Many important biological processes occur near or in membranes. The role of membranes is not merely confined to compartmentalization, they also form the matrix for membrane associated proteins and are of functional importance. Membrane associated proteins on the other hand require specific membrane properties for proper function. The interactions between membranes and proteins are thus of paramount importance and are at the focus of this work. To draw valid conclusions about the nature of such interactions the membrane mimetics required in biophysical methods must faithfully mimic crucial properties of biological membranes. To this end, new types of small isotropic bicelles which mimic plant and bacterial membranes were characterized by their size and lipid dynamics using solution-state NMR. Small isotropic bicelles are specifically well suited for solution-state NMR studies since they maintain a bilayer while being sufficiently small to conduct interpretable experiments at the same time. Monogalactosyl diacylglycerol and digalactosyl diacylglycerol, which are highly abundant in thylakoid membranes, were successfully incorporated into bicelles. Also, it was possible to make bicelles containing a lipid mixture extracted from Escherichia coli cells. A fundamental physical property of lipids in bilayers is their phase behaviour and thus the dynamics that lipids undergo in a membrane. Here, the dynamics of 13C-1H bonds in lipids were studied by nuclear spin relaxation. From such studies it was found that the glycerol backbone of lipids in bicelles is rigid while the flexibility of the acyl chain increases towards its end. Bulky head groups are rigid, while smaller head groups are more dynamic than the glycerol backbone. Acyl chain modifications, like unsaturations or cyclopropane moities, that are typically found in E. coli lipids, locally increase the rigidity of the acyl chain. Membrane interactions of a putative membrane anchor of the glycosyltransferase WaaG, MIR-WaaG, were studied by fluorescence methods, circular dichroism and solution-state NMR. It was found that MIR-WaaG binds to vesicles that mimic the anionic charge of E. coli inner membranes and that α-helical structure is induced upon interaction. The NMR-structure of MIR-WaaG agrees well with the crystal structure and from paramagnetic relaxation enhancement studies it could be concluded that a central part of MIR-WaaG is immersed in the membrane mimetic. Based on these results a model of the membrane interaction of WaaG is proposed where MIR-WaaG anchors WaaG to the cytosolic leaflet of the E. coli inner membrane via electrostatic interactions. These are potentially enhanced by membrane interactions of Tyr residues at the membrane interface and of hydrophobic residues inside the membrane.

La dystrophine est une grande protéine membranaire périphérique qui assure un rôle de soutien du sarcolemme permettant aux cellules musculaires de résister aux stress mécaniques engendrés lors des processus de contraction/élongation. Des mutations génétiques conduisent à sa production sous forme tronquée voire à un déficit total en protéine engendrant de sévères myopathies actuellement incurables. Concevoir des thérapies adaptées passe par une meilleure compréhension du rôle biologique de la dystrophine. Par une approche structure/fonction, notre objectif est de déterminer les bases moléculaires impliquées dans les interactions de la dystrophine avec les lipides membranaires du sarcolemme. Grâce à une approche de diffusion aux petits angles (SAXS et SANS) combinée à de la modélisation moléculaire, nous montrons dans un premier temps que les bicelles constituent un modèle expérimental particulièrement adapté aux analyses de structures de protéines qui y sont associées. Ce développement méthodologique original a été exploité dans un deuxième temps pour caractériser les modifications structurales subies par la dystrophine lorsqu’elle interagit avec les lipides. Nous montrons particulièrement que la liaison aux lipides induit l’ouverture significative de la structure en triple hélice « coiled-coil » de la répétiton 1 du domaine central, et proposons en conclusion un modèle tout atome de la protéine en présence de bicelles. Ces travaux de thèse (i) constituent un apport méthodologique significatif pour l’étude de protéines membranaires, (ii) contribuent à une meilleure compréhension du rôle biologique de la dystrophine en vue de thérapies dédiées aux patients atteints de myopathies. / Dystrophin is a large peripheral membrane protein that provides a supporting role for sarcolemma allowing muscle cells to withstand the mechanical stresses generated during contraction / elongation processes. Genetic mutations lead to dystrophin production in truncated form or even to a total deficit in the protein leading to severe myopathies currently incurable. Designing adapted therapies requires a huge knowledge of the biological role of dystrophin. Using a structure / function approach, our aim is to determine the molecular bases involved in the interactions of dystrophin with the membrane lipids of the sarcolemma. Using a small-angle scattering approach (SAXS and SANS) combined with molecular modeling, we show that bicelles constitute a versatile membrane mimic that is particularly adapted to analyze the structure of membrane proteins. This original methodological development was exploited to characterize the structural changes undergone by dystrophin upon lipid binding. We highlight in particular that the lipid binding induces a significant opening of the coiled-coil structure of the repeat 1 of the central domain and, in conclusion, we propose an all-atom model of the protein bound to a bicelle. These thesis works (i) constitute a significant methodological contribution for the study of membrane proteins, (ii) contribute to a better understanding of the biological role of dystrophin for therapies dedicated to patients with myopathies.

