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Oldham, Alexis Jean. "Modulation of lipid domain formation in mixed model systems by proteins and peptides." View electronic thesis, 2008. http://dl.uncw.edu/etd/2008-1/r1/oldhama/alexisoldham.pdf.
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Hubert, Anne Kasyoka. "Interactions between membrane transport proteins and lipids." Thesis, University of Leeds, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.432304.
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Ariöz, Candan. "Exploring the Interplay of Lipids and Membrane Proteins." Doctoral thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-102675.
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The interplay between lipids and membrane proteins is known to affect membrane protein topology and thus have significant effect (control) on their functions. In this PhD thesis, the influence of lipids on the membrane protein function was studied using three different membrane protein models. A monotopic membrane protein, monoglucosyldiacylglyecerol synthase (MGS) from Acholeplasma laidlawii is known to induce intracellular vesicles when expressed in Escherichia coli. The mechanism leading to this unusual phenomenon was investigated by various biochemical and biophysical techniques. The results indicated a doubling of lipid synthesis in the cell, which was triggered by the selective binding of MGS to anionic lipids. Multivariate data analysis revealed a good correlation with MGS production. Furthermore, preferential anionic lipid sequestering by MGS was shown to induce a different fatty acid modeling of E. coli membranes. The roles of specific lipid binding and the probable mechanism leading to intracellular vesicle formation were also investigated. As a second model, a MGS homolog from Synechocystis sp. PCC6803 was selected. MgdA is an integral membrane protein with multiple transmembrane helices and a unique membrane topology. The influence of different type of lipids on MgdA activity was tested with different membrane fractions of Synechocystis. Results indicated a very distinct profile compared to Acholeplasma laidlawii MGS. SQDG, an anionic lipid was found to be the species of the membrane that increased the MgdA activity 7-fold whereas two other lipids (PG and PE) had only minor effects on MgdA. Additionally, a working model of MgdA for the biosynthesis and flow of sugar lipids between Synechocystis membranes was proposed. The last model system was another integral membrane protein with a distinct structure but also a different function. The envelope stress sensor, CpxA and its interaction with E. coli membranes were studied. CpxA autophosphorylation activity was found to be positively regulated by phosphatidylethanolamine and negatively by anionic lipids. In contrast, phosphorylation of CpxR by CpxA revealed to be increased with PG but inhibited by CL. Non-bilayer lipids had a negative impact on CpxA phosphotransfer activity. Taken together, these studies provide a better understanding of the significance of the interplay of lipids and model membrane proteins discussed here.
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Danial, John Shokri Hanna. "Imaging lipid phase separation on droplet interface bilayers." Thesis, University of Oxford, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.711943.
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Polozov, Ivan V. "Interactions of class A and class L amphipathic helical peptides with model membranes." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0006/NQ30110.pdf.
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Clogston, Jeffrey. "Applications of the lipidic cubic phase from controlled release and uptake to in meso crystallization of membrane proteins /." Connect to resource, 2005. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1117564268.
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Thesis (Ph.D.)--Ohio State University, 2005.Title from first page of PDF file. Document formatted into pages; contains xxii, 352 p.; also includes graphics. Includes bibliographical references (p. 346-352). Available online via OhioLINK's ETD Center
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Naughton, Fiona. "Interactions of perihperal membrane proteins with phosphatidylinositol lipids : insights from molecular dynamics simulations." Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:d7bb7b03-3eda-40f2-85fc-f5a314ae3c44.
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Interactions between proteins and membranes are central to many signalling pathways and other cellular processes. Phosphatidylinositol phosphates (PIPs) are a family of lipids often acting as second messengers and targeted by peripheral proteins in these processes. A pipeline was developed combining the molecular dynamics (MD) approaches of umbrella sampling and coarse-grain modelling, and used to quantify and compare the interactions with PIP-containing model membranes of 13 pleckstrin homology (PH) domains, a common lipid-binding domain found in many proteins showing varied affinities and specificities for PIPs. Lipid selectivity generally agreed with previous observations. Several membrane-binding modes were identified, revealing PIP interactions through a secondary site are more common than suggested experimentally and appear to be related to overall affinity. Results suggest that simultaneous binding of multiple PIP lipids is required to achieve the high affinities characteristic of PH domains. Multiscale MD, combining coarse-grain binding simulations and atomistic refinement, was used to investigate PTEN, a tumour suppressor catalysing interconversion of PIPs and associated with many cancers and other disorders. Regions often ignored in previous studies were revealed to favour productive binding, largely via electrostatics. PIP clustering by bound PTEN and membrane insertion in the productive mode were demonstrated. Existence of an N-terminal PIP-binding site was supported, with this region appearing disordered, rather than helical as previously suggested. Changes in interdomain orientation when bound and with the clinically-relevant R173C mutation further suggest the importance of the interdomain interface for productive binding. Together, this work demonstrates the important contributions MD can make towards understanding protein/membrane interactions, particularly in the context of managing the diseases caused by their disruption.
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Nordlund, Gustav. "Membrane-mimetic systems : Novel methods and results from studies of respiratory enzymes." Doctoral thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-94554.
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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 & 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).At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.
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Putta, Priya. "The Tale/ Head of Two Membrane Lipids Through Protein Interactions." Kent State University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=kent1524311387080992.
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Mulet, Xavier. "Phosphatidylinositol Lipids and the role of Membrane Curvature in Regulation of Membrane-Associated Proteins." Thesis, Imperial College London, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.498508.
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Chiaradia, Laura. "Isolement et caractérisation de la mycomembrane des mycobactéries." Thesis, Toulouse 3, 2018. http://www.theses.fr/2018TOU30015.
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Les mycobactéries, dont les plus connues sont Mycobacterium tuberculosis et Mycobacterium leprae, agents étiologiques de la tuberculose et de la lèpre respectivement, présentent une enveloppe complexe et atypique faisant l'objet de nombreuses études dans le cadre de la lutte contre ces pathologies. Cette enveloppe est composée d'une couche externe aussi appelée capsule dans le cas de bactéries pathogènes, d'une paroi (mycomembrane - arabinogalactane (AG) - peptidoglycane (PG)) et d'une membrane plasmique. La membrane externe des mycobactéries, appelée mycomembrane est composée de protéines et majoritairement d'acides mycoliques, acides gras à très longues chaînes a-ramifiés et ß-hydroxylés. Ces derniers sont retrouvés covalemment liés, d'une part au complexe AG-PG dans le feuillet interne de la mycomembrane, et d'autre part au tréhalose au niveau du feuillet externe de la mycomembrane. On trouve également en fonction des mycobactéries des lipides complexes dont la localisation exacte dans l'enveloppe n'est à ce jour pas clairement connue et reste sujette à débats. Ce travail a permis de mettre au point un protocole, en deux étapes majeures, permettant le fractionnement cellulaire de deux espèces mycobactériennes, M. aurum et M. smegmatis. Le but étant d'isoler les deux membranes mycobactériennes afin de déterminer leur composition en terme de lipides mais aussi de protéines. Tout d'abord, des culots enrichis en mycomembranes (liées à l'AG-PG) ou en membranes plasmiques sont obtenus par ultracentrifugations différentielles puis purifiés sur gradients de densité discontinus de saccharose. L'absence de contaminations des membranes entre elles est vérifiée grâce à des marqueurs spécifiques. Il a été montré que les phospholipides qui sont les composants majoritaires de la membrane plasmique sont également présents dans la mycomembrane à côté des mycolates de tréhalose. De plus ce travail a permis de montrer que les lipoglycanes, lipoarabinomannanes et lipomannanes, lipides possédant des propriétés antigéniques, sont retrouvés dans les deux fractions membranaires. Ce travail de fractionnement a été le point de départ d'une étude de protéomique afin d'identifier les protéines retrouvées spécifiquement au niveau de la mycomembrane-AG-PG mais également les protéines de la membrane plasmique, les protéines sécrétées et les protéines solubles, provenant des cytosol et périplasme. Une étude de dynamique par RMN sur les fractions membranaires natives menée conjointement avec l'étude protéomique, devrait permettre de mieux comprendre l'organisation de l'enveloppe cellulaire des mycobactéries ainsi que certains des mécanismes impliqués dans la pathogénicitéMycobacteria, including Mycobacterium tuberculosis and Mycobacterium leprae, etiological agents of tuberculosis and leprosy respectively, are composed of a complex and atypical cell wall, which is the focus of numerous studies in the context of the fight against these pathologies. This cell envelope, to which many biological properties have been attributed, is composed of three entities: an outer layer also called capsule in the case of pathogenic species, a cell wall and a plasma membrane. Within the mycobacterial cell wall, the outer membrane, called mycomembrane, is mainly composed of proteins and mycolic acids, very long chain a-branched and ß-hydroxylated fatty acids. These mycolic acids are found in the inner leaflet of the mycomembrane, covalently linked to the arabinogalactan-peptidoglycan complex (AG-PG), and in the outer leaflet where they are linked to trehaloses. Complex lipids are also known in mycobacteria, and may vary depending on the species, however their exact localization within the cell envelope is not yet clearly known and remains open to debate. In order to better delineate the composition of the two mycobacterial membranes, mycomembrane and plasma membrane, a two-step protocol was developed for cell fractionation of two mycobacterial species, M. aurum and M. smegmatis. Firstly, pellets enriched in mycomembranes (linked to AG-PG) or plasma membranes are obtained by differential ultracentrifugations. Then, these membrane pellets are purified using a sucrose step density gradient. To ensure the absence of cross-contaminations of the membranes, specific markers of each membranes are used. Phospholipids, which are the major components of the plasma membrane, are also found in the mycomembrane with trehalose mycolates. Moreover, this study allowed us to demonstrate that immunogenic lipoglycans, lipoarabinomannans and lipomannans, are found in the two mycobacterial membranes. Once the fractionation successfully achieved, it was possible to initiate proteomic studies in order to identify proteins that are specific of the mycomembrane-AG-PG but also those secreted or present in the soluble fraction, derived from the cytosol and periplasm compartments. Future NMR dynamic studies, to be performed on the native membranes, combined with the proteomic studies will help deciphering the organization of the mycobacterial cell envelope as well as the mechanisms involved in pathogenicity
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Zheng, Hong. "Designing Peptides to Target Membrane Lipids and to Evaluate Fluorination of Proteins." Thesis, Boston College, 2012. http://hdl.handle.net/2345/3682.
