Studying lipid interactions of specific myelin proteins using nanoscaled model membrane-mimics and nanoparticles

Postponed access: the file will be accessible after 2020-11-20The 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.Masteroppgave i nanovitenskapMAMN-NANONANO39

Structural characterization of two nanobodies targeting the ligand-binding pocket of human Arc

The activity-regulated cytoskeleton-associated protein (Arc) is a complex regulator of synaptic plasticity in glutamatergic neurons. Understanding its molecular function is key to elucidate the neurobiology of memory and learning, stress regulation, and multiple neurological and psychiatric diseases. The recent development of anti-Arc nanobodies has promoted the characterization of the molecular structure and function of Arc. This study aimed to validate two anti-Arc nanobodies, E5 and H11, as selective modulators of the human Arc N-lobe (Arc-NL), a domain that mediates several molecular functions of Arc through its peptide ligand binding site. The structural characteristics of recombinant Arc-NL-nanobody complexes were solved at atomic resolution using X-ray crystallography. Both anti-Arc nanobodies bind specifically to the multi-peptide binding site of Arc-NL. Isothermal titration calorimetry showed that the Arc-NL-nanobody interactions occur at nanomolar affinity, and that the nanobodies can displace a TARPγ2-derived peptide from the binding site. Thus, both anti-Arc-NL nanobodies could be used as competitive inhibitors of endogenous Arc ligands. Differences in the CDR3 loops between the two nanobodies indicate that the spectrum of short linear motifs recognized by the Arc-NL should be expanded. We provide a robust biochemical background to support the use of anti-Arc nanobodies in attempts to target Arc-dependent synaptic plasticity. Function-blocking anti-Arc nanobodies could eventually help unravel the complex neurobiology of synaptic plasticity and allow to develop diagnostic and treatment tools.publishedVersio

Studying lipid interactions of specific myelin proteins using nanoscaled model membrane-mimics and nanoparticles

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

Cryo-EM, X-ray diffraction, and atomistic simulations reveal determinants for the formation of a supramolecular myelin-like proteolipid lattice

Myelin protein P2 is a peripheral membrane protein of the fatty acid–binding protein family that functions in the formation and maintenance of the peripheral nerve myelin sheath. Several P2 gene mutations cause human Charcot-Marie-Tooth neuropathy, but the mature myelin sheath assembly mechanism is unclear. Here, cryo-EM of myelin-like proteolipid multilayers revealed an ordered three-dimensional (3D) lattice of P2 molecules between stacked lipid bilayers, visualizing supramolecular assembly at the myelin major dense line. The data disclosed that a single P2 layer is inserted between two bilayers in a tight intermembrane space of ∼3 nm, implying direct interactions between P2 and two membrane surfaces. X-ray diffraction from P2-stacked bicelle multilayers revealed lateral protein organization, and surface mutagenesis of P2 coupled with structure-function experiments revealed a role for both the portal region of P2 and its opposite face in membrane interactions. Atomistic molecular dynamics simulations of P2 on model membrane surfaces suggested that Arg-88 is critical for P2-membrane interactions, in addition to the helical lid domain. Negatively charged lipid headgroups stably anchored P2 on the myelin-like bilayer surface. Membrane binding may be accompanied by opening of the P2 β-barrel structure and ligand exchange with the apposing bilayer. Our results provide an unprecedented view into an ordered, multilayered biomolecular membrane system induced by the presence of a peripheral membrane protein from human myelin. This is an important step toward deciphering the 3D assembly of a mature myelin sheath at the molecular level

Neuropathy-related mutations alter the membrane binding properties of the human myelin protein P0 cytoplasmic tail

Schwann cells myelinate selected axons in the peripheral nervous system (PNS) and contribute to fast saltatory conduction via the formation of compact myelin, in which water is excluded from between tightly adhered lipid bilayers. Peripheral neuropathies, such as Charcot-Marie-Tooth disease (CMT) and Dejerine-Sottas syndrome (DSS), are incurable demyelinating conditions that result in pain, decrease in muscle mass, and functional impairment. Many Schwann cell proteins, which are directly involved in the stability of compact myelin or its development, are subject to mutations linked to these neuropathies. The most abundant PNS myelin protein is protein zero (P0); point mutations in this transmembrane protein cause CMT subtype 1B and DSS. P0 tethers apposing lipid bilayers together through its extracellular immunoglobulin-like domain. Additionally, P0 contains a cytoplasmic tail (P0ct), which is membrane-associated and contributes to the physical properties of the lipid membrane. Six CMT- and DSS-associated missense mutations have been reported in P0ct. We generated recombinant disease mutant variants of P0ct and characterized them using biophysical methods. Compared to wild-type P0ct, some mutants have negligible differences in function and folding, while others highlight functionally important amino acids within P0ct. For example, the D224Y variant of P0ct induced tight membrane multilayer stacking. Our results show a putative molecular basis for the hypermyelinating phenotype observed in patients with this particular mutation and provide overall information on the effects of disease-linked mutations in a flexible, membrane-binding protein segment. Using neutron reflectometry, we additionally show that P0ct embeds deep into a lipid bilayer, explaining the observed effects of P0ct on the physical properties of the membrane