<p>Isotropic bicelles are disc-shaped aggregates of lipids and detergents, and are suitable model systems for high-resolution NMR studies of membrane-interacting peptides. In this thesis the structures for the two peptides motilin and transportan were determined by homonuclear ¹H methods in the presence of bicelles, and the structure of the bovine prion protein peptide (bPrPp) was solved in the presence of DHPC micelles. All of these peptides were found to be largely a-helical when bound to the model membranes. In subsequent experiments both motilin and transportan were shown to reside on the surface of the bicelles, whereas bPrPp is more likely to have a transmembrane configuration. </p><p>NMR translational diffusion experiments revealed that the isotropic bicelles studied here are very large objects compared to what is regularly indicated by high-resolution NMR spectroscopy. Furthermore, these studies showed that all three peptides examined interact strongly with bicelles. Investigation of the NMR-relaxation of labeled sites in the peptides motilin and penetratin demonstrated that the overall rotational correlation times for these peptides do not reflect the bicellar size. Such decoupling of NMR relaxation from the dependence of overall size is also seen for the dynamics of the lipid molecules in the bicelles. It is therefore concluded that the overall size is not the sole determinant of the linewidths in NMR spectra, but that extensive motions within the bicelles also exert significant effects. </p><p>Another interesting observation is that the membrane-bound structures of the peptides motilin, transportan, penetratin and bPrPp are very similar, even though these peptides have very different biological functions. In contrast, considerably more variation is observed in the membrane-positioning and molecular dynamics of these peptides. Since the bicelles have been found to induce differences in membrane positioning and molecular dynamics compared to micelles, these model membranes are likely to be important in order to enhance our understanding of the biological function of membrane interacting peptides.</p>

Isotropic bicelles are disc-shaped aggregates of lipids and detergents, and are suitable model systems for high-resolution NMR studies of membrane-interacting peptides. In this thesis the structures for the two peptides motilin and transportan were determined by homonuclear 1H methods in the presence of bicelles, and the structure of the bovine prion protein peptide (bPrPp) was solved in the presence of DHPC micelles. All of these peptides were found to be largely a-helical when bound to the model membranes. In subsequent experiments both motilin and transportan were shown to reside on the surface of the bicelles, whereas bPrPp is more likely to have a transmembrane configuration. NMR translational diffusion experiments revealed that the isotropic bicelles studied here are very large objects compared to what is regularly indicated by high-resolution NMR spectroscopy. Furthermore, these studies showed that all three peptides examined interact strongly with bicelles. Investigation of the NMR-relaxation of labeled sites in the peptides motilin and penetratin demonstrated that the overall rotational correlation times for these peptides do not reflect the bicellar size. Such decoupling of NMR relaxation from the dependence of overall size is also seen for the dynamics of the lipid molecules in the bicelles. It is therefore concluded that the overall size is not the sole determinant of the linewidths in NMR spectra, but that extensive motions within the bicelles also exert significant effects. Another interesting observation is that the membrane-bound structures of the peptides motilin, transportan, penetratin and bPrPp are very similar, even though these peptides have very different biological functions. In contrast, considerably more variation is observed in the membrane-positioning and molecular dynamics of these peptides. Since the bicelles have been found to induce differences in membrane positioning and molecular dynamics compared to micelles, these model membranes are likely to be important in order to enhance our understanding of the biological function of membrane interacting peptides.

No description available.

No description available.