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Thesis advisor: Jianmin GaoMy graduate research has used engineered peptides to perturb the non-covalent interactions in protein folding, protein-protein association and protein-membrane association. We have focused on understanding the fundamental principles of molecular recognition behind protein-protein and protein-membrane interactions, and further using these principles in protein engineering. This thesis includes three projects. I) Towards Small Molecule Receptors for Membrane Lipids: A Case Study on Phosphatidylserine The lipid composition and distribution of cell membranes play important roles in regulating the physiology of the cell. The lipid composition of plasma membranes is one characteristic feature that can be used to identify cell types and functions. Molecules that specifically recognize a particular lipid are useful as imaging probes for targeting cells or tissues of interest. Protein based lipid binding probes have intrinsic limitations due to their large size and poor pharmacokinetic properties such as slow clearance rate and poor in vivo stability. A plausible strategy to achieve a probe with small size and high binding affinity and selectivity is to use a peptide to mimic the protein lipid-binding domains. As a case study, a cyclic peptide that specifically targets phosphatidylserine containing membranes has been developed. This cyclic peptide is potentially capable of imaging apoptosis in vivo, and the strategy of developing this cyclic peptide can be generalized to the design of peptide-based probes for other lipid species. My research has pointed out a challenging but feasible way to design a peptide that achieves specificity and affinity similar to lipid-binding proteins. (II) Study of Apoptotic Cell Membrane (ACM) Permeant Molecules Noninvasive imaging of apoptosis is highly desirable for the diagnosis of a variety of diseases, as well as for the early prognosis of anticancer treatments. One characteristic feature of apoptotic cells that has been targeted for developing specific biomarkers is enhanced membrane permeability compared to that of healthy cells. Several unrelated molecules that are capable of selectively penetrating the apoptotic cell membrane (ACM) have recently been reported. However, the origin of the altered ACM permeability is poorly understood, as is the scope of molecular structures that can permeate through the ACM. Herein, we report a systematic investigation on the altered ACM permeability. Our results show that simple modifications of commonly used dyes (e.g. fluorescein) afford specific entry into cells at the early stages of apoptosis. The ACM appears to be permeable to molecules of various functional groups and charge, but does discriminate against molecules of large size. The new findings reported here greatly expand the pool of small molecules for imaging cell death, thus facilitating the development of noninvasive imaging agents for apoptosis. (III) Study of Aromatic-Fluorinated Aromatic Interactions in Peptide Systems Therapeutic proteins have been through a remarkable expansion in the last two decades. A general problem that they are facing is poor stability. Protein engineering focuses on solving this problem by incorporating unnatural amino acids into protein sequences to purposefully modify protein structures. Fluorinated aliphatic amino acids have been demonstrated to be effective in stabilizing protein structures and functioning as recognition motifs. In contrast, fluorinated aromatic amino acids are less studied. We investigated the effect of perturbation of fluorination on aromatic residues on the stability of protein model systems, as well as the influence on protein-protein association behavior. The results of this study provided a fundamental understanding of aromatic interactions in protein systems, and guidelines for protein engineering with fluorinated aromatics for stabilizing protein structures or directing specific protein-protein interactions
Thesis (PhD) — Boston College, 2012
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry
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Tjörnhammar, Richard. "Classical and Quantum Descriptions of Proteins, Lipids and Membranes." Doctoral thesis, KTH, Teoretisk biologisk fysik, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-151396.
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In this thesis the properties of proteins and membranes are studied by molecular dynamics simulations. The subject is decomposed into parts addressing free energy calculations in proteins, mechanical inclusion models for lipid bilayers, phase transitions and structural correlations in lipid bilayers and atomistic lipid bilayer models. The work is based on results from large scale computer simulations, quantum mechanical and continuum models. Efficient statistical sampling and the coarseness of the models needed to describe the ordered and disordered states are of central concern. Classical free energy calculations of zinc binding, in metalloproteins, require a quantum mechanical correction in order to obtain realistic binding energies. Classical electrostatic polarisation will influence the binding energy in a large region surrounding the ion and produce reasonable equilibrium structures in the bound state, when compared to experimental evidence. The free energy for inserting a protein into a membrane is calculated with continuum theory. The free energy is assumed quadratic in the mismatch and depend on two elastic constants of the membrane. Under these circumstances, the free energy can then be written as a line tension multiplied by the circumference of the membrane inclusion. The inclusion model and coarse grained particle simulations of the membranes show that the thickness profile around the protein will be an exponentially damped oscillation. Coarse-grained particle simulations of model membranes containing mixtures of phospholipid and cholesterol molecules at different conditions were performed. The gel-to-liquid crystalline phase transition is successively weakened with increasing amounts of cholesterol without disappearing even at a concentration of cholesterol as high as 60%. A united atom parameterization of diacyl lipids was constructed. The aim was to construct a new force field that retains and improves the good agreement for the fluid phase and at the same time produces a gel phase at low temperatures, with properties coherent with experimental findings. The global bilayer tilt obtains an azimuthal value of 31◦ and is aligned between lattice vectors in the bilayer plane. It is also shown that the model yield a correct heat of melting as well as heat capacities in the fluid and gel phase of DPPC.QC 20140919
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Raychaudhuri, Pinky. "Bilayer formation with fluorinated amphiphiles and applications in membrane protein studies." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:f8d7ec23-7b2f-4610-b7c8-395b2660464a.
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Every cell is enclosed by a membrane which gives structure to the cell and allows for the passage of nutrients and wastes into and out of the cell. Membranes are made up of amphiphilic lipid molecules, with one water-soluble end, and one hydrophobic end. Naturally occurring and synthetic membranes are made up of double-chained amphiphiles derived from hydrocarbons. Recently, a novel class of amphiphilic molecules derived from fluorocarbons have been reported. The properties of fluorinated amphiphiles are very different to that of hydrocarbon based amphiphiles. Fluorinated amphiphiles have been previously reported to be useful in the studies of membrane proteins. In this thesis, we explore some novel uses of fluorinated amphiphiles. Chapter one: Provides a comprehensive review of the properties of fluorocarbon-based amphiphiles and discusses the existing uses of fluorinated amphiphiles in biochemical and biomedical research. Chapter two: Describes some of the important materials and methods used in this thesis including a detailed description of the proteins used and the working principles behind the techniques used in the study. Chapter three: Looks at the stability of pre-formed planar lipid bilayers in the presence of fluorinated amphiphiles (F-amphiphiles), and characterizes the behaviour of alpha-haemolysin and other proteins in liposomes and planar lipid bilayers in the presence of F-amphiphiles. We found that F-amphiphiles have an inhibitory effect on the insertion of protein into lipid bilayers, and this property has been exploited to control the number of proteins in the bilayer. Chapter four: Using droplet interface bilayers, we investigate the electrical properties and behaviour of protein(s) in bilayers formed by F-amphiphiles. The results obtained with fluorinated bilayers are compared with results obtained in conventional DPhPC lipid bilayers. This is the first ever report to carry out such an investigation and it provides insights into the formation, stability and utility of fluorinated bilayers. Chapter five: In Chapter five, we explore another aspect of droplet interface bilayers: the feasibility of using droplet interface bilayers to screen for membrane protein libraries. I have chosen to focus on certain fundamental aspects of the screening process that are sufficient to establish the feasibility of the method and to act as the proof of concept. Chapter six: Summarizes all the important results in the thesis and discusses some possible future directions of this project.
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Münzberg, Eileen [Verfasser], and Dieter [Gutachter] Schinzer. "Of proteins and lipids : a molecular dynamics study of membrane-bound Rab5 / Eileen Münzberg ; Gutachter: Dieter Schinzer." Magdeburg : Universitätsbibliothek Otto-von-Guericke-Universität, 2019. http://d-nb.info/1219937762/34.
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Cai, Yingqi. "Lipogenic Proteins in Plants: Functional Homologues and Applications." Thesis, University of North Texas, 2018. https://digital.library.unt.edu/ark:/67531/metadc1404563/.
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Although cytoplasmic lipid droplets (LDs) are the major reserves for energy-dense neutral lipids in plants, the cellular mechanisms for packaging neutral lipids into LDs remain poorly understood. To gain insights into the cellular processes of neutral lipid accumulation and compartmentalization, a necessary step forward would be to characterize functional roles of lipogenic proteins that participate in the compartmentalization of neutral lipids in plant cells. In this study, the lipogenic proteins, Arabidopsis thaliana SEIPIN homologues and mouse (Mus Musculus) fat storage-inducing transmembrane protein 2 (FIT2), were characterized for their functional roles in the biogenesis of cytoplasmic LDs in various plant tissues. Both Arabidopsis SEIPINs and mouse FIT2 supported the accumulation of neutral lipids and cytoplasmic LDs in plants. The three Arabidopsis SEIPIN isoforms play distinct roles in compartmentalizing neutral lipids by enhancing the numbers and sizes of LDs in various plant tissues and developmental stages. Further, the potential applications of Arabidopsis SEIPINs and mouse FIT2 in engineering neutral lipids and terpenes in plant vegetative tissues were evaluated by co-expressing these and other lipogenic proteins in Nicotiana benthamiana leaves. Arabidopsis SEIPINs and mouse FIT2 represent effective tools that may complement ongoing strategies to enhance the accumulation of desired neutral lipids and terpenes in plant vegetative tissues. Collectively, our findings in this study expand our knowledge of the broader cellular mechanisms of LD biogenesis that are partially conserved in eukaryotes and distinct in plants and suggest novel targets that can be introduced into plants to collaborate with other factors in lipid metabolism and elevate oil content in plant tissues.
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Szpryngiel, Scarlett. "Structure and lipid interactions of membrane-associated glycosyltransferases : Cationic patches and anionic lipids regulate biomembrane binding of both GT-A and GT-B enzymes." Doctoral thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-131084.
