pLGICs are embedded. This dissertation explores the various ways in which lipids interact with and modulate the function of pLGIC. Potential mechanisms and biological consequences of this modulation will be presented and discussed within the context of our current state of knowledge of pLGIC and nervous system function.

Understanding the molecular basis of protein function is a challenging task that lays the foundation for the pharmacological intervention in many diseases originating in altered structural states of the involved proteins. Dissecting a complex functional machinery into modules is a promising approach to protein function. The motivation for this work was to identify minimal requirements for “local” switching processes in the function of multidomain proteins that can adopt a variety of structural substates of different biological activity or representing intermediates of a complex reaction path. For example, modular switches are involved in signal transduction, where receptors respond to ligand-activation by specific conformational changes that are allosterically transmitted to “effector recognition sites” distant from the actual ligand-binding site. Heptahelical receptors have attracted particular attention due to their ubiquitous role in a large variety of pharmacologically relevant processes. Although constituting switches in their own right, it has become clear through mutagenesis and functional studies that receptors exhibit substates of partial active/inactive structure that can explain biological phenotypes of different levels of activity. Here, the notion that microdomains undergo individual switching processes that are integrated in the overall response of structurally regulated proteins is addressed by studies on the molecular basis of proton-dependent (chemical) and force-dependent (mechanical) conformational transitions. A combination of peptide synthesis, biochemical analysis, and secondary structure sensitive spectroscopy (Infrared, Circular dichroism, Fluorescence) was used to prove the switching capability of putative functional modules derived from three selected proteins, in which conformational transitions determine their function in transmembrane signaling (rhodopsin), transmembrane transport (bacteriorhodopsin) and chemical force generation (kinesin-1). The data are then related to the phenotypes of the corresponding full length-systems. In the first two systems the chemical potential of protons is crucial in linking proton exchange reactions to transmembrane protein conformation. This work addresses the hypothesized involvement of lipid protein interactions in this linkage (1). It is shown here that the lipidic phase is a key player in coupling proton uptake at a highly conserved carboxylic acid (DRY motif located at the C-terminus of helix 3) to conformation during activation of class-1 G protein coupled receptors (GPCRs) independently from ligand protein interactions and interhelical contacts. The data rationalize how evolutionary diversity underlying ligand-specifity can be reconciled with the conservation of a cytosolic ‘proton switch’, that is adapted to the general physical constraints of a lipidic bilayer described here for the prototypical class-1 GPCR rhodopsin (2). Whereas the exact sequence of modular switching events is of minor importance for rhodopsin as long as the final overall active conformation is reached, the related heptahelical light-transducing proton pump bacteriorhodopsin (bR), requires the precise relative timing in coupling protonation events to conformationtional switching at the cytosolic, transmembrane, and extracellular domains to guarantee vectorial proton transport. This study has focused on the cytosolic proton uptake site of this retinal protein whose proton exchange reactions at the cytosolic halfchannel resemble that of rhodopsin. It was a prime task in this work to monitor in real time the allosteric coupling between different protein regions. A novel powerful method based on the correlation of simultaneously recorded infrared absorption and fluorescence emission changes during bR function was established here (3), to study the switching kinetics in the cytosolic proton uptake domain relative to internal proton transfer reactions at the retinal and its counter ion. Using an uptake-impaired bR mutant the data proves the modular nature of domain couplings and shows that the energy barrier of the conformational transition in the cytosolic half but not its detailed structure is under the control of proton transfer reactions at the retinal Schiff base and its counter ion Asp85 (4). Despite the different functions of the two studied retinal proteins, the protonation is coupled to local switching mechanisms studied here at two levels of complexity, [a] a single carboxylic acid side chain acting as a lipid-dependent proton switch [b] a full-length system, where concerted modular regions orchestrate the functional coupling of proton translocation reactions. Switching on the level of an individual amino acid is shown to rely on localizable chemical properties (charge state, hydrophobicity, rotamer state). In contrast, switching processes involving longer stretches of amino acids are less understood, less generalizable, and can constitute switches of mechanical, rather than chemical nature. This applies particularly to molecular motors, where local structural switching processes are directly involved in force generation. A controversy exists with respect to the structural requirements for the cooperation of many molecular motors attached to a single cargo. The mechanical properties of the Hinge 1 domain of kinesin-1 linking the “neck” and motor domain to the “tail” were addressed here to complement single molecule data on torsional flexibility with secondary structure analysis and thermal stability of peptides derived from Hinge 1 (5). It is shown that the Hinge 1 exhibits an unexpected helix-forming propensity that resists thermal forces but unfolds under load. The data resolve the paradox that the hinge is required for motor cooperation, whereas it is dispensable for single motor processivity, clearly emphasizing the modular function of the holoprotein. However, the secondary-structural data reveal the functional importance of providing high compliance by force-dependent unfolding, i.e. in a fundamentally different way than disordered domains that are flexible but yet do not support cooperativity.

