The degradation of myelin in the CNS is the hallmark of multiple sclerosis. Reduction in the net positive charge of myelin basic protein (MBP), through deimination, correlates strongly with disease severity and may mediate myelin instability and loss of compaction. Using Cys scanning, spin labeling, EPR spectroscopy, and site-specific proteolysis, we show that in the membrane-bound state the primary immunodominant epitope, V83-T92, of the less cationic recombinant murine MBP C8 mimic (rmC8) forms a more highly surface-exposed and shorter amphipathic ␣-helix than in the unmodified form, recombinant murine MBP C1 mimic (rmC1), analogous to the most cationic and abundant isomer of MBP in normal myelin. Moreover, cathepsin D digested lipid-associated rmC8 3-fold faster than rmC1, and cleavage at F86 -F87 occurred more readily in rmC8 than rmC1. These findings suggest a mechanism for initial loss of myelin stability and the autoimmune pathogenesis of multiple sclerosis.electron paramagnetic resonance ͉ site-directed spin labeling ͉ lipid bilayer ͉ ␣-helix ͉ cathepsin D T he human inflammatory demyelinating disease multiple sclerosis (MS) is characterized by the active degradation of the myelin sheath in the CNS, resulting in significant neurological deficit (1-4). The resultant lesions contain T cells, B cells, and macrophages that are reactive against myelin antigens. Of the candidate autoantigens, myelin basic protein (MBP) (5) has been widely studied, and its importance to the immunopathogenesis of MS has been extensively reviewed (3, 6, 7). MBP and its peptides are highly encephalitogenic and induce experimental autoimmune encephalomyelitis, used widely as a model for MS, in rodents and primates.The primary role of MBP in the CNS is generally considered to be maintenance of compaction of the myelin sheath, bringing together the apposing faces of the cytoplasmic leaflet of the oligodendrocyte membrane, by virtue of its extreme net positive charge and through synergistic protein-lipid interactions (8-10). Alteration of MBP cationicity may represent a regulatory mechanism for normal myelin assembly or a degradative mechanism in MS. In this regard, MBP shows extensive posttranslational modifications with varying degrees of deimination, phosphorylation, deamidation, methylation, and N-terminal acylation (5, 11). Such modifications give rise to charge variants denoted C1 to C8, of which the C1 component represents the least modified and the most cationic isomer and is the most abundant form of MBP in adult humans (11,12). The least cationic component is C8, with extensive deimination of arginyl residues (13), and it is found in elevated levels in both adults with MS and infants (12), suggesting a role in the early stages of myelinogenesis and in demyelination in MS.The severity of MS correlates strongly with the degree of arginine loss caused by deimination in fulminating Marburg's syndrome: Ͼ90% of the MBP is deiminated, compared with 45% in chronic MS and 20% in normal brain (12, 14). Deiminated MBP is structurally l...