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This thesis concerns work on structure and membrane interactions of enzymes involved in lipid synthesis, biomembrane and cell wall regulation and cell defense processes. These proteins, known as glycosyltransferases (GTs), are involved in the transfer of sugar moieties from nucleotide sugars to lipids or chitin polymers. Glycosyltransferases from three types of organisms have been investigated; one is responsible for vital lipid synthesis in Arabidopsis thaliana (atDGD2) and adjusts the lipid content in biomembranes if the plant experiences stressful growth conditions. This enzyme shares many structural features with another GT found in gram-negative bacteria (WaaG). WaaG is however continuously active and involved in synthesis of the protective lipopolysaccharide layer in the cell walls of Escherichia coli. The third type of enzymes investigated here are chitin synthases (ChS) coupled to filamentous growth in the oomycete Saprolegnia monoica. I have investigated two ChS-derived MIT domains that may be involved in membrane interactions within the endosomal pathway. From analysis of the three-dimensional structure and the amino-acid sequence, some important regions of these very large proteins were selected for in vitro studies. By the use of an array of biophysical methods (e.g. Nuclear Magnetic Resonance, Fluorescence and Circular Dichroism spectroscopy) and directed sequence analyses it was possible to shed light on some important details regarding the structure and membrane-interacting properties of the GTs. The importance of basic amino-acid residues and hydrophobic anchoring segments, both generally and for the abovementioned proteins specifically, is discussed. Also, the topology and amino-acid sequence of GT-B enzymes of the GT4 family are analyzed with emphasis on their biomembrane association modes. The results presented herein regarding the structural and lipid-interacting properties of GTs aid in the general understanding of glycosyltransferase activity. Since GTs are involved in a high number of biochemical processes in vivo it is of outmost importance to understand the underlying processes responsible for their activity, structure and interaction events. The results are likely to be useful for many applications and future experimental design within life sciences and biomedicine.At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.
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Lidman, Martin. "The role of the mitochondrial membrane system in apoptosis : the influence of oxidative stress on membranes and their interactions with apoptosis-regulating Bcl-2 proteins." Doctoral thesis, Umeå universitet, Kemiska institutionen, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-110701.
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Apoptosis is a crucial process in multicellular organisms in sculpting them, especially during embryogenesis. In addition, apoptosis is responsible for the clearance of harmful or damaged cells which can otherwise be detrimental to the organism. The Bcl-2 family proteins are key players in the regulation of the intrinsic pathway of the apoptotic machinery. This family consists of three subfamilies with B-cell CLL/lymphoma 2 (Bcl-2) protein itself representing anti-apoptotic members, the Bcl-2-associated X protein (Bax), and pro-apoptotic BH3-only signaling proteins. The interplay between pro- and anti-apoptotic proteins on the mitochondrial membranes is central to the balance between the life and death decision of whether the membrane should be permeabilized or not. The cytosolic Bax protein can upon cellular stress translocate to the mitochondrial membrane where it can either carry out its action of forming homo-oligomers that cause outer membrane permeabilization or be inhibited there by the anti-apoptotic membrane protein Bcl-2. Upon mitochondrial outer membrane permeabilization (MOMP) apoptogenic factors leak out from the intermembrane space (IMS) of the mitochondria, leading to caspase activation and ultimately cell death. A common stress signal initiating apoptosis is an increased formation of reactive oxygen species (ROS in the mitochondria, who can cause oxidative damage to lipid membranes. This membrane damage presumably influences the lipid landscape and the membrane features and hence the interactions of the Bcl-2 family proteins with each other and the mitochondrial outer membrane (MOM). To investigate the significance of membrane oxidation on the behavior of the Bcl-2 family proteins, especially Bax, synthetically produced oxidized phospholipids (OxPls) were incorporated in MOM-mimicking vesicles. Differential scanning calorimetry (DSC), nuclear magnetic resonance (NMR) spectroscopy and circular dichroism (CD) spectroscopy revealed a major perturbation in membrane organization in the presence of OxPls. These changes in membrane properties increase the affinity of Bax to its target membrane and enable its partial penetration and formation of pores, as fluorescence leakage assays confirmed. However, in the absence of BH3-only proteins these pores are not sufficiently large for the release of apopototic factors such as cytochrome C (CytC). To understand the inhibition of Bax by the full-length Bcl-2 protein, suitable detergent solubilizing conditions were carefully chosen to enable the measurement of their direct binding to each other outside the membrane, by an antimycin A2 fluorescence assay. The observed protein-protein interaction was confirmed by surface plasmon resonance (SPR). An established protocol for the reconstitution of Bcl-2 into stable proteoliposomes now paves the way for structural studies of this key protein, in its membrane environment near physiological conditions; information essential for understanding its function, on a molecular level, and its potential as a cancer drug target.
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Srour, Batoul. "Emerging roles for natural and artificial lipids in shaping the catalytic function, stability and oligomeric state of membrane proteins." Thesis, Strasbourg, 2015. http://www.theses.fr/2015STRAF068/document.
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L'étude des membranes biologiques nécessite l'examen des différentes propriétés de ses composantes principales: les lipides et les protéines. Dans ce manuscrit, l'interaction lipide- lipide et lipide-protéine ont été suivies par spectroscopie vibrationnelle (Raman, Infrarouge). Nous sommes intéressés en premier lieu à l'étude de la structure et l'organisation des phospholipides dans leur phase gel et leur phase cristalline liquide en utilisant la spectroscopie moyen infrarouge. En outre, l'effet de la composition du groupement hydrophiles des lipides sur le comportement de la liaison hydrogène des mélanges lipidiques a été sondé en utilisant la spectroscopie lointain infrarouge. Dans la seconde partie, l'interaction de la protéine NADH ubiquinone oxydoréductase et du mutant NuoL (D563N) avec le zinc ont été étudiés par spectroscopie différentielle et les changements conformationnels induits par la liaison du zinc avec les protéines ont été examinés. Enfin, les vibrations métal-ligand des groupements fer-soufre dans le mutant de NuoB (C64A G100C) à différents pH ont été analysées par spectroscopie RamanThe study of biological membranes involves the examination of the different properties of its main components: as lipids and proteins. In this manuscript, the lipid-lipid interaction and the lipid-protein interaction were monitored by vibrational spectroscopy (Raman and Infrared). We have been interested in the first part in studying the structure and organization of phospholipids in the gel phase and the liquid crystalline phase using mid infrared spectroscopy. In addition, the effect of the head group composition on the hydrogen bonding behaviour of lipid mixtures was probed using far infrared spectroscopy. In the second part, the interaction of the NADH ubiquinone oxidoreductase protein and NuoL mutant (D563N) with zinc was investigated through FTIR difference spectroscopy where the conformational changes upon zinc binding were monitored. Finally, the metal-ligand vibrations of the iron- sulfur clusters in NuoB mutants (C64A G100C) at different pH were analysed using Raman spectroscopy
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Chang, Hsiu-Ming Samuel. "Interactions between membrane lipids and integral proteins: Effects of bilayer structure on the reconstituted calcium-activated potassium channel from rat brain." Diss., The University of Arizona, 1994. http://hdl.handle.net/10150/186738.
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The plasma membrane isolates the interior of cells from the external environment. In addition to acting as an insulating barrier, it permits selective interactions across the cell membrane through the presence of membrane-integral proteins which playa major role in the regulation of the internal environment of the cell. One class of membrane-integral proteins forms channels through which solutes pass in and out of the cell. In this dissertation, the properties of one such channel--the high conductance calcium-activated potassium (BK) channel--are examined subsequent to reconstituting the channel into bilayer membranes whose structure is experimentally altered. The central theme of this dissertation is to ask whether an alteration in membrane lipids, which results in different structural properties, provides information on the regulation of membrane-integral proteins such as ion channels. It is known that the lipid composition of cell membranes changes during development and aging, during adaptation to different temperatures, and in some disease states. A better understanding of lipid-channel interactions is therefore likely to provide key information concerning cellular homeostasis. Natural and induced changes in membrane lipid composition, and hence membrane structure, alter the physico-chemical environment at the membrane--protein interface. I propose in this dissertation that changes in membrane structure are ultimately expressed as physical changes that affect ion channel behavior in predictable ways. To test the hypothesis that the lipid environment modifies channel function, I examined the properties of the BK channel reconstituted from rat brain into lipid bilayers of different compositions. The bilayer was modified with phospholipids of different headgroups (altering charge and size), or with phospholipids which have different fatty acid chains (altering the order parameter). Changing the bilayer surface charge is expected to change the concentration of ions near the channel thereby. Therefore the properties of BK channels is expected to be changed due to the interactions of calcium and potassium ions with the surface charge. Furthermore, the interaction between negatively charged lipid and calcium is known to order the lipid structure and may introduce structure stress in bilayers. This structural stress in bilayers may act on the BK channel and modify its properties. Altering the size of phospholipid headgroups and the order of fatty acid chains are likely to change the packing of lipids in the bilayer. Also, such structural alteration in lipid changes the lateral elastic and curvature stress within the bilayer. Addition of cholesterol to phospholipid bilayers is known to increase the orderliness of the fatty acid chains and increase the modulus of compressibility of the membrane, thus increasing the lipid structural stress. Adding general anesthetics into bilayers has been shown to disorder the lipid structure but also increase the lipid structural stress. These physical changes in bilayers may, in tum, act on the channel protein and altering its properties. The results showed that increasing the negatively charged lipid in the bilayer surface resulted in an increase in channel mean opentime, open probability and conductance of the BK channel. Increasing the lipid structural stress, in general, reduces channel mean opentime and open probability. In the case of cholesterol, the conductance is also reduced in addition to mean opentime and open probability. Taking together, these results suggest that the lipid environment plays a profound role in shaping ion channel properties.
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Misquitta, Yohann Reynold. "The rational design of monoacylglycerols for use as matrices for the crystallization of membrane proteins." Columbus, Ohio : Ohio State University, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1141940412.
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Laliberte, Jason P. "Role of Host Cellular Membrane Raft Domains in the Assembly and Release of Newcastle Disease Virus: A Dissertation." eScholarship@UMMS, 2008. https://escholarship.umassmed.edu/gsbs_diss/360.
