DOCSLIB.ORG

Les Récepteurs Couplés Aux Protéines G

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Les Récepteurs Couplés Aux Protéines G Université de Montréal Structure quaternaire des récepteurs de chimiokines CXCR4 et CCR2 et interaction avec leurs effecteurs. par Sylvain Armando Département de Biochimie Faculté de médecine Thèse présentée à la Faculté des études supérieures en vue de l’obtention du grade de docteur en Biochimie Décembre 2010 © Armando, 2010 ii Université de Montréal Faculté des études supérieures Cette thèse intitulée : Structure quaternaire des récepteurs de chimiokines CXCR4 et CCR2 et interaction avec leurs effecteurs présentée par : Sylvain Armando a été évaluée par un jury composé des personnes suivantes : Gérardo Ferbeyre, président-rapporteur Richard Leduc, examinateur externe, rapporteur Michel Bouvier, directeur de recherche Laurent Prézeau, directeur de recherche Pascal Chartrand, membre du jury Bernard Mouillac, représentant de l’université Montpellier 2 Graciela Pineyro, représentante du doyen de la FES 1 Résumé Les récepteurs couplés aux protéines G (RCPG) sont une famille très diversifiée de protéines membranaires capables de répondre à un grand nombre de signaux chimiques tels que des photons, des molécules odorantes, ou des hormones. En plus de cette diversité, l’étude des RCPG montre que des associations protéiques spécifiques multiplient les possibilités de signalisation de chacun de ces récepteurs. En permettant d’atténuer, de potentialiser, ou de générer une nouvelle voie de signalisation, l’association des RCPG en oligomères s’avère une importante source de diversité. L’utilisation du transfert d’énergie de résonance de bioluminescence (BRET) qui permet de détecter les interactions protéiques a révélé de nombreuses associations de RCPG. Durant cette thèse, des outils ont été développés pour combiner efficacement le BRET à des essais de complémentation de protéines (PCA) dans le but de savoir si l’oligomérisation des RCPG pouvait impliquer plus de deux récepteurs. Les résultats présentés montrent que les récepteurs de chimiokines CXCR4 et CCR2 forment des homo et hétéro tétramères, et que l’activation d’un dimère CCR2 peut moduler la conformation d’un dimère CXCR4 par un changement conformationnel trans-récepteur. La coopérativité négative de liaison de ligand qui a été démontrée auparavant entre CXCR4 et CCR2 dans des lymphocytes T CD4+ exprimant les récepteurs de manière endogène confirme la validité biologique de cette interaction. Les données présentées suggèrent également que ces complexes peuvent engager les effecteurs Gαi et β-arrestine2, indiquant qu’ils représentent la forme fonctionnelle de ces récepteurs. Enfin, nous avons pu confirmer que chaque récepteur de l’hétérodimère CXCR4-CCR2 est impliqué dans l’engagement des effecteurs lors de l’activation de CCR2. Un autre niveau de complexité dans la signalisation des RCPG est atteint par leur capacité à coupler de multiples protéines G. La liaison du facteur dérivé des cellules stromales (SDF-1) au récepteur CXCR4 permet la migration des lymphocytes T par une voie de signalisation dépendante de la protéine Gαi. Nous avons pu démontrer en revanche que la 2 migration des cellules de cancer du sein était initiée par un couplage de CXCR4 à la voie Gα13-Rho pour former des métastases dans des organes distants. Enfin, un dernier niveau de régulation des RCPG a été abordé par l’étude de la phosphorylation de CXCR4 suite à son activation, qui permet la désensibilisation du récepteur et l’engagement de voies de signalisation dépendantes de la β-arrestine. Il apparaît que la désensibilisation de la voie du calcium serait médiée par la phosphorylation de CXCR4 par les kinases des RCPG (GRK) GRK2 et GRK6 et le recrutement de β- arrestine2, alors GRK3, GRK6 et la β-arrestine1 potentialiseraient l’activation des kinases régulées par les signaux extracellulaires (ERK1/2). Nous suggérons également que c’est la phosphorylation de l’extrémité C-terminale de CXCR4 qui permettrait son association avec la β-arrestine. Mots-clés : Récepteurs couplés aux protéines G (RCPG), récepteur de chimiokine CXCR4, CCR2, transfert d’énergie de résonance de bioluminescence (BRET), dimère, tétramère, oligomère, essai de complémentation de protéines (PCA), complémentation bimoléculaire de luminescence (BiLC), complémentation bimoléculaire de fluorescence (BiFC), protéines G, β-arrestines, cancer, métastases, phosphorylation, kinases des RCPG (GRK). 3 Abstract G protein-coupled receptors (GPCRs) are a diverse family of membrane proteins capable of responding to a large number of extracellular stimuli including photons, odorant molecules and hormones. In addition to this diversity, it has been shown that GPCRs form specific protein:protein interactions, multiplying the signalling possibilities of each of these receptors. With the ability to diminish, to potentiate or even generate new signalling pathways, the oligomeric association of GPCRs plays an important role in generating this diversity. The use of bioluminescence resonance energy transfer (BRET), which allows the detection of interactions among proteins, has revealed numerous associations between GPCRs. During this thesis, tools have been developed that effectively combine BRET with protein complementation assays (PCA) with the goal of determining if interactions between GPCRs could involve more than two receptors. The results show that the chemokine receptors CXCR4 and CCR2 form both homo and hetero tetramers, and that the activation of a dimer of CCR2 can modulate the conformation of a CXCR4 dimer through a trans- receptor conformational change. Negative