The electrostatic field created by the inward pointing dipole moments of an oriented membrane leaflet has never been measured directly, but is thought to have an important influence on membrane function. Here we present the first direct measurement of the membrane dipole field in lipid bicelles using vibrational Stark effect spectroscopy which is based on the sensitivity of a nitrile oscillator’s vibrational frequency to its local electrostatic environment. The nitrile probe was introduced as the artificial amino acid p-cyanophenylalanine (CN-Phe) in four different locations of a α-helical peptide composed of alternating alanine and leucine residues. This peptide was intercalated into bicelles composed of mixtures of the long chain lipids 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and the short chain lipid 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) formed in two different sizes, 5 nm and 15 nm in radius. Formation of the bicelle above the phase transition temperature of the lipid mixture was confirmed by ³¹P NMR, and the structure of the [alpha]-helix within the bicelle was confirmed by circular dichroic spectroscopy. The absorption energy of the nitrile probe at 4 positions along the helical axis was measured by Fourier transform infrared spectroscopy, from which we estimate the magnitude of the membrane dipole electrostatic field to be -6 MV/cm. Then we successfully manipulated the dipole field in q = 0.5 DMPC/DHPC bicelles by incorporating the small molecule phloretin into the membrane and measured the corresponding ratiometric fluorescence signal of the co-intercalated voltage gated dye di-8-ANEPPS. We measured 0.7 ± 0.2 cm⁻¹ blue shift in absorption energy of the nitrile probe due to the decrease in dipole field caused by phloretin, corresponding to a dipole field of -4.2 MV/cm. This change was essentially identical to what has been estimated through ratiometric fluorescence methods, indicating that VSE spectroscopy will be useful tool for measurement of the biological effects of electrostatic fields in lipid membranes. / text

There are two contributions of this thesis. The first contribution, described in chapters one through six, involves studing the relationship between the protein packing structure of bacteriorhodopsin (bR) and its function as a proton pump. In 2002, a novel crystallization method published by Bowie and Farham resulted in an unusual antiparallel monomeric packing structure of bicelle bacteriorhodopsin (bcbR) crystals, the spectroscopic properties of which had not been studied. In this thesis, these bicelle bR crystals are investigated to better understand how the changes in the protein tertiary structure affect the function. Specifically: Does the retinal Schiff base retain its ability to isomerize in this unusual protein packing structure of bR? How is the hydration of its binding pocket affected? Does the protein retain the ability to undergo the photocycle and pump protons? If so, how are the rates of the deprotonation/reprotonation of the Schiff base affected by the antiparallel monomer packing structure of the protein? Is Asp85 still the proton acceptor during the deprotonation process of the photocycle? The second contribution of the thesis, described in chapter seven, describes the surface attachment and growth of the biofilm formed by the pathogenic bacterium Streptococcus pneumoniae using attenuated total reflection/Fourier transform infrared spectroscopy (ATR/FTIR). This organism was chosen for its clinical significance; it is one of the organisms suspected in forming biofilms in individuals who develop otitis media, one of the most common causes of ear infections of childhood. In contrast to previous ATR/FTIR experiments examining the formation of biofilms on surfaces, this method is unique in that it combines two techniques - ATR/FTIR and Epifluorescence microscopy which when used together allow for the simultaneous monitoring of the IR spectrum of the S. pneumoniae biofilm as it develops and as provides a method for quantifying total and viable cell counts at various stages during the development.

Biological membranes form a protective barrier around cells and cellular compartments. A broad range of biochemical processes occur in or at membranes demonstrating that they are not only of structural but also of functional importance. One important class of membrane proteins are membrane-associated glycosyltransferases. WaaG is a representative of this class of proteins; its function is to catalyze one step in the synthesis of lipopolysaccharides, which are outer membrane lipids found in Gram-negative bacteria. To study protein-membrane complexes by biophysical methods, one must employ membrane mimetics, i.e. simplifications of natural membranes. One type of membrane mimetic often employed in solution-state NMR is small isotropic bicelles, obloid aggregates formed from a lipid bilayer that is dissolved in aqueous solvent by detergent molecules that make up the rim of the bicelle. In this thesis, fast dynamics of lipid atoms in bicelles containing lipid mixtures that faithfully mimic plant and bacterial membranes were investigated by NMR relaxation. Lipids were observed to undergo a broad range of motions; while the glycerol backbone was found to be rigid, dynamics in the acyl chains were much more rapid and unrestricted. Furthermore, by employing paramagnetic relaxation enhancements an ‘atomic ruler’ was developed that allows for measurement of the immersion depths of lipid carbon atoms. WaaG is a membrane-associated protein that adopts a GT-B fold. For proteins of this type, it has been speculated that the N-terminal domain anchors tightly to the membrane via electrostatic interactions, while the anchoring of the C-terminal domain is weaker. Here, this model was tested for WaaG. It was found by a set of circular dichroism, fluorescence, and NMR techniques that an anchoring segment located in the N-terminal domain termed MIR-WaaG binds electrostatically to membranes, and the structure and localization of isolated MIR-WaaG inside micelles was determined. Full-length WaaG was also found to bind membranes electrostatically. It senses the surface charge density of the membrane whilst not discriminating between anionic lipid species. Motion of the C-terminal domain could not be observed under the experimental conditions used here. Lastly, the affinity of WaaG to membranes is lower than expected, indicating that WaaG should not be classified as a monotopic membrane protein but rather as a peripheral one. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 5: Manuscript.</p>