No description available.

Les peptides et protéines extracteurs de lipides (PEL) se lient aux membranes lipidiques puis en extraient des lipides en formant de plus petits auto-assemblages, un phénomène qui peut aller jusqu'à la fragmentation des membranes. Dans la nature, cette extraction se produit sur une gamme de cellules et entraîne des conséquences variées, comme la modification de la composition de la membrane et la mort de la cellule. Cette thèse se penche sur l’extraction lipidique, ou fragmentation, induite par le peptide mélittine et la protéine Binder-of-SPerm 1 (BSP1) sur des membranes lipidiques modèles. Pour ce faire, des liposomes de différentes compositions sont préparés et incubés avec la mélittine ou la BSP1. L'association aux membranes est déterminée par la fluorescence intrinsèque des PEL, tandis que l'extraction est caractérisée par une plateforme analytique combinant des tests colorimétriques et des analyses en chromatographie en phase liquide et spectrométrie de masse (LCMS). La mélittine fait partie des peptides antimicrobiens cationiques, un groupe de PEL très répandu chez les organismes vivants. Ces peptides sont intéressants du point du vue médical étant donné leur mode d’action qui vise directement les lipides des membranes. Plusieurs de ceux-ci agissent sur les membranes des bactéries selon le mécanisme dit « en tapis », par lequel ils s’adsorbent à leur surface, forment des pores et ultimement causent leur fragmentation. Dans cette thèse, la mélittine est utilisée comme peptide modèle afin d’étudier le mécanisme par lequel les peptides antimicrobiens cationiques fragmentent les membranes. Les résultats montrent que la fragmentation des membranes de phosphatidylcholines (PC) est réduite par une déméthylation graduelle de leur groupement ammonium. L'analyse du matériel fragmenté révèle que les PC sont préférentiellement extraites des membranes, dû à un enrichissement local en PC autour de la mélittine à l'intérieur de la membrane. De plus, un analogue de la mélittine, dont la majorité des résidus cationiques sont neutralisés, est utilisé pour évaluer le rôle du caractère cationique de la mélittine native. La neutralisation augmente l'affinité du peptide pour les membranes neutres et anioniques, réduit la fragmentation des membranes neutres et augmente la fragmentation des membranes anioniques. Malgré les interactions électrostatiques entre le peptide cationique et les lipides anioniques, aucune spécificité lipidique n'est observée dans l'extraction. La BSP1 est la protéine la plus abondante du liquide séminal bovin et constitue un autre exemple de PEL naturel important. Elle se mélange aux spermatozoïdes lors de l’éjaculation et extrait des lipides de leur membrane, notamment le cholestérol et les phosphatidylcholines. Cette étape cruciale modifie la composition lipidique de la membrane du spermatozoïde, ce qui faciliterait par la suite la fécondation de l’ovule. Cependant, le contact prolongé de la protéine avec les spermatozoïdes endommagerait la semence. Cette thèse cherche donc à approfondir notre compréhension de ce délicat phénomène en étudiant le mécanisme moléculaire par lequel la protéine fragmente les membranes lipidiques. Les résultats des présents travaux permettent de proposer un mécanisme d’extraction lipidique en 3 étapes : 1) L'association à l’interface des membranes; 2) La relocalisation de l’interface vers le cœur lipidique; 3) La fragmentation des membranes. La BSP1 se lie directement à deux PC à l'interface; une quantité suffisante de PC dans les membranes est nécessaire pour permettre l'association et la fragmentation. Cette liaison spécifique ne mène généralement pas à une extraction lipidique sélective. L'impact des insaturations des chaînes lipidiques, de la présence de lysophosphatidylcholines, de phosphatidyléthanolamine, de cholestérol et de lipides anioniques est également évalué. Les présentes observations soulignent la complexe relation entre l'affinité d'un PEL pour une membrane et le niveau de fragmentation qu'il induit. L'importance de la relocalisation des PEL de l'interface vers le cœur hydrophobe des membranes pour permettre leur fragmentation est réitérée. Cette fragmentation semble s'accompagner d'une extraction lipidique préférentielle seulement lorsqu'une séparation de phase est induite au niveau de la membrane, nonobstant les interactions spécifiques PEL-lipide. Les prévalences des structures amphiphiles chez certains PEL, ainsi que de la fragmentation en auto-assemblages