Characterizing the Effect of Substitutions Mimicking Deimination and Phosphorylation of 18.5-kDa Myelin Basic Protein (MBP) on its Structure and Dynamics in Myelin-Like Membranes

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Multiple sclerosis (MS) is a severely debilitating neurological disease of unknown origin and high clinical variability. The disease hallmark is central nervous system demyelination, i.e. loss of the protective, lipid-rich myelin sheath that enwraps nerve axons and enables them to transmit impulses efficiently leading to formation of plaques, neurological debilitation, and progressive degeneration. Neither the initial trigger for the disease is known, nor is there any cure. Myelin structure and homeostasis are maintained by the myelin basic protein (MBP) family, of which the 18.5-kDa isoform predominates in adult brain. This protein has been shown to be intrinsically disordered and multifunctional, it adheres the cytoplasmic leaflets of oligodendrocytes, associates with a large number of other proteins, undergoes partial induced folding in all interactions (including molecular recognition fragments and switches), and its targeting and associations are modulated by post-translational modifications (PTMs). In MS, PTMs reduce the net positive charge of a large fraction of MBP (from +19 to +13) and contribute to physical demyelination and induction of autoimmunity. The 18.5-kDa MBP isoform (the family-prototype) has preferred tertiary contacts and self-assembles in myelin, allowing it to be a versatile networking protein. This proposal aims at characterizing and understanding PTM-induced changes in these interactions that directly alter MBP structure and self-assembly in myelin. We use an approach based on Electron Paramagnetic Resonance (EPR) and Infrared Reflection-Absorption (IRRA) spectroscopies, combined where necessary with other approaches. The goal is to form a detailed model of the conformational ensemble of MBP and its distribution within myelin, to understand how site-specific modifications, in this case charge reductions mimicking deimination and phosphorylation in the protein, regulate its associations with membranes, its tertiary structure ensemble and dynamics in myelin-like membranes. To this end, 8 recombinant variants of 18.5-kDa MBP (168 residues in mouse), representing different functional forms, will be expressed and studied using the combined EPR/IRRA spectroscopic approaches (for EPR after double spin-labeling with nitroxides). After reconstitution of MBP with membranes, mainly the MBP tertiary will be studied by a combination of diverse EPR approaches and is complemented by the IRRAS method that allows studying orientation and secondary structure changes at the lipid/air interface. The overall goal is to directly correlate the PTM-mimicking changes, the lipid membrane composition and the availability of divalent metal ions with the different myelin-compacting capabilities of the different MBP isoforms. We hope to shed light on the molecular bases on the early steps of demyelination in MS and to aid the search for new approaches to therapy.