cooperativity of ligand binding has previously been demonstrated between CXCR4 and CCR2 in CD4+ T lymphocytes endogenously expressing the receptors, confirming the biological validity of this interaction. The data presented also suggests that these complexes can engage the effector proteins Gαi and β- arrestin 2, indicating that they represent a functional form of the receptors. Furthermore, we have confirmed that each receptor of the CXCR4-CCR2 heterodimer is implicated in the engagement of effectors during the activation of CCR2. An additional level of complexity in GPCR-promoted signaling exists in their capacity to couple of multiple G proteins. Binding of stromal cell-derived factor-1 (SDF-1) to CXCR4 is known to promote T lymphocyte migration through a Gαi-dependent signalling pathway. In addition to this mechanism, we have demonstrated that breast cancer cell migration can initiated by a coupling of CXCR4 to the Gα13-Rho pathway, leading to the formation of metastases in distant organs. 4 Finally, a novel level of GPCR regulation was revealed through the study of CXCR4 phosphorylation following its activation, which leads to the desensitization of the receptor and the engagement of β-arrestin-dependent signalling pathways. It appears that the desensitization of calcium signalling is mediated through the phosphorylation of CXCR4 by the GPCR kinases (GRKs) GRK2 and GRK6 and the recruitment of β-arrestin 2, whereas GRK3, GRK6 and β-arrestin 1 potentiate the activation of extracellular regulated kinase (ERK1/2). We also propose that the phosphorylation of the far C-terminal tail of CXCR4 is required for the interaction between the receptor and β-arrestin. Keywords : G protein coupled receptors (RCPG), chemokine receptor CXCR4, CCR2, bioluminescence resonance energy transfer (BRET), dimer, tetramer, oligomer, protein complémentation assay (PCA), bimolecular luminescence complementation (BiLC), bimolecular fluorescence complementation (BiFC), G proteins, β-arrestins, cancer, metastasis, phosphorylation, GPCR kinases (GRK). 5 6 Table des matières Introduction ................................................................................................................................ 16 Communication cellulaire. ............................................................................................................... 16 Les récepteurs couplés aux protéines G. ..................................................................................... 17 Généralités sur les RCPG. .............................................................................................................................. 17 Les différentes classes de RCPG. ................................................................................................................ 19 Structure et activation des RCPG .............................................................................................................. 21 Structure ............................................................................................................................................................................ 21 Activation des RCPG et couplage aux protéines G ........................................................................................... 23 Famille des protéines G hétérotrimériques. ......................................................................................... 24 Oligomérisation des RCPG ................................................................................................................ 40 Liaison de ligand radiomarqué, micro-agrégation et complémentation fonctionnelle ................. 41 Co-immunoprécipitation ............................................................................................................................................ 42 Cas particulier des récepteurs de classe C : ........................................................................................................ 44 Méthodes de transfert d’énergie par résonance de Förster et par bioluminescence
Load more

Recommended publications
  • 3. Lipid Rafts
    UNIVERSITÀ DEGLI STUDI DI TRIESTE XXX CICLO DEL DOTTORATO DI RICERCA IN NANOTECNOLOGIE Lipid raft formation and protein-lipid interactions in model membranes Settore scientifico-disciplinare: FIS/03 DOTTORANDO Fabio Perissinotto COORDINATORE PROF. Lucia Pasquato SUPERVISORE DI TESI Dr. Loredana Casalis CO-SUPERVISORE DI TESI Dr. Denis Scaini ANNO ACCADEMICO 2016/2017 TABLE OF CONTENTS ABSTRACT ......................................................................................................................................... 4 INTRODUCTION ............................................................................................................................... 6 1. Biological membranes ............................................................................................................... 6 1.1 Lipid composition of cellular membranes ............................................................................ 7 1.2 Membrane proteins ........................................................................................................... 11 2. Physical and structural properties of cell membranes ........................................................... 12 2.1 Lipid-lipid interactions and phase separation .................................................................... 12 2.2 Membrane asymmetry ....................................................................................................... 14 2.3 Lipid diffusion ....................................................................................................................