discoïdaux sont discutées. Finalement, le rôle des interactions électrostatiques entre les peptides antimicrobiens cationiques et les membranes bactériennes anioniques est nuancé : les résidus chargés diminueraient l'association des peptides aux membranes neutres suite à l'augmentation de leur énergie de solvatation. / Lipid-extracting peptides and proteins (LEPs) bind to lipid membranes, extract lipids in the form of smaller auto-assemblies, and ultimately fragment membranes. In nature, this lipid extraction occurs in many different cell systems and causes various consequences, such as a modification of the membrane lipid composition or the cell death. This thesis focuses on the lipid extraction, or fragmentation, induced by the peptide melittin and the protein Binder-of-SPerm 1 (BSP1) on model lipid membranes. To this end, liposomes of different composition are prepared and incubated with melittin or BSP1. The association to membranes is determined by the LEPs intrinsic fluorescence, while the extraction is characterized by a combination of colorimetric phosphorus assays and liquid chromatography-mass spectrometry analyses (LCMS). Melittin is a cationic antimicrobial peptide, a very common category of LEP found in living organisms. Cationic antimicrobial peptides are interesting to medicine because they directly target membrane lipids. The action of many of these peptides is described by the carpet-like mechanism, by which they adsorb to membrane surface, induce the formation of pores and then cause the fragmentation of the membranes. In this thesis, melittin is used as a model peptide in order to study the mechanism by which cationic antimicrobial peptides fragment lipid membranes. Results show that the phosphocholine (PC) membrane fragmentation is reduced by a gradual demethylation of the ammonium group. Analysis of the fragmented material reveals that PC are preferentially extracted from membranes, due to a local enrichment in PC near melittin in the membrane. Furthermore, a melittin analogue, for which a majority of its cationic residues were neutralized, is used to investigate the role of the cationic character of native melittin. The neutralization increases the peptide affinity for neutral and anionic membranes, reduces fragmentation of neutral membranes and increases fragmentation of anionic membranes. Despite electrostatic interactions between the cationic peptide and the anionic lipids, no lipid specificity is observed in the extraction. BSP1 is the most abundant protein of the bovine seminal plasma and constitutes another example of important LEP found in nature. Upon ejaculation, it mixes with spermatozoa and extracts membrane lipids, such as cholesterol and phosphatidylcholines. This crucial process modulates the lipid composition of sperm membranes, which would then facilitate egg fertilization. However, a prolonged contact between the protein and spermatozoa could damage the semen. This thesis is looking to deepen our understanding of this delicate phenomenon by studying the molecular mechanism by which this protein fragments lipid membranes. Results of the present work suggest a 3-step mechanism for the extraction: 1) Association to membrane interface; 2) Relocation towards the lipid core; 3) Fragmentation of membranes. BSP1 binds directly to two interfacial PC; a sufficient quantity of PC in membranes is necessary for protein association and fragmentation. This specific binding generally does not lead to specificity in the lipid extraction. The impact of unsaturation of the lipid chains, of the presence of lysophosphatidylcholines, of phosphatidylethanolamines, of cholesterol and of anionic lipids is also studied. The present observations underline the complex relationship between a LEP affinity for membranes and the level of fragmentation it induces. The importance of LEP relocation, from the interface to the hydrophobic core of the membranes, for fragmentation is reiterated. This fragmentation seems to be lipid specific only when a phase separation of the lipids occurs in the membrane, notwithstanding specific LEP-lipid interactions. The prevalence of amphipathic structures in certain LEPs, as well as of the auto-assembled discoidal structures resulting from fragmentation is discussed. Finally, the role of electrostatic interactions between cationic antimicrobial peptides and anionic bacterial membranes is detailed: charged residues lower peptide association to neutral membrane due to an increase of their free energy of solvation.