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Newcastle disease virus (NDV) belongs to the Paramyxoviridae, a family of enveloped RNA viruses that includes many important human and animal pathogens. Although many aspects of the paramyxovirus life cycle are known in detail, our understanding of the mechanisms regulating paramyxovirus assembly and release are poorly understood. For many enveloped RNA viruses, it has recently become apparent that both viral and host cellular determinants coordinate the proper and efficient assembly of infectious progeny virions.
Utilizing NDV as a model system to explore viral and cellular determinants of paramyxovirus assembly, we have shown that host cell membrane lipid raft domains serve as platforms of NDV assembly and release. This conclusion was supported by several key experimental results, including the exclusive incorporation of host cell membrane raftassociated molecules into virions, the association of structural components of the NDV particle with membrane lipid raft domains in infected cells and the strong correlation between the kinetics of viral protein dissociation from membrane lipid raft domains and incorporation into virions. Moreover, perturbation of infected cell membrane raft domains during virus assembly resulted in the disordered assembly of abnormal virions with reduced infectivity. These results further established membrane raft domains as sites of virus assembly and showed the integrity of these domains to be critical for the proper assembly of infectious virions.
Although specific viral protein-protein interactions are thought to occur during paramyxovirus assembly, our understanding of how these interactions are coordinated is incomplete. While exploring the mechanisms underlying the disordered assembly of non-infectious virions in membrane raft-perturbed cells, we determined that the integrity of membrane raft domains was critical in the formation and virion incorporation of a complex consisting of the NDV attachment (HN) and fusion (F) proteins. The reduced virus-to-cell membrane fusion capacity of particles released from membrane raft-perturbed cells was attributed to an absence of the HN – F glycoprotein-containing complex within the virion envelope. This result also correlated with a reduction of these glycoprotein complexes in membrane lipid raft fractions of membrane raft-perturbed cells. Specifically, it was determined that the formation of newly synthesized HN and F polypeptides into the glycoprotein complex destined for virion incorporation was dependent on membrane lipid raft integrity.
Finally, a novel virion complex between the ribonucleoprotein (RNP) structure and the HN attachment protein was identified and characterized. Unlike the glycoprotein complex, the detection of the RNP – HN protein-containing complex was not affected by membrane raft perturbation during virus assembly in the cell. The biological importance of this novel complex for the proper assembly of an infectious progeny virion is currently under investigation.
The results presented in this thesis outline the role of host cell membrane lipid raft domains in the assembly and release processes of a model paramyxovirus. Furthermore, the present work extends our understanding of how these particular host cell domains mechanistically facilitate the ordered assembly and release of an enveloped RNA virus.
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Corey, Elizabeth Ann. "Characterization of the Relationship Between Measles Virus Fusion, Receptor Binding, and the Virus-Specific Interaction Between the Hemagglutinin and Fusion Glycoproteins: a Dissertation." eScholarship@UMMS, 2006. https://escholarship.umassmed.edu/gsbs_diss/221.
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Measles (MV) virions, like those of other enveloped viruses, enter cells by fusing their lipid membranes with those of the target host cells. Additionally, infected tissues often possess giant multinucleate cells, known as syncytia, which are formed by fusion of infected cells with uninfected neighbors. Expression of both the MV attachment (H) and fusion (F) proteins is required for membrane fusion. MV H mediates receptor binding in order to bring the two membranes into close proximity prior to F activation and is thought to trigger F activation through a specific interaction between the two proteins.
Although measles H and F are efficiently transported to the cell surface when expressed independently, evidence has been reported in support of an intracellular interaction between the two proteins that can be detected using an ER co-retention approach. However, it was not determined if the putative co-retention was specific to the two measles glycoproteins, as is their ability to complement each other for efficient fusion promotion. Thus, in this thesis, the formation of an intracellular complex between MV H and F was re-examined. Consistent with the formation of an intracellular complex, cell surface expression and receptor binding of untagged wt MV H is slightly reduced by co-expression of an excess of ER-tagged MV F compared to co-expression with wt F. However, the reduction in surface expression is non-specific in that it can also be induced with heterologous proteins of NDV, which lack significant homology with those of MV. Although this approach did not detect a specific intracellular interaction between MV H and F, it cannot be ruled out that there is a weak association of the proteins that is undetectable by this method. This led to the use of an alternative approach to investigate the cellular site(s) of interaction between the measles H and F proteins.
Consistent with a cell surface interaction between MV H and F, the combination of surface biotinylation and co-immunoprecipitation detects formation of a virus-specific H-F complex. Approximately, 21% of the total amount of MV H at the cell surface can be captured with MV F using an antibody against the latter protein. Two complementary approaches were used to address the relationship between this cell surface interaction and receptor recognition by MV H. First, the proteins were co-immunoprecipitated from the surface of Chinese hamster ovary (CHO) cells, which do not express either MV receptor, CD46 or CD150. Similar levels of MV H can be co-immunoprecipitated with F from the surfaces of parental CHO cells and stably transfected cells that express, human CD46 (CHO-CD46), indicating that binding to CD46 is not the trigger for the H-F interaction. Second, MV H proteins, carrying mutations that dramatically reduce CD46 binding, were shown to co-immunoprecipitate efficiently with F from the surface of HeLa cells. Significantly, these results indicate that MV H and F interact in the absence of, and thus prior to, receptor binding. This is in direct contrast to the NDV HN-F cell surface interaction, which is thought to be triggered by receptor binding.
Identification of the domains of the para myxovirus attachment and fusion proteins that mediate membrane fusion activities is an essential part of understanding the mechanism of fusion. As a result of the H-F interaction prior to receptor binding, MV H attachment to its cellular receptor must result in conformational changes that trigger activation of the F protein. Site-directed mutagenesis analyses of two regions of MV H indicate that a HR domain in the stalk of the attachment protein is essential to the ability of H to activate F. However, either it is not the only region of H that interacts with F or it is indirectly involved in F activation because mutations in the HR do not disrupt MV H-F complex formation at the cell surface. Additionally, the functional interaction between MV H and F may be mediated, at least in part, by Loop 1 of the amino terminus of the C-rich region of the fusion protein. However, the exact role of this region of the F protein in fusion promotion remains to be determined. Importantly, the cell surface interaction between MV H and F proteins appears to be mediated by more that one region of each protein. In contrast to NDV, in no case has a definitive link between any single amino acid difference in MV H or F and an inability to form the cell surface H-F complex been established.
In conclusion, the data presented in this dissertation support a model of measles membrane fusion in which the Hand F proteins form a complex prior to receptor recognition. This complex may hold F in its meta-stable pre-fusion state until binding of H to receptors at the cell surface triggers dissociation of the complex, releasing F to assume its fusogenic form. Importantly, these data also indicate that, although paramyxoviruses may all use the same general process. for promotion of membrane fusion, the mechanism may vary in multiple aspects. A more complete understanding of the means by which measles promotes membrane fusion may direct the development of specific strategies aimed at interfering with the early stages of infection.
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Kriegel, Sébastien [Verfasser], and Thorsten [Akademischer Betreuer] Friedrich. "Transformation of a membrane protein from the respiratory chain into a sensor for the analysis of substrates, inhibitors and lipids = Umwandlung eines Proteins der Atmungskette in einen Sensor für die Analyse von Substraten, Inhibitoren und Lipiden." Freiburg : Universität, 2014. http://d-nb.info/1115813668/34.
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Roselló, Busquets Cristina. "Paper de la Sintaxina-1 i els lipid rafts en guia axonal i regeneració neural." Doctoral thesis, Universitat de Barcelona, 2019. http://hdl.handle.net/10803/668211.
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Durant el desenvolupament del sistema nerviós cal que les cèl·lules formades a una zona concreta migrin fins a la seva posició final o bé que projectin els seus axons per tal de contactar amb les seves cèl·lules diana. En aquest procés participen multitud de molècules de guia axonal i els seus receptors, que produiran l’atracció o la repulsió de l’axó, guiant-lo fins a la seva posició final. En aquest procés el conus de creixement té un paper clau ja que és on es localitzen la majoria d’aquests receptors i on es dona el recanvi de membrana a través de l’exocitosi i endocitosi, necessàries pel creixement de l’axó. En aquests processos de fusió de membrana participen les proteïnes SNARE, entre elles la Sintaxina-1. Una de les molècules guia més importants durant el desenvolupament del sistema nerviós és la Netrina-1. Aquesta molècula té diferents receptors i segons al receptor que s’uneixi provocarà atracció o repulsió. Estudis previs demostren que la la Sintaxina-1 interacciona amb un dels receptors de la Netrina-1, el DCC, i que aquesta interacció és necessària, durant el desenvolupament de la medul·la espinal, per a l’atracció de les neurones comissurals in vitro. Hi ha pocs estudis on s’investigui el paper de les proteïnes SNARE en guia axonal in vivo. Per aquesta raó, en aquesta tesi estudiem el paper de les proteïnes SNARE, específicament de la Sintaxina-1, en la guia axonal de les neurones comissurals, durant el desenvolupament de la línia mitja i la medul·la espinal, en tres models animals diferents (mosca, pollastre i ratolí). Per mitjà de la supressió dels gens de diferents proteïnes SNARE, demostrem que la Sintaxina-1 és necessària per la correcta guia axonal de les neurones comissurals i el correcte desenvolupament de la medul·la a les tres espècies. A més, confirmem que la pèrdua de funció de la Sintaxina-1 comporta una pèrdua de la sensibilitat de les neurones comissurals a les molècules de guia axonal Netrina-1 i Slit-2 i que la funció de la Sintaxina-1 està conservada en diferents processos de guia axonal com la repulsió depenent de Slit/ROBO a les neurones comissurals, i la repulsió depenent de Netrina-1/UNC5 a les neurones de la EGL.