    [Show full text]
  • Doxorubicin and Paclitaxel Cause Different Changes In
    CHANGES IN PLASMA MEMBRANE FLUIDITY OF MCF-7 CELLS 135 POSTÊPY BIOLOGII KOMÓRKI TOM 36, 2009 SUPLEMENT NR 25 (135152) Rola komórek dendrytycznych w odpowiedzi transplantacyjnej* The role of dendritic cells in transplantation Maja Budziszewska1, Anna Korecka-Polak1, Gra¿yna Korczak-Kowalska1,2 1Zak³ad Immunologii, Wydzia³ Biologii, Uniwersytet Warszawski 2 Instytut Transplantologii, Warszawski Uniwersytet Medyczny *Dofinansowanie z grantu MNiSzW nr N N402 268036 Streszczenie: Komórki dendrytyczne (DC) s¹ najwa¿niejszymi komórkami prezentuj¹cymi antygenDOXORUBICIN (APC) limfocytom AND T. StopieñPACLITAXEL dojrza³oci komórki DC ma kluczowe znaczenie dla rodzaju odpowiedzi limfocytów T. Niedojrza³a komórka DC indukujeCAUSE stan DIFFERENT tolerancji, podczas CHANGES gdy dojrza³a IN komórkaPLASMA DC MEMBRANE- pe³n¹ odpowied immunologiczn¹.FLUIDITY Ma toOF ogromne MCF-7 znaczenie BREAST w transplantologii,CANCER CELLS a zw³aszcza w reakcjach odrzucania przeszczepu po transplantacjach narz¹du. Komórka DC dawcy prezentujeDOKSORUBICYNA antygen w sposób I PAKLITAKSEL bezporedni, natomiast POWODUJ¥ komórka RÓ¯NE DC ZMIANYbiorcy drog¹ poredni¹.W P£YNNOCI Komórki B£ONY DC niedojrza³ePLAZMATYCZNEJ lub o w³aciwociach KOMÓREK RAKA tolerogennych PIERSI MCF-7 mog¹ wyd³u¿yæ prze¿ycie przeszczepu allogenicznego. Takie oddzia³ywanie na funkcjê komórekKarolina MATCZAKDC, aby by³y1, Aneta one niewra¿liweKOCEVA-CHYLA na sygna³y1, Krzysztof dojrzewania GWOZDZINSKI in vivo lub2, aktywowanie komórek DC charakteryzuj¹cychZofia JÓWIAK1 siê trwa³ymi w³aciwociami tolerogennymi
    [Show full text]
  • Molecular Dynamics Simulations of Tight Junction Proteins
    Molecular Dynamics Simulations of Tight Junction Proteins Eleni Fitsiou This dissertation is submitted for the degree of Doctor of Philosophy August 2020 Department of Chemistry I would like to dedicate this dissertation to my husband Antonios and sons Dimitrios and Ioannis. ii “Wisdom begins in wonder” Socrates iii Declaration I, Eleni Fitsiou, declare that this thesis titled ‘Molecular Dynamics Simulations of Tight Junction Proteins’ has not been submitted in support of an application for another degree at this or any other university. It is the result of my own work and includes nothing that is the outcome of work done in collaboration except where specifically indicated. Where I have quoted from the work of others, the source is always given. Lancaster University, UK iv Abstract Tight junctions (TJs) are specialised cell-cell structures that serve primarily as a barrier to molecular transport through the intercellular space between the cells. The claudin family of proteins are the main structural and functional components of the TJ strands that circumscribe the cells. The detailed molecular organisation at the TJs is not entirely resolved, being relatively inaccessible by current experimental methods. Here, we have employed molecular dynamics simulations using both atomistic and coarse-grained models to investigate the TJ structure formed by claudin-1 using self-assembly coupled with free energy calculations and enhanced sampling techniques. A feature of the studies is that the self-assembly simulations have been carried out using atomistic detail (a first) by simulating only the extracellular domains of claudin-1 in an implied membrane. The results show that the cis-interaction can occur in the absence of trans-interacting partners and that a claudin dimer is the smallest stable unit.
