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Myelin basic protein is a candidate autoantigen in multiple sclerosis. One of its dominant antigenic epitopes is segment Pro 85 to Pro 96 (human sequence numbering, corresponding to Pro 82 to Pro 93 in the mouse). There have been several, contradictory predictions of secondary structure in this region; either ␤-sheet, ␣-helix, random coil, or combinations thereof have all been proposed. In this paper, molecular dynamics and site-directed spin labeling in aqueous solution indicate that this segment forms a transient ␣-helix, which is stabilized in 30% trifluoroethanol. When bound to a myelin-like membrane surface, this antigenic segment exhibits a depth profile that is characteristic of an amphipathic ␣-helix, penetrating up to 12 Å into the bilayer. The ␣-helix is tilted ϳ9°, and the central lysine is in an ideal snorkeling position for side-chain interaction with the negatively charged phospholipid head groups.Multiple sclerosis (MS) 1 is thought to be an autoimmune disease characterized by chronic inflammatory response against myelin in the central nervous system. There is significant evidence that myelin basic protein (MBP) is a candidate antigen for T-cells and autoantibodies in MS (1). The 18.5-kDa isoform of MBP maintains the compaction of the myelin sheath in the central nervous system by anchoring the cytoplasmic face of the two apposing bilayers (2, 3). The mechanism and sites that are important for membrane adhesion are not known.The level of anti-MBP antibodies is increased in the cerebrospinal fluid of patients with active MS (4), as well as in 96% of patients with relapsing and chronic MS (5). An MBP region between Pro 85 and Pro 96 (human sequence numbering) was identified to be a minimal B cell epitope and a T cell epitope for HLA DR2b (DRB1*1501)-restricted T cells that recognize the protein (1, 6). This epitope overlaps with the DR2a-restricted epitope for T-cells reactive to MBP residues 87-106 (7). Experimental treatments for MS based on peptide mimetics of MBP have focused on this region of the protein (8).In solution, MBP is "intrinsically unstructured" (or "natively unfolded") (9). Upon binding to detergents or lipids, the levels of ␤-sheet and especially ␣-helical structure increase dramatically (10, 11). Presently the tertiary structure of MBP is unknown, with the most detailed predictions coming from Martenson (12) and Stoner (13) in the mid-1980s. In these models, as well as in a newer model based upon further research on an electron microscopy single-particle reconstruction, residues 86 -92 are found in a ␤-sheet (14, 15). Using [ 1 H]NMR and circular dichroic spectropolarimetry of various MBP peptides with detergent micelles, Mendz et al. (16) suggested that there were discrete interaction sites in the protein, one of which could be a helix between residues 87 and 97. Based on the arrangement of hydrophobic and hydrophilic residues, Warren et al. (6) predicted that this epitope of MBP was an amphipathic ␣-helix located at the interface between the oligodendrocyte cytoplasm and the me...

