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Lipid Raft Formation and Peptide-Lipid Interactions in Myelin Model Membranes

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Wilfrid Laurier University Scholars Commons @ Laurier Theses and Dissertations (Comprehensive) 2012 Lipid raft formation and peptide-lipid interactions in myelin model membranes Ashtina R. Appadu Wilfrid Laurier University, [email protected] Follow this and additional works at: https://scholars.wlu.ca/etd Part of the Biochemistry Commons, and the Biophysics Commons Recommended Citation Appadu, Ashtina R., "Lipid raft formation and peptide-lipid interactions in myelin model membranes" (2012). Theses and Dissertations (Comprehensive). 1124. https://scholars.wlu.ca/etd/1124 This Thesis is brought to you for free and open access by Scholars Commons @ Laurier. It has been accepted for inclusion in Theses and Dissertations (Comprehensive) by an authorized administrator of Scholars Commons @ Laurier. For more information, please contact [email protected]. Lipid raft formation and peptide-lipid interactions in myelin model membranes By Ashtina Appadu B.Sc. Biochemistry/Biotechnology, Wilfrid Laurier University, 2009 THESIS Submitted to the Department of Chemistry In partial fulfillment of the requirements for Master of Science in Chemistry Wilfrid Laurier University 2012 Ashtina Appadu ©2012 Abstract Multiple sclerosis (MS), a demyelinating disease affecting 75,000 Canadians and almost 400,000 Americans, is one of the most prevalent diseases in young adults. Unfortunately, there exist no known cures to date and the pathways involved in the progression of the disease remain relatively obscure. The demyelination process triggered by the onset of MS, affects the lipid composition of the myelin membrane and causes a loss in viable myelin which can in turn greatly impact the proper functioning of the central nervous system (CNS). The cholesterol content of myelin fluctuates during MS and consequently this could affect the fluidity as well as the lipid microdomain profile in the membranes. Using model membranes such as a fluid POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) system and the “canonical’ raft system (POPC/sphingomyelin/cholesterol 1:1:1) to represent the two extremes of the fluidity scale, as well as myelin-mimicking membranes (healthy and diseased) which have intermediate lipid fluidities, the lipid domains in each of the systems were probed using fluorescence resonance energy transfer (FRET). FRET analysis indicated that the myelin membrane models contained lipid domains than were overall smaller that those found in the raft system. The addition of melittin, a positively-charged (+6) peptide, to these lipid systems triggered lipid rearrangement to yield larger lipid domains in the myelin systems, whereas in the raft system a change from large domains to smaller ones in the presence of the peptide was observed. Melittin interacted differently with each of the lipid systems where the lipid composition determined the overall conformation of the peptide which, according to circular dichroism (CD) spectroscopy, was shown to be mostly α-helical in membrane environments. It is believed that melittin can exist in a number of different states; a monomer at low concentrations, and as the concentration increases self-association results in either dimers consisting of two parallel α-helices or even a tetramer which would be a dimer of dimers (four α-helices). Tryptophan fluorescence indicated that melittin had a strong affinity for the myelin mimicking models which were negatively-charged and the peptide appeared to be buried relatively deep into the hydrocarbon core of the bilayer, whereas melittin was located nearer the interfacial region in the raft system which was not ii charged. Furthermore, melittin appeared to interact more strongly with the healthy myelin model compared to the diseased model which contained a higher level of cholesterol. This would indicate that melittin’s interaction is strongly dictated by electrostatic interactions as well as the presence of cholesterol which would affect the fluidity and physical properties of the lipid bilayer, thus making it harder for the peptide to penetrate deeply. Since the lipid composition is affected during demyelination and the cholesterol content has been shown to increase, this could suggest that similarly to melittin, other basic proteins found in the CNS could also suffer reduced lipid interactions which would affect their overall structure and consequently their function. The proteolipid protein (PLP) is amongst one of the most abundant proteins found in the myelin membrane. This extremely hydrophobic integral membrane protein consisting of four transmembrane α-helices is known to induced encephalomyelitis in experimental animals and is consequently of great interest in MS research. Among