    [Show full text]
  • Structural Elucidation of the Interaction Between Neurodegenerative Disease-Related Tau Protein with Model Lipid Membranes Emmalee Jones
    University of New Mexico UNM Digital Repository Nanoscience and Microsystems ETDs Engineering ETDs 1-28-2015 Structural Elucidation of the Interaction Between Neurodegenerative Disease-Related Tau Protein with Model Lipid Membranes Emmalee Jones Follow this and additional works at: https://digitalrepository.unm.edu/nsms_etds Recommended Citation Jones, Emmalee. "Structural Elucidation of the Interaction Between Neurodegenerative Disease-Related Tau Protein with Model Lipid Membranes." (2015). https://digitalrepository.unm.edu/nsms_etds/16 This Dissertation is brought to you for free and open access by the Engineering ETDs at UNM Digital Repository. It has been accepted for inclusion in Nanoscience and Microsystems ETDs by an authorized administrator of UNM Digital Repository. For more information, please contact [email protected]. Emmalee M. Jones Candidate Nanoscience and Microsystems Engineering Department This dissertation is approved, and it is acceptable in quality and form for publication: Approved by the Dissertation Committee: Eva Chi Chairperson Steven Graves Andrew Shreve Deborah Evans i STRUCTURAL ELUCIDATION OF THE INTERACTION BETWEEN NEURODEGENERATIVE DISEASE-RELATED TAU PROTEIN WITH MODEL LIPID MEMBRANES BY EMMALEE M. JONES B.S., Applied Physics, Brigham Young University, 2009 M.S., Nanoscience and Microsystems Engineering, University of New Mexico, 2013 DISSERTATION Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Nanoscience and Microsystems Engineering The University of New Mexico Albuquerque, New Mexico December, 2014 ii ACKNOWLEDGMENTS I wish to gratefully acknowledge the support of my academic advisor, Dr. Eva Y. Chi of the Department of Chemical and Nuclear Engineering. She is an inspiring and patient mentor who has devoted many hours to me.
    [Show full text]
  • Interplay Between Phospholipids and Digalactosyldiacylglycerol in Phosphate Limited Oats
    University of Gothenburg Faculty of Science 2009 Interplay Between Phospholipids and Digalactosyldiacylglycerol in Phosphate Limited Oats Henrik Tjellström Akademisk avhandling för filosofie doktorsexamen in växtmolekylärbiologi, som enligt beslut i lärarförslagsnämnden i biologi kommer att offentligt försvaras fredagen den 27:e mars 2009, klockan 10.15 i föreläsningssalen, Institutionen för Växt-och Miljövetenskaper, Carl Skottbergsgata 22B, 413 19 Göteborg Examinator: Professor Adrian K. Clarke Fakultetsopponent: Dr Sébastien Mongrand, CNRS, Université Victor Segalen Bordeaux 2, Bordeaux, France Göteborg, Mars 2009 ISBN: 978-91-85529-27-8 Interplay Between Phospholipids and Digalactosyldiacylglycerol in Phosphate Limited Oats Henrik Tjellström University of Gothenburg Dept of Plant and Environmental Sciences Box 461, SE-405 30 Gothenburg, Sweden ABSTRACT Phosphate is an essential nutrient. In most soils it is limiting, which has resulted in that phosphate is supplied as fertilizer to increase crop yield. Through evolution, plants have adapted several mechanisms to increase phosphate uptake from the soil and to household with acquired phosphate. A recent discovered house-holding mechanism is that plants utilize the phosphate bound in the headgroups of phospholipids: under phosphate-limiting conditions, phospholipids can be replaced by the non-phosphate containing lipid digalactosyldiacylglycerol (DGDG), previously assumed to reside in plastid membranes. The extra-plastidial phospholipid-to-DGDG replacement occurs in plasma membrane, tonoplast and mitochondria and has led to discoveries of new enzymes and metabolic pathways in plants. This thesis reports that phosphate limitation-induced biochemical and lipid compositional changes in oat root plasma membranes occur prior to any morphological changes in the oat. The phospholipase kinetics suggests that the plasma membrane is continuously supplied with phospholipids and that the products of plasma membrane lipase activities, phosphatidic acid and diacylglycerol, both are removed from the membrane.