La majoria dels mecanismes involucrats en la guia axonal durant el desenvolupament són molt similars als processos que es donen durant la regeneració. Gran part dels receptors que participen en guia axonal i creixement estan localitzats en microdominis de membrana rics en esfingolípids i colesterol, anomenats lipid rafts, i la seva funcionalitat depèn de la correcta localització en aquests microdominis. A més el colesterol proporciona ordre i rigidesa a les membranes. Aquí demostrem com al disminuir el colesterol de les membranes incrementem el creixement neurític, l’àrea dels conus de creixement, la densitat dels fil·lopodis i la ramificació de neurones immadures del sistema nerviós central i perifèric cultivats in vitro. A més, demostrem que la pèrdua de colesterol afavoreix la regeneració de neurones hipocampals axotomitzades in vitro, de la via perforant en cultius organotípics i del nervi ciàtic in vivo. Finalment, també demostrem que la Nistatina, una droga àmpliament utilitzada per eliminar els lipid rafts de les membranes, incrementa la regeneració in vitro de neurones d’hipocamp activant l’enzim òxid nítric sintasa i, en conseqüència, incrementant els nivells d’òxid nítric.During the nervous system development neurons have to migrate and extend their axons to connect with their targets. In this process a huge amount of axon guidance molecules and their receptors participate to produce axon attraction or repulsion, guiding them to their final position. The growth cone has a key role in this process because the majority of receptors are localized in its surface and is where membrane turnover takes place. SNARE proteins are important components for membrane fusion in both, exocytosis and endocytosis, and their participation in axon guidance has been recently described. One important molecule during the nervous system development is Netrin-1. This guidance cue has different receptors and has the ability to produce attraction or repulsion depending on the receptor it binds. Recent studies have demonstrated that one of the SNARE proteins, Syntaxin-1, interacts with the Netrin-1 receptor DCC and that this interaction is necessary for the attraction of the commissural neurons. In this thesis we study the role of Syntaxin-1 during the commissural neuron guidance in three different animal models (fly, chicken and mouse). Deleting SNARE genes, we demonstrate that Syntaxin-1 is necessary for the correct commissural axon guidance and the correct spinal cord development in the three species. The mechanisms that regulate axon growth during development are very similar to processes that take place during axon regeneration. A variety of axon guidance receptors are localized in membrane microdomains enriched in cholesterol, termed lipid rafts, whose functionality depends on the proper localization within these microdomains. Here, we demonstrate that cholesterol depletion increases neurite growth, growth cone area, filopodia density and branching in immature neurons of the central and peripheral nervous system in vitro. Moreover, cholesterol depletion enhances axon regeneration after axotomy in vitro in dissociated hippocampal neurons, in slice organotypic cultures and in sciatic nerve in vivo. Finally, we also demonstrate that hippocampal regeneration in vitro is increased when nitric oxide synthase is activated by Nystatin treatments, a well-known drug used to disrupt lipid rafts from the cell membrane.
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Angius, Federica. "Molecular basis of membrane protein production and intracellular membranes proliferation in E. coli." Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCC217/document.
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Le système d’expression le plus utilisé pour la production des protéines membranaires, est le système basé sur l’ARN polymérase T7 (ARNpol T7) (Hattab et al., 2015). L'inconvénient de ce système est néanmoins que la vitesse de transcription de l’ARNpol T7 est dix fois plus rapide que celle de l’enzyme bactérienne. Depuis l’isolement de mutants spontanés, notamment C41 (DE3) et C43 (DE3) (Miroux et Walker, 1996) et l’identification de leurs mutations dans le génome, il apparaît clairement que la toxicité provoquée par la surproduction des protéines membranaires est liée à la quantité trop élevée d’ARNpol T7 dans la cellule (Wagner et al., 2008 ; Kwon et al., 2015). Les protéines membranaires ont besoin d’une vitesse de transcription/traduction plus basse pour se replier correctement dans la membrane de la bactérie. Le premier objectif de ma thèse était d’étendre l’amplitude du promoteur du système T7 sur laquelle est basée l’expression des protéines. Pour cela, nous avons isolé et caractérisé de nouvelles souches bactériennes dans lesquelles le niveau d’ARNpol T7 était efficacement régulé par un mécanisme non transcriptionnel très favorable à l’expression des protéines membranaires (Angius et al., 2016). Le deuxième objectif était de comprendre la prolifération des membranes intracellulaires chez E. coli suite à la surexpression de la protéine AtpF, une sous unité membranaire du complexe de l’ATP synthétase (Arechaga et al., 2000). Pour mieux comprendre les voies métaboliques impliquées dans la biogenèse, la prolifération et l’organisation des membranes, nous avons utilisé une approche de séquençage d’ARN à haut débit à différents temps après induction de la surexpression de la sous-unité AtpF dans la souche C43 (DE3). Ensuite, et en collaboration avec Gerardo Carranza and Ignacio Arechaga (Université de Cantabria, Espagne), nous avons construit et étudié des mutants de C43 (DE3) déficients pour les trois gènes codants pour des enzymes de la biosynthèse des cardiolipides afin d’évaluer leur participation dans la biogénèse des membranes intracellulairesThe most successful expression system used to produce membrane proteins for structural studies is the one based on the T7 RNA polymerase (T7 RNAP) (Hattab et al., 2015). However, the major drawback of this system is the overtranscription of the target gene due to the T7 RNAP transcription activity that is over ten times faster than the E. coli enzyme. Since the isolation of spontaneous mutants, namely C41(DE3) and C43(DE3) (Miroux and Walker, 1996) and the identification of their mutation in the genome, it becomes clear that reducing the amount of the T7 RNAP level removes the toxicity associated with the expression of some membrane proteins (Wagner et al., 2008; Kwon et al., 2015). Also, some membrane proteins require a very low rate of transcription to be correctly folded at the E. coli membrane. The first objective of my PhD was to extend the promoter strength coverage of the T7 based expression system. We used genetic and genomic approaches to isolate and characterize new bacterial strains (Angius et al., 2016) in which the level of T7 RNAP is differently regulated than in existing hosts. A second objective was to understand intracellular membrane proliferation in E. coli. Indeed it has been shown that over-expression of membrane proteins, like overexpression of AtpF of E. coli F1Fo ATP synthase is accompanied by the proliferation of intracellular membranes enriched in cardiolipids (Arechaga et al., 2000). To understand metabolic pathways involved in membrane biogenesis, proliferation and organization, we used a RNA sequencing approach at several time point upon over-expression of the F-ATPase b subunit in C43(DE3) host. On the other hand, in collaboration with Gerardo Carranza and Ignacio Arechaga (University of Cantabria, Spain) we studied C43(DE3) cls mutants, in which the cardiolipids genes A, B and C are deleted, to test how they participate to intracellular membranes structuration
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Lauwers, Elsa. "Role of sphingolipids and polyubiquitin chains in intracellular trafficking of the yeast GAP1 permease." Doctoral thesis, Universite Libre de Bruxelles, 2007. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/210648.
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In the past fifteen years, ubiquitin has emerged as a central regulator of membrane protein trafficking. In this context, covalent attachment of this small protein to lysine residues of cargo proteins, a reversible modification termed ubiquitylation, provides a signal for their targeting to the vacuolar/lysosomal lumen where they are degraded, both in yeast and higher eukaryotes. Ubiquitylation is also used as a means of controlling the function of specific proteins in several trafficking machineries. The role of lipids - and in particular of membrane domains named lipid rafts - in controlling the intracellular trafficking of membrane proteins has also been the subject of intense investigation in recent years.One of the membrane proteins of the yeast Saccharomyces cerevisiae whose intracellular trafficking has been extensively studied is the general amino acid permease Gap1. Yet some aspects of the function of ubiquitin in the nitrogen-dependent control of this protein remain controversial. Moreover, the potential role of lipid rafts in regulating the functional properties and traffic of the Gap1 permease had not been investigated before this thesis work.
The first part of our work readdresses the role of Gap1 ubiquitylation, and more precisely of the modification of the permease with polyubiquitin chains linked through the lysine 63 of ubiquitin, in controlling the fate of this protein in the secretory pathway. Our observations indicate that nitrogen-induced ubiquitylation of newly synthesised Gap1 occurs in the trans-Golgi complex. However, contrary to the generally accepted view, this modification is not necessary for the permease to exit this compartment en route to the endosome but only for its subsequent targeting to the vacuolar lumen via the multivesicular body (MVB) pathway. Our results also provide evidence that K63-linked polyubiquitylation is important mostly at the late endosomal level, for proper sorting of Gap1 into the MVB pathway, whether the permease comes from the cell surface by endocytosis or directly from the secretory pathway.
In the second part of this work, we present a set of data providing novel insights into the controversial question of the exact nature of lipid rafts in yeast. We first showed that the Gap1 permease is associated with detergent-resistant membranes (DRMs) - the proposed biochemical equivalent of lipid rafts - when it is located at the cell surface. Our data further suggest that this may be true for most if not all yeast plasma membrane proteins. Moreover, we found that Gap1 production must be coupled to de novo synthesis of sphingolipids (SLs), major constituents of rafts, in order for the newly synthesised permease to be correctly folded, active, associated with DRMs, and stable at the cell surface. We propose a model where Gap1 would associate with newly synthesised SLs during its biogenesis and/or secretion, this association shaping the permease into its native conformation and ensuring its incorporation and stabilisation in specific lipid domains at the plasma membrane. Failure of Gap1 to acquire this lipidic microenvironment in turns leads to its ubiquitin-dependent degradation by a quality-control mechanism. This model might be valid for many other plasma membrane proteins and might account for their lateral distribution between distinct membrane domains.
Doctorat en Sciences
info:eu-repo/semantics/nonPublished
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LADHA, PARAG. "POLYMERIC MEMBRANE SUPPORTED BILAYER LIPID MEMBRANES RECONSTITUTED WITH BIOLOGICAL TRANSPORT PROTEINS." University of Cincinnati / OhioLINK, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1145901880.
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Kemayo, Koumkoua Patricia. "Structural characterisation of highly specific membrane protein-lipid interactions involved in cellular function." Thesis, Strasbourg, 2015. http://www.theses.fr/2015STRAF055/document.