the different loops that connect the helices, the C-terminus located on the cytoplasmic side of the membrane, is of particular interest since it has been hypothesized that it could be involved in guiding and properly positioning PLP in myelin, as well as potentially interacting with other myelin proteins. A PLP C-terminus peptide (residues 258- 277 in the human sequence) was synthesized and its interaction and secondary structure were studied using CD spectroscopy. CD measurements indicated that this loop region adopted different conformations in the different lipid systems, but all in all, it appeared to be mostly randomly coiled and it had a limited amount of interaction with the lipid vesicles. The structure of the peptide also seemed unaffected by the change in lipid composition of the healthy and diseased myelin model systems. This would suggest that the lack of structure of the peptide could allow it to interact with other cytosolic myelin proteins. These studies on the lipid domains in myelin membrane models as well as their interactions with melittin and the PLP C-terminus peptide suggest that within the myelin sheath protein structure and protein-lipid interactions are affected during MS. iii Acknowledgements I would like to express my gratitude and thanks to my wonderful supervisor Dr Lillian DeBruin, for being an amazing mentor and giving me the opportunity to work on this project. Without her guidance and continuous support, this project would have been impossible to complete. I would also like to thank Dr Masoud Jelokhani for his advice and help in understanding and analyzing my data and for allowing me to use the equipment in his lab. I would also like thank my good friend Patrick Hoang for all the help he has provided me in the lab as well as my lab mates Baindu Kosia, Matthew Nichols, Vatsal Patel and Miljan Kuljanin for their company and help during the long hours spent in the lab. Finally I would also like to thank my family, specially my mother, who have always supported me in my studies and encouraged me do better. iv Declaration of work performed All data presented and described in this thesis are the results of my own work. Furthermore, I hereby declare that I am the sole author of this thesis that includes all final revisions, as accepted by my examiners. v TABLE OF CONTENTS ABSTRACT ii ACKNOWLEDGEMENTS iv DECLARATION OF WORK PERFORMED v TABLE OF CONTENTS vi LISTS OF FIGURES x LISTS OF TABLES xii LIST OF ABBREVIATIONS xiii Chapter 1- The myelin membrane and lipid rafts 1 1.1 A brief introduction to the myelin membrane 2 1.1.1 Structure, function and morphology 2 1.1.2 Lipid and protein composition of the myelin membrane 3 1.2 Demyelinating diseases – multiple sclerosis (MS) 5 1.3 Lipid Rafts 7 1.3.1 Lipid phases in the cell membrane 7 1.3.2 Membrane rafts: properties and function 8 1.3.3 Biosynthesis of raft clusters 12 1.3.4 Techniques used to study microdomains 13 1.4 Lipid rafts and the myelin membrane 14 1.5 Purpose of study 15 Chapter 2- Biophysical techniques for the study of peptide-lipid interactions and lipid domain formation 18 2.1 Model membranes 19 2.1.1 Size-dependent vesicle preparation 20 2.1.2 Characterization of myelin domains using model membrane systems 21 vi 2.2. Protein lipid interactions and consequential secondary conformational changes 23 2.2.1 Melittin as a model peptide 24 2.2.2 Myelin proteolipid protein 28 2.3 FRET as a method to monitor changes in microdomain formation 31 2.4 Tryptophan fluorescence and melittin 35 2.5 Circular dichroism (CD) spectroscopy for the determination of peptide and protein secondary structure 36 2.6 Isothermal titration calorimetry (ITC) for the determination of binding affinities 37 Chapter 3 – Materials and Methods 40 3.1 Materials 41 3.2 Methods 42 3.2.1 Vesicle preparation 42 3.2.2 FRET measurements 43 3.2.3 Tryptophan fluorescence measurement 45 3.2.4 CD spectroscopy 45 3.2.5 Isothermal titration calorimetry (ITC) measurements 46 Chapter 4-characterization of lipid microdomains using fluorescence resonance energy transfer (FRET) 4.1 Fluorescence resonance energy transfer (FRET) for the study of lipid rafts 49 4.2 FRET characterization of model membrane systems 50 4.2.1 Fluorescence of donor probe in model membrane systems 50 4.2.2 Energy transfer of in model membranes 55 4.3 FRET analysis and characterization of lipid domains in model membranes 59 4.4 The impact of cholesterol levels on membrane domains 66 vii Chapter 5- Peptide–lipid interactions of melittin and the myelin specific PLP peptide 68 5.1 The effects of lipid composition on the secondary structure of melittin 69 5.1.1 Concentration-dependent structure of melittin in buffer 70 5.1.2 Concentration-dependent structure of melittin in POPC 72 5.1.3 Concentration-dependent structure of melittin in the raft “canonical mixture” membrane
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