    [Show full text]
  • Cholesterol Increases Kinetic, Energetic, and Mechanical Stability
    Cholesterol increases kinetic, energetic, and PNAS PLUS mechanical stability of the human β2-adrenergic receptor Michael Zochera, Cheng Zhangb, Søren G. F. Rasmussenb, Brian K. Kobilkab,1, and Daniel J. Müllera,1 aDepartment of Biosystems Science and Engineering, Eidgenössiche Technische Hochschule (ETH) Zurich, 4058 Basel, Switzerland; and bDepartment of Molecular and Cellular Physiology, Stanford University School of Medicine, Palo Alto, CA 94305 Edited by Wolfgang Baumeister, Max Planck Institute of Biochemistry, Martinsried, Germany, and approved October 10, 2012 (received for review July 18, 2012) The steroid cholesterol is an essential component of eukaryotic Similar to other GPCRs, cholesterol modulates the physio- membranes, and it functionally modulates membrane proteins, logical function of β2AR (26, 28). Furthermore, cholesterol and including G protein-coupled receptors. To reveal insight into how the more water-soluble cholesterol analog cholesteryl hemi- cholesterol modulates G protein-coupled receptors, we have used succinate (CHS) enhance the thermal stability of β2AR (26, 29). dynamic single-molecule force spectroscopy to quantify the me- Additionally, cholesterol facilitates interactions between GPCRs fl chanical strength and exibility, conformational variability, and and seems to be helpful in crystallizing β2AR (9). A recently kinetic and energetic stability of structural segments stabilizing published X-ray crystallography model of human β2AR showed β β the human 2-adrenergic receptor ( 2AR) in the absence and pres- cholesterol to fit into a shallow surface groove formed by ence of the cholesterol analog cholesteryl hemisuccinate (CHS). transmembrane α-helices H1, H2, H3, and H4 (30). This struc- CHS considerably increased the kinetic, energetic, and mechanical tural model unraveled possible interactions between cholesterol fi stability of almost every structural segment at suf cient magni- and β2AR.
    [Show full text]
  • Design, Synthesis and Formulation by Mbuso Faya
    Novel Antimicrobial Peptides for enhanced antimicrobial activity against Methicillin Resistant Staphylococcus aureus: Design, Synthesis and Formulation by Mbuso Faya (MSc Health Science – University of KwaZulu-Natal) Submitted as fulfilment of the requirements for the degree of Doctor of Philosophy in Pharmaceutics at the Discipline of Pharmaceutical Sciences of the School of Health Sciences at the University of KwaZulu-Natal Supervisor: Professor Thirumala Govender (PhD, University of Nottingham, United Kingdom) Date submitted: December 6th 2018 “A candle loses nothing by lighting another candle’’ - James Keller - I “This thesis is dedicated to my family who have consistently pushed beyond boundaries and refused earthly definitions to press on to discover new realities”. ii Declaration 1 – Plagiarism I, Mr. Mbuso Faya, declare that 1. The research data reported in this thesis, except where otherwise indicated is my own original work. 2. This thesis has not been submitted for any degree or examination at any other university. 3. This thesis does not contain data, pictures, graphs, or other information belonging to other people, unless specifically acknowledged as being sourced from other people. 4. This thesis does not contain any other persons’ writing, unless specifically acknowledged as being sources from other researchers. Where other written sources have been quoted, then: a. Their words have been rephrased but the general information attributed to them has been referenced; b. Where their exact words have been used, their writing has been placed inside quotation marks, and referenced. 5. Where I have reproduced a publication of which I am an author, co-author, or editor, I have indicated in detail which part of the publication was written by myself alone and have fully referenced such publications.