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Les membranes cellulaires sont des systèmes complexes composés de lipides variés qui interagissent avec les protéines pour accomplir une fonction. Leur adressage spécifique dans la cellule est crucial pour le fonctionnement cellulaire. Les vésicules COP (coat protein) sont impliquées dans leur transport dans les premières étapes de la voie de sécrétion. Récemment, une interaction très spécifique a été identifiée entre le domaine transmembranaire de la protéine p24 (p24TMD) abondante dans la membrane des vésicules COP et la sphingomyéline C18:0. Cette spécificité a été identifiée dans le cas d’interaction protéine-protéine et protéine-acide nucléique comme impliquée dans la régulation de fonctions cellulaires, C’est pourquoi nous avons décidé d'étudier sur cette interaction. A cet effet, le p24TMD a été obtenu par synthèse chimique et sa structure étudiée par RMN du solide en présence de la sphingomyèline avec pour but ultime de comprendre la fonctionCell membranes are complex systems composed of variety of lipids that interacts with proteins to trigger cellular function. The delivery of these lipids to the right compartment is crucial for cells to work efficiently. The coat protein (COP) complex vesicles are involved in lipids traffic in the early stages of the secretory pathway. Recently, a highly specific interaction has been found between the transmembrane domain of p24 protein (p24TMD) abundant in COPI membrane and sphingomyelin C18:0. As such highly specific interaction have been reported for protein-protein and protein-nucleic acid interactions to be involved in regulation of cell functions, we decide to investigate this specific interaction. The p24TMD was obtained chemically and investigated by solid state NMR in presence of sphingomyelin with the ultimately goal to understand the function behind
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Lee, Chongsoo. "Raman spectroscopy of supported lipid bilayers and membrane proteins." Thesis, University of Oxford, 2005. http://ora.ox.ac.uk/objects/uuid:76f4be6e-b7d3-46c5-a2a1-3dcc7a399410.
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Off-resonance unenhanced total internal reflection (TIR) Raman Spectroscopy was explored to investigate supported single lipid bilayers with incorporated membrane peptides/proteins at water/solid interface. A model membrane was formed on a planar supported lipid layer (pslb) by the fusion of the reconstituted small unilamellar vesicles (SUVs), and the intensity of bilayer was confirmed by a comparison of Raman spectral intensity in the C-H stretching modes with C16TAB. With prominent Raman sensitivity attained, we studied the 2-D phase transition of DMPC and DPPC pslbs and the temperature-dependent polarised spectra revealed a broad transition range of ca. 10 °C commencing at the calorimetric phase transition temperature. We applied polarised TIR-Raman Spectroscopy to pslbs formed by DMPC SUVs reconstituted with a model membrane-spanning peptide gramicidin D. A preferential channel structure formed by dissolution of trifluoroethanol could be probed by polarised Raman Spectroscopy qualitatively showing an antiparallel β-sheet conformation (different from "standard" one) and our Raman spectra by correlation with NMR and CD data confirmed single-stranded π6.3 β-helical channel structure in the single bilayer. We also studied the membrane-penetrating peptide indolicidin in the presence of DMPC pslb over the chain melting temperature and a β-turn structure was dominantly observed concomitant with membrane perturbation. Dynamic adsorption of DPPC to form pslb from a micellar solution of n-dodecyl-β- D-maltoside could be examined with high sensitivity of every 1-min acquisition. Finally we used polarised TIR-Raman scattering to porcine pancreatic phospholipase A2 hydrolytic activity on DPPC pslbs and revealed lipid-active conformation different from that of the enzyme alone.
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Neuberger, Arthur. "On the structure and function of multidrug efflux pumps." Thesis, University of Cambridge, 2019. https://www.repository.cam.ac.uk/handle/1810/288243.
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Infections arising from multidrug-resistant pathogenic bacteria are spreading rapidly throughout the world and threaten to become untreatable. The origins of resistance are numerous and complex, but one underlying factor is the capacity of bacteria to rapidly export drugs through the intrinsic activity of efflux pumps. In this work, a summary is provided of our current understanding of the structures and molecular mechanisms of multidrug efflux pumps in bacteria (Chapter 1). The emerging picture of the structure, function and regulation of efflux pumps suggests opportunities for countering their activities. Although this thesis primarily explores structure and function, it also elucidates the hidden regulatory mechanism (post-translational) behind the association of a small protein called AcrZ with the tripartite complex AcrAB/TolC, in connection with the lipid environment, and the resulting changes in the latter's functionality (Chapter 2). A regulatory role of the native membrane lipid environment as well as of small proteins for efflux pump activity have previously been hypothesised. I present the first example of a function-regulating role of the lipid cardiolipin in combination with a small protein binding partner (AcrZ) for the substrate selectivity and transport activity of an efflux pump protein (AcrB). This regulation happens through induced structural changes which have remained unseen so far. Alongside with these results, a nanodisc reconstitution method was experimentally adapted for a structure-function investigation of an efflux pump (complex) using cryo-EM (Chapter 2). Beyond some fundamental regulatory insights, hidden intrinsic transport mechanisms for some transporters have also remained to be explored and studied. The discovery of a mechanism for active influx by a prominent efflux pump model system (Chapter 3) provides hope that this phenomenon is more common amongst multidrug transporters and that it could be utilised for drug discovery purposes. This novel feature explains the contradictory findings on this transporter in the past and raises new questions about the little-known physiological role and evolution of efflux pumps. The development and evolution of antimicrobial resistance has frequently shown to be a multifactorial and fast-moving process. One of these factors is the evolution of pumps itself towards an altered functionality (e.g. towards a broader or altered substrate spectrum or higher efflux rates). Against this background, the role of key carboxylate residues for efflux-energising proton trafficking was investigated for a prominent study model of a secondary-active transporter (Chapter 4). The re-allocation and/or addition of acidic residues was demonstrated to result in the preservation of wild type activity or the generation of hyper-efflux activity, respectively. These findings suggest that rapid emergence of antimicrobial resistance could be enhanced by the 'plasticity' in the location of key carboxylate residues with a role in proton coupling. It also demonstrates the necessity of antimicrobial drug design programmes to anticipate possible trajectories of an adaptive evolution of efflux pump. The 'cryo-EM revolution' has boosted the pace at which new structural and functional insights into multidrug efflux pumps are gained. Nevertheless, in order to derive the structure of individual pump components or of a full assembly, it is sometimes necessary to identify and characterise homologues and mutants, which would allow the application of cryo-EM for obtaining near-atomic maps. Functional analyses presented in this work helped to characterise a homologue and mutants of the MacAB/TolC tripartite complex to justify the obtained protein structures and strategies for further functional characterisation (Chapter 5). Given (1) the unusual stoichiometry of a MacB dimer in complex with a hexameric membrane-fusion protein (MacA), which leads to a seeming leakiness of the assembly, and (2) the fact that substrate has to pass through a narrow aperture in the membrane-fusion protein for extrusion, it is rather surprising that MacB was previously shown to transport an entire toxin. An experimental approach was developed that could enable the structure determination of a toxin-bound full assembly of MacAB/TolC (Chapter 5). Finally, the role of multidrug efflux pumps for the evolution of multidrug resistance is yet to be studied and better explored. For instance, evolutionary trajectories of pump overexpression, as compared to those of regular expression or no expression, are unknown yet could have the potential to reveal useful insights for spread prevention and drug design. The outline of an experimental design with some preliminary validating data is presented in Chapter 6.
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Hartshorn, Christopher M. "Studies of the molecular effects of a solid support upon lipid membranes and membrane bound proteins." Pullman, Wash. : Washington State University, 2009. http://www.dissertations.wsu.edu/Dissertations/Fall2009/c_Hartshorn_101209.pdf.
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Lelong-Rebel, Isabelle. "Etude de l'evolution de l'architecture membranaire des neurones d'embryons de poulet en culture : composition et topologie des proteines et des lipides polaires au cours du developpement cellulaire." Université Louis Pasteur (Strasbourg) (1971-2008), 1989. http://www.theses.fr/1989STR13089.
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L'isolement d'une fraction hautement purifiee de membranes plasmiques et l'emploi de radioiodations enzymatiques ont permis de suivre la composition des proteines et de certains lipides polaires, ainsi que leur representation a la surface cellulaire
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Krammer, André Thomas. "Computational studies of protein-membrane interactions and forced unfolding of proteins /." Thesis, Connect to this title online; UW restricted, 2000. http://hdl.handle.net/1773/9697.
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Chwastek, Grzegorz. "Interactions of FCHo2 with lipid membranes." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2013. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-129388.
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Endocytosis is one of the most fundamental mechanisms by which the cell communicates with its surrounding. Specific signals are transduced through the cell membrane by a complex interplay between proteins and lipids. Clathrin depended endocytosis is one of important signalling pathways which leads to budding of the plasmalemma and a formation of endosomes. The FCHo2 is an essential protein at the initial stage of the this process. In is a membrane binding protein containing BAR (BIN, Amphiphysin, Rvs) domain which is responsible for a membrane binding. Although numerous valuable work on BAR proteins was published recently, the mechanistic description of a BAR domain functionality is missing. In present work we applied in vitro systems in order to gain knowledge about molecular basis of the activity of the FCHo2 BAR domain. In our studies we used supported lipid bilayers (SLBs) and lipid monolayers as s model membrane system.
The experiments were carried out with a minimal number of components including the purified FCHo2 BAR domain. Using SLBs we showed that the BAR domain can bind to entirely flat bilayers. We also demonstrated that these interactions depend on the negatively charged lipid species incorporated in the membrane. We designed an assay which allows to quantify the membrane tubulation. We found out that the interaction of the FCHo2 BAR domain with the lipid membrane is concentration dependent. We showed that an area of the bilayer deformed by the protein depends on the amount of the used BAR domain.
In order to study the relation between the mobility of lipids and the activity of FCHo2 BAR domain we designed a small-volume monolayer trough. The design of this micro-chamber allows for the implementation of the light microscopy. We demonstrated that the measured lipid diffusion in the monolayer by our new approach is in agreement with literature data. We carried out fluorescence correlation spectroscopy (FCS) experiments at different density of lipids at the water-air interface.We showed that the FCHo2 BAR domain binding affinity is proportional to the mean molecular area (MMA). We additionally demonstrated that the increased protein binding is correlated with the higher lipid mobility in the monolayer.
Additionally, by curing out high-speed atomic force microscopy (hsAFM) we acquired the structural information about FCHo2 BAR domains orientation at the membrane with a high spatio-temporal resolution. Obtained data indicate the BAR domains interact witheach other by many different contact sites what results in a variety of protein orientations in a protein assemble.