Myelin basic protein (MBP) maintains the compaction of the myelin sheath in the central nervous system by anchoring the cytoplasmic face of the two apposing bilayers and may also play a role in signal transduction. Site-directed spin labeling was done at eight matching sites in each of two recombinant murine MBPs, qC1 (charge ؉19) and qC8 charge (؉13), which, respectively, emulate the native form of the protein (C1) and a posttranslationally modified form (C8) that is increased in multiple sclerosis. When interacting with large unilamellar vesicles, most spin-labeled sites in qC8 were more mobile than those in qC1. Depth measurement via continuous wave power saturation indicated that the Nterminal and C-terminal sites in qC1 were located below the plane of the phospholipid headgroups. In qC8, the C-terminal domain dissociated from the membrane, suggesting a means by which the exposure of natural C8 to cytosolic enzymes and ligands might increase in vivo in multiple sclerosis. The importance of two Phe-Phe pairs in MBP to its interactions with lipids was investigated by separately mutating each pair to Ala-Ala. The mobility at F42A/F43A and especially F86A/F87A increased significantly. Depth measurements and helical wheel analysis indicated that the Phe-86/Phe-87 region could form a surface-seeking amphipathic ␣-helix.The 18.5-kDa isoform of myelin basic protein (MBP) 1 is a stabilizing factor in the myelin sheath. A major function of MBP is to bind to the apposing cytoplasmic faces of the myelin membrane and maintain compaction for efficient nerve transmission (1, 2), but it may also be involved in signal transduction (3). Because of a diversity of post-translational modifications, MBP exists as a number of charge isomers denoted C1-C8 with a net positive charge decreasing from ϩ19 to ϩ13 at pH 7.0 (4 -6). The C8 component is characterized by the enzymatic deimination of arginine to citrulline. Each conversion results in the loss of one positive charge, and C8 is thus the least basic form of the protein and has a diminished ability to cause adhesion of lipid bilayers (7-9). Component C8 occurs in greater amounts in patients with the demyelinating disease, multiple sclerosis (9, 10).We have previously produced and characterized a recombinant murine 18.5-kDa MBP (11). Here, we will denote this protein quasi-C1 (qC1), because it is unmodified post-translationally (with the exception of an LEH 6 tag) and emulates the least-modified, most basic charge isomer C1. We have also generated by site-directed mutagenesis a quasi-deiminated form of recombinant murine 18.5-kDa MBP that we call qC8, since it was designed to mimic the less cationic natural form C8. The recombinant qC8 consists of Arg/Lys 3 Gln substitutions at the same deimination sites in human MBP that predominate in chronic multiple sclerosis and has properties similar to those of natural C8 (7). The net charge of qC1 is ϩ19 at neutral pH, whereas that of qC8 is ϩ13 as for their natural counterparts.In this study, we investigated the electrostatic and hydro...

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Myelin basic protein (MBP) binds to negatively charged lipids on the cytosolic surface of oligodendrocyte membranes and is most likely responsible for adhesion of these surfaces in the multilayered myelin sheath. It can also polymerize actin, bundle F-actin filaments, and bind actin filaments to lipid bilayers through electrostatic interactions. MBP consists of a number of posttranslationally modified isoforms of varying charge, including C8, in which six arginines are deiminated to the uncharged residue citrulline. The deiminated form decreases with development, but is increased in patients with the demyelinating disease multiple sclerosis. Here we investigate the effect of decreased net positive charge of MBP on its interaction with actin in vitro by comparing a recombinant murine form, rmC1, of the most highly charged unmodified isoform, C1, and a recombinant analogue of C8 in which six basic residues are converted to glutamine, rmC8. The dissociation constant of the less charged isoform rmC8 for actin was a little greater than that of rmC1, and rmC8 had somewhat reduced ability to polymerize actin and bundle F-actin filaments than rmC1. Moreover, rmC8 was more readily dissociated from actin by Ca(2+)-calmodulin than rmC1, and the ability of the deiminated isoform to bind actin to lipid bilayers was reduced. These results indicate that electrostatic forces are the primary determinant of the interaction of MBP with actin. The spin labeled side chains of a series of rmC1 and rmC8 variants containing single Cys substitutions at seven sites throughout the sequence all became motionally restricted to a similar degree on binding F-actin, indicating that the entire sequence is involved in interacting with actin filaments or is otherwise structurally constrained in actin bundles. Thus, this posttranslational modification of MBP, which occurs early in life and is increased in multiple sclerosis, attenuates the ability of MBP to polymerize and bundle actin, and to bind it to a negatively charged membrane.