    [Show full text]
  • A Model System for Studying Biologically Compatible Collodial Probes for Cancer Research
    A MODEL SYSTEM FOR STUDYING BIOLOGICALLY COMPATIBLE COLLODIAL PROBES FOR CANCER RESEARCH By Samantha M Brandon A thesis submitted to the Johns Hopkins University in conformity with the requirements for the degree of Masters of Science in Chemical & Biomolecular Engineering Baltimore, Maryland June 29, 2015 ABSTRACT A Model System for Studying Biologically Compatible Colloidal Probes for Cancer Research Samantha Mercedes Brandon, B.S., Johns Hopkins University Co-Chairs of Advisory Committee: Dr. Michael A Bevan & Dr. Frechette A key challenge in the development of targeted drug delivery techniques has been to further understand the weak, specific interactions between proteins and carbohydrates. The intermolecular forces between these biomolecules influence critical biological functions such as cell-cell adhesion, motility, signaling, proliferation, and metastasis. In the scope of cancer research, the CD44-Hyaluronic Acid (HA) complex is tightly coupled with many essential cellular functions and has been pointed to as a marker in cancer metastasis. While this complex has been studied thoroughly, there still lacks substantial measurements of the multivalent binding properties. This work attempts to fill that knowledge gap by utilizing lipid bilayers as a model cell system to study CD44-Hyaluronic Acid interactions. In order to develop a controlled model of a cellular system, we utilized lipid bilayers integrated with a polymer cushion. With confocal microscopy, we were able to verify that the lipid bilayers formed planar monolayers on glass slides. Fluorescence Recovery After Photobleaching (FRAP) confirmed that the lipid bilayers maintained fluidity similar to that of in vivo cellular systems. We then determined the colloidal stability of the system by studying the interaction potentials of PEG (Polyethylene Glycol) coated and HA coated particles with the bilayer.
    [Show full text]
  • Lipid Bilayers As Surface Functionalizations for Planar and Nanoparticle Biosensors
    Lipid Bilayers as Surface Functionalizations for Planar and Nanoparticle Biosensors by Shell Y. Ip A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Chemistry University of Toronto © Copyright by Shell Y. Ip 2010 Lipid Bilayers as Surface Functionalizations for Planar and Nanoparticle Biosensors Shell Y. Ip Doctor of Philosophy Department of Chemistry University of Toronto 2010 Abstract Many biological processes, pathogens, and pharmaceuticals act upon, cellular membranes. Accordingly, cell membrane mimics are attractive targets for biosensing, with research, pathology, and pharmacology applications. Lipid bilayers represent a versatile sensor functionalization platform providing antifouling properties, and many receptor integration options, uniquely including transmembrane proteins. Bilayer-coated sensors enable the kinetic characterization of membrane/analyte interactions. Addressed theoretically and experimentally is the self-assembly of model membranes on plasmonic sensors. Two categories of plasmonic sensors are studied in two parts. Part I aims to deposit raft-forming bilayers on planar nanoaperture arrays suitable for multiplexing and device integration. By vesicle fusion, planar bilayers are self-assembled on thiol-acid modified flame-annealed gold without the need for specific lipid head-group requirements. Identification of coexisting lipid phases is accomplished by AFM imaging and force spectroscopy mapping. These methods are successfully extended to metallic, plasmon-active nanohole arrays, nanoslit arrays and annular aperture arrays, with coexisting phases observed among the holes. Vis-NIR transmission spectra of the arrays are measured before and after deposition, indicating bilayer detection. Finally, the extraction of membrane proteins from cell cultures and incorporation into model supported bilayers is demonstrated. These natural membrane proteins potentially act as lipid-bound surface receptors.
    [Show full text]
  • STABILIZED LIPID BILAYER NANODISCS William Michael Penny
    University of Montana ScholarWorks at University of Montana Graduate Student Theses, Dissertations, & Graduate School Professional Papers 2018 DETERMINATION OF LIPID BILAYER AFFINITIES FOR SMALL MOLECULES USING CAPILLARY ELECTROPHORESIS AND COPOLYMER – STABILIZED LIPID BILAYER NANODISCS William Michael Penny Let us know how access to this document benefits ouy . Follow this and additional works at: https://scholarworks.umt.edu/etd Recommended Citation Penny, William Michael, "DETERMINATION OF LIPID BILAYER AFFINITIES FOR SMALL MOLECULES USING CAPILLARY ELECTROPHORESIS AND COPOLYMER – STABILIZED LIPID BILAYER NANODISCS" (2018). Graduate Student Theses, Dissertations, & Professional Papers. 11255. https://scholarworks.umt.edu/etd/11255 This Dissertation is brought to you for free and open access by the Graduate School at ScholarWorks at University of Montana. It has been accepted for inclusion in Graduate Student Theses, Dissertations, & Professional Papers by an authorized administrator of ScholarWorks at University of Montana. For more information, please contact [email protected]. DETERMINATION OF LIPID BILAYER AFFINITIES FOR SMALL MOLECULES USING CAPILLARY ELECTROPHORESIS AND COPOLYMER – STABILIZED LIPID BILAYER NANODISCS By William Michael Penny Bachelor of Science with Honors in Chemistry, Seton Hall University, South Orange, New Jersey, 2012 Dissertation Presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Analytical Chemistry The University of Montana Missoula, MT August 2018 Approved by: Scott Whittenburg, Dean of the Graduate School Graduate School Christopher P. Palmer, Ph.D. Department of Chemistry and Biochemistry Orion B. Berryman, Ph.D. Department of Chemistry and Biochemistry Michael D. DeGrandpre, Ph.D. Department of Chemistry and Biochemistry J.B. Alexander Ross, Ph.D. Department of Chemistry and Biochemistry J.