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Köhler, Sebastian. "Etude de bicouches lipidiques amarrées destinées à l'incorporation de protéines membranaires." Thesis, Université Grenoble Alpes, 2020. http://www.theses.fr/2020GRALS023.
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Cette thèse a pour but l’étude de la structure des membranes lipidiques bicouches attachées, qui sont des bicouches lipidiques greffées chimiquement sur une surface plane. Ces systèmes, comparés à d'autres systèmes de membranes modèles, présentent une stabilité accrue, mais leur structure est plus complexe, nécessitant une étude approfondie. La structure des bicouches lipidiques attachées est fortement influencée par l'architecture moléculaire des composés utilisés pour greffer la bicouche lipidique à la surface. Cette thèse porte sur l’étude de deux systèmes d'attache des bicouches lipidiques. Le premier système est une bicouche lipidique attachée à une surface d’or utilisant une seule chaîne alkyle pour ancrer la membrane. Le second système utilise une structure moléculaire synthétique semblable à un lipide pour ancrer la membrane et lie à une surface de silicium par chimie du silane. Cette thèse a trois objectifs pour ces systèmes d'attache: (i) étudier les bicouches attachées avec des fractions élevées de cardiolipine lipidique, (ii) étudier la structure des membranes attachées sur une surface via l’utilisation de différentes molécules de greffage, et (iii) étudier l'incorporation de la protéine membranaire NhaA dans ces systèmes. Le lipide cardiolipine a une structure unique contenant quatre chaînes d'acides gras. Cela conduit à des propriétés intéressantes des bicouches le contenant ainsi qu'à une interaction avec plusieurs protéines membranaires. Nous avons étudié la structure des membranes captives avec des fractions de cardiolipine élevées pouvant atteindre 80%. Les deux systèmes d'attache pourraient être utilisés pour former des bicouches lipidiques avec des fractions élevées de cardiolipine. En comparaison, le cardiolipine est généralement présent dans les membranes naturelles à de faibles fractions de l’ordre de 10%. La nanostructure des membranes avec différentes fractions de cardiolipine a été structurellement caractérisée par réflectométrie neutronique, et leurs propriétés électrophysiologiques ont été étudiées par spectroscopie d'impédance électrochimique. Ces membranes attachées de cardiolipine ont montré une structure très condensée avec de hautes propriétés d'étanchéité électrique. Ces résultats sont importants pour d'autres utilisations technologiques des bicouches lipidiques captives. Enfin, l'incorporation de la protéine membranaire NhaA dans de tels systèmes membranaires a été étudiée. NhaA a été incorporée avec succès dans les deux systèmes d'architecture d'attache à des fractions élevées sans perturber de manière significative la structure bicouche lipidique attachée. Cela fait des membranes attachées contenant des quantités élevées de cardiolipine un système de membrane modèle prometteur pour l'incorporation de protéinesThis thesis investigates the structure of tethered lipid bilayer membranes, which are lipid bilayers chemically grafted to a planar surface. Compared to other model membrane systems they show increased stability, but have a higher complexity in structure, which requires thorough investigation. The structure of tethered lipid bilayers is strongly influenced by the molecular architecture of the grafting molecules. Within the framework of this thesis, two different types of tether architecture were investigated. The first was a lipid bilayer tethered to a gold surface and using a single alkyl chain to anchor the membrane, while the second used a synthetic lipid-like molecular structure and was bound to a silicon surface by silane chemistry. Three main objectives for those systems were pursued: (i) to investigate tethered bilayers with high fractions of the lipid cardiolipin, (ii) to investigate the structural differences between tethered membranes having different surface grafting molecules, and (iii) to investigate the incorporation of the membrane protein NhaA into these systems. Cardiolipin has a unique structure characterized by four fatty acid chains, which leads to interesting effects on bilayer structure, as well as interaction with several membrane proteins. Tethered membranes with high cardiolipin fractions of up to 80 % were structurally characterized by neutron reflectometry, and their electrophysiological properties were investigated by electrochemical impedance spectroscopy. In both tethered bilayer architectures, cardiolipin containing membranes showed a very condensed structure with high electrical sealing properties. Differences in tether architecture mainly affected the structure of the water-filled sub-membrane space. The tethered bilayers grafted to a gold surface showed very low water content below the membrane, while the sub-membrane space of bilayers grafted to a silicon surface was highly hydrated. Finally, NhaA was successfully incorporated into both tether architecture systems at high fractions without damaging the tethered lipid bilayer structure significantly. This makes tethered membranes containing elevated amounts of cardiolipin a promising model membrane system for protein incorporation and offers possibilities for further technological uses of tethered lipid bilayers
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Allen, Victoria Louise. "Photoactivated lipids as modulators of membrane protein folding." Thesis, University of Bristol, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.528101.
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Botelho, Ana Vitoria. "Lipid-protein interactions: Photoreceptor membrane model." Diss., The University of Arizona, 2005. http://hdl.handle.net/10150/280765.
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G-protein coupled receptors (GPCRs) are transmembrane proteins capable of recognizing an astonishing variety of biological signals, ranging from photons of light to hormones, odorants, and neurotransmitters involved in key biological signaling processes. The aim of this work is to identify how lipid-protein interactions involving the membrane bilayer ultimately affect such vital biological functions. Here the relationship between the bilayer thickness, hydrophobic mismatch, and protein aggregation are investigated by expanding the framework of membrane-receptor interactions in terms of a new flexible surface model. Previously, we have shown how coupling of the elastic stress-strain due to mismatch of the spontaneous curvature and hydrophobic thickness at the lipid/protein interface can govern the conformational transitions of membrane proteins. This approach has now been extended to include coupling of the lateral organization of the GPCR rhodopsin to the curvature and area stress and strain of the proteolipid membrane. Rhodopsin was labeled with site-specific fluorophores, and a FRET technique was employed to probe protein association in different lipid environments. Moreover, UV-visible spectroscopy was used for thermodynamic characterization of the conformational change of rhodopsin. Lastly, the deformation of the lipids with and without rhodopsin was probed in terms of acyl chain order parameters and relaxation rates by solid-state NMR methods, giving insight into the lipid deformation. The results showed that optimal receptor activation occurs in phosphatidylcholine bilayers of 20-carbon acyl chain length, hence one can say that metarhodopsin II is likely to adopt an elongated shape. Lipids promoting aggregation, or below their gel to liquid crystalline transition temperature all favor formation of metarhodopsin I. The data also showed that association and activation of rhodopsin do not always correlate. In terms of the extended flexible surface model, the stress due to hydrophobic mismatch is coupled via the effective number of lipids surrounding the protein due to the lateral organization of the membrane. The measured changes in rhodopsin-rhodopsin interactions and membrane influences on the conformation of the protein after photoisomerization may be crucial to understanding physiological regulation of the rod disk membranes. They are relevant to understanding the complexity of biomembranes involved in many cellular mechanisms, including signal transduction.
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Dos, Santos Morais Raphael. "Interaction dystrophine-membrane : structure 3D de fragments de la dystrophine en présence de phospholipides." Thesis, Rennes 1, 2017. http://www.theses.fr/2017REN1B062/document.
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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 myopathiesDystrophin 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
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Bacle, Amélie. "Etude in silico des gouttelettes lipidiques et de leur interaction avec des protéines périphériques via des hélices amphipathiques." Thesis, Sorbonne Paris Cité, 2016. http://www.theses.fr/2016USPCC235/document.
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Les gouttelettes lipidiques (GL) sont des organites intracellulaire qui jouent un rôle central dans le métabolisme des lipides. Elles sont également impliquées dans des maladies telles que l'obésité ou le diabète. Les GL ont une structure unique : une monocouche de phospholipides (PL) qui entoure un cœur de lipide neutre composé de triglycérides (TAG) et d'esters de cholestérol (CE). Certaines protéines sont recrutées sur les GL mais également à la surface d'autres organites, alors que d'autres protéines ciblent spécifiquement la surface des GL. Il a été montré que quelques une de ces protéines seraient sensibles à une haute tension de surface, soit une augmentation de l'aire par lipide, dans des GL reconstituées. Comment les propriétés de surface d'une GL diffèrent d'une membrane ? Comment la surface d'une GL répond à l'augmentation de la tension de surface ?Comment les protéines interagissent avec la surface des GL ? Nous avons réalisé des simulations de dynamique moléculaire atome-unifié de système tricouche qui mime la surface d'une GL afin de caractériser les propriétés de surface de cet organite. Plusieurs simulations ont été effectuées à différentes tension de surface en augmentant l'aire par lipide. Les propriétés de surface ont été caractérisées en terme de défauts de \textit{packing} (i.e. vides interfaciaux à l'interface membrane/eau). Aucune différence n'a été observé avec une bicouche à l'équilibre. Cependant, la tension de surface promeut l'insertion de lipides neutres dans la monocouche et augmente significativement les défauts de \textit{packing}. Des simulations préliminaires sur l'interaction d'une protéine modèle, la périlipine 4, qui se lie aux GLs \textit{in vivo} via une longue hélice amphipathique 11/3 ont été faites. Les premiers résultats montrent que la protéine adopte une structure plus flexible dans une interface huile/eau que dans une interface membrane/eau. Des essais de dimérisation montrent que la répartition des résidus chargés serait importante pour le processus d'oligomérisation. Pris globalement, ces résultats apportent une compréhension moléculaire quantitative sur l'effet de la tension de surface sur la monocouche de GL et des résultats préliminaires sur l'interaction protéine/GL. Notre travail constitue une première étape vers la description du comportement et de la structure des propriétés de surface des GL et peut être utile à la compréhension du ciblage protéique vers la surface de GLLipid droplets (LD) are intracellular organelles that have a central role in lipid metabolism andimplication in diseases such as obesity and diabetes. LDs have a unique architecture: aphospholipid (PL) monolayer that surrounds a neutral lipid core composed of triacylglycerols (TAG)and cholesteryl esters (CE). Some proteins are recruited both to LDs and to other cellularorganelles, whereas others are targeted specifically to the surface of LDs. It has been shown thatsome of these proteins could be sensitive to a high surface tension (ST), increase in the area perlipid, in reconstituted LD. How do surface properties differ between a membrane and an LD? Howdoes the LD surface respond to an increase in ST? How do proteins interact with LDs? Weperformed united-atom molecular dynamics simulations on trilayer systems that mimic the LDsurface to investigate the surface properties of this organelle. Several simulations were performedat different ST by increasing the area per lipid. Surface properties were characterized in terms ofpacking defects (i.e interfacial voids at the membrane-water interface). No difference was observedwith a bilayer at equilibrium. However, high ST promoted the insertion of neutral lipids into themonolayer and a significant increase of packing defects. Preliminary simulations has been done oninteraction of a model protein called perilipin 4, which binds to LDs \textit{in vivo} using a long 11/3amphipathic helix. The first results show that the protein adopts a more flexible conformation on oilwaterinterface than in bilayer-water interface. Attempts of dimerisation show that the localization ofthe charged residues may be involved in the oligomerisation process. Taken together, our resultsprovide a quantitative molecular understanding of how ST affects the LD surface and preliminaryresults on protein-LD interaction. Our work constitutes a first step towards characterizing thebehavior and structure of LD surface properties and will be useful for a better understanding onhow some specific proteins are targeted to LD
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Bruzzese, Novoa Agustín Alberto. "Investigation of the influence of the membrane lipid environment on g protein-coupled receptor activation by molecular dynamics simulations." Doctoral thesis, Universitat Autònoma de Barcelona, 2021. http://hdl.handle.net/10803/671812.