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The effects of deimination (conversion of arginyl to citrullinyl residues) of myelin basic protein (MBP) on its binding to calmodulin (CaM) have been examined. Four species of MBP were investigated: unmodified recombinant murine MBP (rmMBP-Cit 0 ), an engineered protein with six quasi-citrullinyl (i.e., glutaminyl) residues per molecule (rmMBP-qCit 6 ), human component C1 (hMBP-Cit 0 ), and human component C8 (hMBP-Cit 6 ), both obtained from a patient with multiple sclerosis (MS). Both rmMBP-Cit 0 and hMBP-Cit 0 bound CaM in a Ca 2+-dependent manner and primarily in a 1:1 stoichiometry, which was verified by dynamic light scattering. Circular dichroic spectroscopy was unable to detect any changes in secondary structure in MBP upon CaM-binding. Inherent Trp fluorescence spectroscopy and a single-site binding model were used to determine the dissociation constants: K d ‫ס‬ 144 ± 76 nM for rmMBP-Cit 0 , and K d ‫ס‬ 42 ± 15 nM for hMBP-Cit 0 . For rmMBP-qCit 6 and hMBP-Cit 6 , the changes in fluorescence were suggestive of a two-site interaction, although the dissociation constants could not be accurately determined. These results can be explained by a local conformational change induced in MBP by deimination, exposing a second binding site with a weaker association with CaM, or by the existence of several conformers of deiminated MBP. Titration with the collisional quencher acrylamide, and steady-state and lifetime measurements of the fluorescence at 340 nm, showed both dynamic and static components to the quenching, and differences between the unmodified and deiminated proteins that were also consistent with a local conformational change due to deimination. Keywords:Myelin basic protein; calmodulin; multiple sclerosis; deimination; citrulline; intrinsic Trp fluorescence; fluorescence lifetime; dynamic light scattering; circular dichroism Myelin basic protein (MBP) is one of the most abundant proteins of the myelin sheath of the central nervous system (CNS), and its primary role is generally considered to be maintenance of the stability of the sheath by holding together the apposing cytoplasmic leaflets of the oligodendrocyte membrane (Smith 1992;Moscarello 1997). However, the MBP family comprises numerous developmentally regulated isoforms, of which the 18.5-kD species is the most abundant in adult human myelin, and has been the most studied (Givogri et al. 2000(Givogri et al. , 2001. This isoform itself undergoes a complex series of posttranslational modifications, giving rise to charge isomers designated as components C1 to C8 (Moscarello 1997;Wood and Moscarello 1997;Zand et al. 1998). In addition to its associations with lipids, MBP Reprint requests to: George Harauz, Department of Molecular Biology and Genetics, University of Guelph, 50 Stone Road East, Guelph, Ontario N1G 2W1, Canada; e-mail: gharauz@uoguelph.ca; fax: (519) 837-2075. 4 These authors contributed equally to this work. Article and publication are at http://www.proteinscience.org/cgi

The cystic fibrosis transmembrane conductance regulator (CFTR) channel interacts with scaffolding and other proteins that are expected to restrict its lateral movement, yet previous studies have reported predominantly free diffusion. We examined the lateral mobility of CFTR channels on live baby hamster kidney cells using three complementary methods. Channels bearing an extracellular biotinylation target sequence were labeled with streptavidin conjugated with fluorescent dyes (Alexa Fluor 488 or 568) or quantum dots (qDot605). Fluorescence recovery after photobleaching and image correlation spectroscopy of the dye-labeled channels revealed a significant immobile population ( approximately 50%), which was confirmed by direct single particle tracking (SPT) of qDot605-labeled CFTR. Adding 10 histidine residues at the C-terminus of CFTR to mask the postsynaptic density 95, Discs large, ZO-1 (PDZ) binding motif abolished its association with EBP50/NHERF1, reduced the immobile fraction, and increased mobility. Other interactions that are not normally detected on this timescale became apparent when binding of PDZ domain proteins was disrupted. SPT revealed that CFTR(His-10) channels diffuse randomly, become immobilized for periods lasting up to 1 min, and in some instances are recaptured at the same location. The impact of transient confinement on the measured diffusion using the three fluorescence techniques were assessed using computer simulations of the biological experiments. Finally, the impact of endosomal CFTR on mobility measurements was assessed by fluorescence correlation spectroscopy. These results reveal unexpected features of CFTR dynamics which may influence its ion channel activity.