Studying lipid interactions of specific myelin proteins using nanoscaled model membrane-mimics and nanoparticles
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The nervous system is a complex and highly specialized network, where rapid conduction of nerve impulses over large distances is required for correct functioning of vertebrate nervous system. Saltatory conduction of electrical signals from one neuron to another is enabled by the myelin sheath, which is a multi-layered proteolipid membrane with unique myelin proteins. Myelin in rich in lipids and proteins that are not common in normal cell membranes, and the proteins specific to the myelin structure is quite unique and differ between the CNS and PNS. Abnormalities in myelin-specific proteins are involved in neurological diseases, leading to demyelination and chronic disability, such as multiple sclerosis (MS) and Charcot-Marie-Tooth disease (CMT). Nanotechnology can be used to investigate protein-membrane interactions, introducing nano-sized model membranes that mimic the native lipid bilayers found in myelin, and also in approaching new treatments for neurological diseases. In this study, the interactions between three specific myelin proteins and lipid membranes were studied with the used of two model membranes; liposomes and bicelles, and at the same time comparing the model systems to see if one could be preferred over the other in future research. The myelin proteins myelin basic protein (MBP) and peripheral myelin protein 2 (P2) and cytoplasmic tail of myelin protein 0 (P0) were expressed and purified and used in this thesis together with a mutant form of the P2 protein. Turbidimetry and small-angle X-ray diffraction (SAXD) were used to investigate proteolipid aggregate stability and structural effects induced by the myelin proteins. Lipid ratio dependencies were examined with co-sedimentation assay while synchrotron radiation circular dichroism (SCRD) measurements of P2wt with the different model membranes were conducted to explore the structural changes of P2 induced by binding to lipids. Transmission electron microscopy (TEM) was used to visually look at how the different protein induced lipid aggregated. Finally, a pilot study with nanoparticles were conducted to gain knowledge about how they can be functionalized and used in studying protein-membrane interactions, and how they in the future can be used in applications targeting the nervous system. Co-sedimentation assays were carried out to analyze protein binding, while ultraviolet-visible spectrophotometry (UV-vis spectrophotometry) assessed together with TEM to get any confirmation of the lipid coating of the gold nanoparticles. In this thesis, several interactions properties of the three myelin proteins MBP, P2 and P0 were found to differ between the model membranes, and highly ordered structures of bicelle aggregates induced by P2wt and P2 F57A were investigated. Examination of lipid coated gold nanoparticles revealed partial coating and that optimization of the protocols are highly needed.
PublisherThe University of Bergen
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