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Els receptors acoblats a proteïnes G (GPCRs) són importants dianes terapèutiques per a nombroses malalties. Si bé els GPCRs s’han estudiat àmpliament en les últimes dècades, els mecanismes moleculars que determinen la seva activació així com la seva modulació al·lostèrica per lípids de membrana no han estat elucidats íntegrament. La present tesi examina els processos de (de)activació de dos GPCRs prototípics de la classe A, β2-adrenèrgic i adenosina A2a, en membranes de diferent composició lipídica mitjançant simulacions de dinàmica molecular.Los receptores acoplados a proteínas G (GPCRs) son importantes dianas terapéuticas para numerosas enfermedades. Si bien los GPCRs se han estudiado ampliamente en las últimas décadas, los mecanismos moleculares que determinan su activación así como su modulación alostérica por lípidos de membrana no han sido elucidados en su totalidad. La presente tesis examina los procesos de (de)activación de dos GPCRs prototípicos de la clase A, β2-adrenérgico y adenosina A2a, en membranas de diferente composición lipídica mediante simulaciones de dinámica molecular.
G-protein-coupled receptors (GPCRs) are important therapeutic targets for numerous diseases. Although GPCRs have been extensively studied in recent decades, the molecular mechanisms that determine their activation as well as their allosteric modulation by membrane lipids have not been fully elucidated. This thesis examines the processes of (de)activation of two prototypical class A GPCRs, β2-adrenergic and adenosine A2a receptors, in membranes of different lipid composition by means of molecular dynamics simulations.
Universitat Autònoma de Barcelona. Programa de Doctorat en Neurociències
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Kobayashi, Aya. "Mechanism of membrane lipid efflux by ABC proteins." Kyoto University, 2007. http://hdl.handle.net/2433/136565.
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Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第13311号
農博第1653号
新制||農||947(附属図書館)
学位論文||H19||N4290(農学部図書室)
UT51-2007-H676
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 植田 和光, 教授 阪井 康能, 教授 植田 充美
学位規則第4条第1項該当
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Witkowski, Thomas, Rainer Backofen, and Axel Voigt. "The influence of membrane bound proteins on phase separation and coarsening in cell membranes." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-139226.
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A theoretical explanation of the existence of lipid rafts in cell membranes remains a topic of lively debate. Large, micrometer sized rafts are readily observed in artificial membranes and can be explained using thermodynamic models for phase separation and coarsening. In live cells such domains are not observed and various models are proposed to describe why the systems do not coarsen. We review these attempts critically and show within a phase field approach that membrane bound proteins have the potential to explain the different behaviour observed in vitro and in vivo. Large scale simulations are performed to compute scaling laws and size distribution functions under the influence of membrane bound proteins and to observe a significant slow down of the domain coarsening at longer times and a breakdown of the self-similarity of the size-distribution functionDieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich
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Pilot, Jeffrey David. "Effects of lipid on membrane protein function." Thesis, University of Southampton, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.390717.
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Witkowski, Thomas, Rainer Backofen, and Axel Voigt. "The influence of membrane bound proteins on phase separation and coarsening in cell membranes." Royal Society of Chemistry, 2012. https://tud.qucosa.de/id/qucosa%3A27814.
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A theoretical explanation of the existence of lipid rafts in cell membranes remains a topic of lively debate. Large, micrometer sized rafts are readily observed in artificial membranes and can be explained using thermodynamic models for phase separation and coarsening. In live cells such domains are not observed and various models are proposed to describe why the systems do not coarsen. We review these attempts critically and show within a phase field approach that membrane bound proteins have the potential to explain the different behaviour observed in vitro and in vivo. Large scale simulations are performed to compute scaling laws and size distribution functions under the influence of membrane bound proteins and to observe a significant slow down of the domain coarsening at longer times and a breakdown of the self-similarity of the size-distribution function.Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.
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Chiantia, Salvatore. "Protein-lipid interactions in raft-exhibiting membranes probed by combined AFM and FCS." Doctoral thesis, [S.l. : s.n.], 2008. http://nbn-resolving.de/urn:nbn:de:bsz:14-ds-1216391330086-30964.
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Wikström, Malin. "Synthesis and protein curing abilities of membrane glycolipids." Doctoral thesis, Stockholm University, Department of Biochemistry and Biophysics, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-1361.
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There are many types of membrane lipids throughout Nature. Still little is known about synthesizing pathways and how different lipids affect the embedded membrane proteins. The most common lipids are glycolipids since they dominate plant green tissue. Glycolipids also exist in mammal cells as well as in most Gram-positive bacteria. Glycosyltransferases (GTs) catalyze the final enzymatic steps for these glycolipids. In the bacteria Acholeplasma laidlawii and Streptococcus pneumonie and in the plant Arabidopsis thaliana, GTs for mono-/di-glycosyl-diacylglycerol (-DAG) are suggested to be regulated to keep a certain membrane curvature close to a bilayer/nonbilayer phase transition. The monoglycosylDAGs are nonbilayer-prone with small headgroups, hence by themselves they will not form bilayer structures.
Here we have determined the genes encoding the main glycolipids of A. laidlawii and S. pneumonie. We have also shown that these GTs belong to a large enzyme group widely spread in Nature, and that all four enzymes are differently regulated by membrane lipids. The importance of different lipid properties were traced in a lipid mutant of Escherichia coli lacking the major (75 %), nonbilayer-prone/zwitterionic, lipid phosphatidylethanolamine. Introducing the genes for the GTs of A. laidlawii and two analogous genes from A. thaliana yielded new strains containing 50 percent of glyco-DAG lipids. The monoglyco-DAG strains contain significant amounts of nonbilayer-prone lipids while the diglyco-DAG strains contain no such lipids. Comparing these new strains for viability and the state of membrane-associated functions made it possible to connect different functions to certain lipid properties. In summary, a low surface charge density of anionic lipids is important in E.coli membranes, but this fails to be supportive if the diluting species have a too large headgroup. This indicates that a certain magnitude of the curvature stress is crucial for the membrane bilayer in vivo.
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Reading, Eamonn. "Structural insights into membrane proteins, membrane protein-lipid interactions and drug metabolites in the gas-phase from ion mobility mass spectrometry." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:f94d42d1-f870-49f9-98b5-42c9b1064e1e.
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Investigating the structures of membrane proteins and their interactions with lipids remains challenging for well-established biophysical techniques. In this thesis the use of mass spectrometry (MS) and ion mobility (IM) spectrometry were explored for the interrogation of membrane proteins, their stoichiometry, stability and interactions with lipids. The techniques used were also applied to the identification of drug metabolites. In the first two chapters reviews of both mass spectrometry methods, and membrane protein biogenesis and membrane protein-lipid interactions are presented. The first challenge for studying membrane proteins by MS was to optimise solution conditions. A detergent screening strategy was developed for this purpose (Chapter 3). The various detergent environments studied revealed dramatic differences in mass spectral quality permitting investigation of membrane protein-lipid interactions. Changes were observed in the electrospray charging of membrane proteins and trends were established from an extensive collection of membrane proteins ejected from a wide variety of detergent environments. The physicochemical principles behind the MS of membrane proteins were deduced and are presented (Chapter 4). The results of these experiments led to a deeper understanding of the ionisation processes and the influence of detergent micelles on both charge state and release mechanisms. Experiments from a range of different micelles also allowed the influence of charge and its effects on the preservation of native-like membrane protein conformations to be monitored by IM-MS. By resolving lipid-protein interactions, and by monitoring the effects of lipid binding on the unfolding of three diverse membrane protein complexes, substantial differences in the selectivity of membrane proteins for different lipids were revealed (Chapter 5). Interestingly lipids that stabilised membrane proteins in the gas-phase were found to induce modifications in structure or function thus providing an approach to assess direct lipid contributions, and to rank order lipids based on their ability to modulate membrane proteins. Using the MS approaches developed here also enabled study of the diversity of oligomeric states of the mechanosensitive channel of large conductance (MscL) (Chapter 6). Results revealed that the oligomeric state of MscL is sensitive to deletions in its C-terminal domain and to its detergent-lipid environment. Additionally, a case study with GlakoSmithKline (GSK) was undertaken using IM-MS technology but in this case applied to the identification of drug metabolites (Chapter 7). The results showed that IM-MS and molecular modelling could inform on the identity of different drug metabolites and highlights the potential of this approach in understanding the structure of various drug metabolites.
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Liu, Wei. "Membrane protein crystallization in the lipid cubic phase testing hypotheses relating to reconstitution /." Columbus, Ohio : Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1196274127.
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Hernandez, Lopez Agustin. "Plasma membrane sterols and fatty acids : effects on membrane properties and H'+-ATPase of Ustilago maydis." Thesis, University of Bristol, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.336825.
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