Ácidos graxos são moléculas hidrofóbicas essenciais para a composição da estrutura física celular, para o metabolismo energético dos seres vivos e também para os caminhos de sinalização molecular no proteoma celular. No caso de deficiência no ácido graxo docosahexaenóico (DHA) e do ácido eicosapentaenoico (EPA) temos a depressão e a mudança do comportamento. O transporte destas moléculas hidrofóbicas no citosol celular é realizado por uma família de proteínas capazes de se ligar a esses ácidos graxos de maneira seletiva, com alta afinidade e de forma reversível. Esta família de proteína é conhecida como FABP, ou proteínas ligantes de ácido graxo. Para realizar esta função, as FABP possuem características únicas tanto na sua estrutura tridimensional quanto na dinâmica experimentada pelos vários elementos estruturais. Diversos trabalhos identificaram regiões relevantes e, com mutações realizadas em resíduos específicos, caracterizaram o mecanismo como a proteína interage com ligantes e com a bicamada lipídica para a realização da sua função, identificando um processo multi-estágio na interação com a bicamada lipídica. Contudo, a não realização de mutações em todos os resíduos da proteína pode deixar não-identificados regiões ou resíduos da proteína também envolvidos na sua função. Além disso, nunca foi caracterizado o que ocorre com os resíduos e com a estrutura da FABP quando a proteína está complexada com uma bicamada lipídica. No presente trabalho, escolhemos a B-FABP para estudar a interação com ligantes e o complexo proteína-membrana desta família de proteínas. Para isto, as técnicas de ressonância magnética nuclear 15N-HSQC e eletrônica (RMN e RPE) foram utilizadas para acompanhar mudanças estruturais e dinâmicas ocorridas quanto de interações moleculares. Com a técnica de RPE e o uso de derivados de ácidos graxos marcados com radicais nitróxidos, monitoramos o sítio de ligação da molécula de ácido graxo e suas alterações quando na presença do surfactante SDS. No caso de RMN, foi usada em proteínas marcadas isotopicamente com 15N na presença de bicelas isotrópicas de DMPC: DHPC na razão igual a um (q = 1), em uma concentração lipídica (CL) de 4%. Nossos resultados além de identificar os mesmos resíduos já conhecidos na interação da FABP com modelos de membrana, também encontrou novos resíduos nunca antes associados à superfície de contato da FABP com a bicamada lipídica. / Fatty acids are hydrophobic molecules essential to the cell structure, to the energetic metabolism of living organisms and to the molecular signaling pathways in the cell proteome. Depression and behavior alteations are two common consequences of deficiencies in docosahexanoic (DHA) and eicosapentaenoic (EPA) acids. The transport of such hydrophobic molecules in the cytosol is the main function of a family of proteins capable of making a selective, high affinity, and reversible binding of fatty acids. This family of proteins is known as FABPs (fatty acid binding proteins). To perform their function, FABPs have unique features in both their tridimensional structure and in the dynamics experienced by the several structural elements. Many reports have identified regions that are relevant to function and, through point mutations of specific residues, have characterized the mechanism used by the protein to bind its ligand and also to interact with lipid bilayers. However, the point mutation strategy relies heavily on the choice of residues such that missing residues can lead to the lack of identification of important elements involved in protein function. Moreover, the characterization of the protein-bilayer complex still deserves a more detailed investigation. In this work, we study the B-FABP protein in terms of its interaction with ligands as well as a membrane model system. We made use of magnetic resonance techniques, nuclear (NMR) and electronic (EPR), to probe structural and dynamical changes occurring upon intermolecular interaction. EPR and spin labeled fatty acids allowed us to monitor the ligand binding site in the protein structure and also its alterations in the presence of the surfactant SDS. NMR HSQC was used to gain information on the conformational changes of isotopically labeled protein in the presence of biceles made of DMPC:DHPC (q = 1 and lipid concentration CL of 4%). Our results confirmed relevant functional residues that had been previously identified and also pointed to new residues that had not been implicated as part of the contact surface before, thus widening our understanding of FABP-bilayer interaction.