    [Show full text]
  • Supported Lipid Bilayers with Phosphatidylethanolamine As the Major Component † † † † † ‡ Anne M
    Article Cite This: Langmuir 2017, 33, 13423-13429 pubs.acs.org/Langmuir Supported Lipid Bilayers with Phosphatidylethanolamine as the Major Component † † † † † ‡ Anne M. Sendecki, Matthew F. Poyton, Alexis J. Baxter, Tinglu Yang, and Paul S. Cremer*, , † ‡ Department of Chemistry and Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802, United States *S Supporting Information ABSTRACT: Phosphatidylethanolamine (PE) is notoriously difficult to incorporate into model membrane systems, such as fluid supported lipid bilayers (SLBs), at high concentrations because of its intrinsic negative curvature. Using fluorescence- based techniques, we demonstrate that having fewer sites of unsaturation in the lipid tails leads to high-quality SLBs because these lipids help to minimize the curvature. Moreover, shorter saturated chains can help maintain the membranes in the fluid phase. Using these two guidelines, we find that up to 70 mol % PE can be incorporated into SLBs at room temperature and up to 90 mol % PE can be incorporated at 37 °C. Curiously, conditions under which three-dimensional tubules project outward from the planar surface as well as conditions under which domain formation occurs can be found. We have employed these model membrane systems to explore the ability of Ni2+ to bind to PE. It was found that this transition metal ion binds 1000-fold tighter to PE than to phosphatidylcholine lipids. In the future, this platform could be exploited to monitor the binding of other transition metal ions or the binding of antimicrobial peptides. It could also be employed to explore the physical properties of PE-containing membranes, such as phase domain behavior and intermolecular hydrogen bonding.
    [Show full text]
  • ABSTRACT Ou, Erkang. Effect of Silica Support on Electrostatics at Membrane-Water Interface and Lipid
    ABSTRACT Ou, Erkang. Effect of Silica Support on Electrostatics at Membrane-Water Interface and Lipid- Protein Interface by EPR-Active Molecular pH Probes. (Under the direction of Prof. Tatyana I. Smirnova). Polarity, electrostatic potentials, and hydration are the major physico-chemical characteristics of lipid membranes that govern membrane-protein and protein-protein interactions as well as transport of small molecules through cellular membranes. At the membrane-water interface, the surface electrostatic potential of a lipid bilayer plays a fundamental role in such key processes of cellular functioning as endo- and exocytosis, membrane fusion and cellular division to name a few. At the lipid-protein interface, local dielectric constant of biomembranes determines stability, folding, and aggregation of membrane receptor proteins that are involved in a myriad of cellular functions. Some of the properties of cellular membranes could be mimicked by supported lipid bilayers (SLB) that serve as very useful model membrane platforms with the lipid bilayer providing a biocompatible interface and the solid support allowing for manipulation of lipid bilayer properties in a controllable manner. Such membrane-mimicking systems are considered to be the promising candidates for a number of biomedical and biotechnological applications. At present, little is understood about the influence of nanostructured support and the nanoconfinement on the properties of the membrane at membrane-water interface and protein- lipid interface. This PhD thesis project reports on employing EPR-active pH sensitive probes to assess the surface electrostatics and to profile a heterogeneous dielectric environment along a transmembrane peptide incorporated into both unsupported unilamellar lipid vesicles and the lipid membranes formed on the surface of silica beads.
    [Show full text]