Lateral self-assembly of 18.5-kDa myelin basic protein (MBP) charge component-C1 on membranes

Myelin basic protein (MBP), particularly the classic 18.5-kDa isoform, is a major structural protein of the myelin sheath of the central nervous system. It is an intrinsically disordered, peripheral membrane protein that shows structural polymorphism in combination with several overlapping interaction sites. Here, double electron-electron resonance (DEER) spectroscopy, in combination with a simplified, semi-quantitative analysis based on Monte Carlo simulations, is used to determine the distance distribution of murine 18.5-kDa MBP, unmodified charge component-C1, on large unilamellar vesicles of a lipid composition mimicking the cytoplasmic leaflet of myelin. Three singly spin-labeled MBP variants and a mixture of singly-labeled MBP variants are used. The MBPs, each bearing only one spin label, exhibit average intermolecular distances that are significantly shorter than the distances expected when assuming a random distribution at the employed lipid-to-protein ratios, indicating self-assembly on the membrane. The distribution of elliptical pervaded areas (hard ellipses) on a two-dimensional surface can serve as a model of the nonspecific self-assembly process. The corresponding pair correlation functions g(r) are determined from Monte Carlo simulations with variation of various parameters such as the ellipses' aspect ratios. Comparing the g(r) values with the DEER-derived distance distributions, the pervaded volume is best characterized by a nearly elliptical projection onto the membrane, with an aspect ratio of approximately 1.5, and with the longer semi-axis of approximately 1.4nm. The approach of using local information from DEER with low-resolution models derived from Monte Carlo simulations can be applied to study the lateral self-assembly properties of other protein complexes on membranes.     

The effects of deimination of myelin basic protein on structures formed by its interaction with phosphoinositide-containing lipid monolayers

The recombinant 18.5-kDa charge isoform of murine myelin basic protein (rmMBP) is unmodified posttranslationally and was used to study the effects of deimination, i.e., the conversion of arginyl to citrullinyl residues, on the protein's interactions with itself and with lipids. The unmodified species rmMBP-Cit(0) (i.e., containing no citrullinyl residues) interacted with binary monolayers containing acidic (phosphatidylinositol) and nickel-chelating lipids to form paracrystalline arrays with 4.8-nm spacing. A sample of protein was deiminated to an average of 9 citrullinyl residues per molecule of protein, yielding rmMBP-Cit(9). Under both low- and high-salt conditions, this species formed better-ordered domains than rmMBP-Cit(0), viz., planar crystalline assemblies. Thus, deimination of MBP resulted in a significant alteration of its lipid-organizing and self-interaction properties that might be operative in myelin in vivo, especially in progression of the autoimmune disease multiple sclerosis. Comparisons of amino acid sequences indicated significant similarities of MBP with filaggrin, a protein that is deiminated in another autoimmune disease, rheumatoid arthritis, suggesting that comparable epitopes could be targeted in both pathologies. In contrast, binary lipid monolayers consisting of phosphatidylinositol-4-phosphate (or phosphatidylinositol-4,5-bisphosphate) and a nickel-chelating lipid formed helical tubular vesicular structures, which appeared to be induced and/or stabilized by rmMBP, especially in its deiminated form. Sequence comparisons with other actin- and phosphoinositide-binding proteins (vinculin, ActA, MARCKS) suggested that the carboxyl-terminal segment of MBP could form an amphipathic alpha helix and was the phosphoinositide binding site.     

Increased Phosphorylation of Myelin Basic Protein During Hippocampal Long-Term Potentiation

Abstract: Hippocampal long-term potentiation (LTP) is a long-lasting and rapidly induced increase in synaptic strength. Previous experiments have determined that persistent activation of protein kinase C (PKC) contributes to the early maintenance phase of LTP (E-LTP). Using the back-phosphorylation method, we observed an increase in the phosphorylation of a 21-kDa PKC substrate, termed p21, 45 min after LTP was induced in the CA1 region of the hippocampus. p21 was found to have the same apparent molecular weight as the 18.5-kDa isoform of myelin basic protein (MBP) and was recognized by an antibody to MBP in western blotting and immunoprecipitation. Furthermore, p21 from control and potentiated hippocampal slices and purified MBP have identical phosphopeptide maps when back-phosphorylated and then digested with either endoproteinase Lys-C or endoproteinase Asp-N, suggesting that p21 and MBP are identical proteins. As there was no observed change in the amount of MBP in LTP, the increase in MBP phosphorylation during LTP cannot be explained by a change in the amount of protein. From these experiments, we conclude that the phosphorylation of the 18.5-kDa isoform of MBP is increased during E-LTP.     

Adhesion of acidic lipid vesicles by 21.5 kDa (recombinant) and 18.5 kDa isoforms of myelin basic protein

Myelin basic protein (MBP) is thought to be responsible for adhesion of the intracellular surfaces of compact myelin to give the major dense line. The 17 and 21.5 kDa isoforms containing exon II have been reported by others to localize to the cytoplasm and nucleus of murine oligodendrocytes and HeLa cells while the 14 and 18.5 kDa isoforms lacking exon II are confined to the plasma membrane. However, we show that the exon II(-) 18.5 kDa form and a recombinant exon II(+) 21.5 kDa isoform both caused similar aggregation of acidic lipid vesicles, indicating that they should have similar abilities to bind to the intracellular lipid surface of the plasma membrane and to cause adhesion of those surfaces to each other. The circular dichroism spectra of the two isoforms indicated that both had a similar secondary structure. Thus, both isoforms should be able to bind to and cause adhesion of the cytosolic surfaces of compact myelin. The fact that they do not could be due to differences in post-translational modification in vivo, trafficking through the cell and/or subcellular location of synthesis, but it is not due to differences in their lipid binding.     

Classic 18.5- and 21.5-kDa Myelin Basic Protein Isoforms Associate with Cytoskeletal and SH3-Domain Proteins in the Immortalized N19-Oligodendroglial Cell Line Stimulated by Phorbol Ester and IGF-1

The 18.5-kDa classic myelin basic protein (MBP) is an intrinsically disordered protein arising from the Golli (Genes of Oligodendrocyte Lineage) gene complex and is responsible for compaction of the myelin sheath in the central nervous system. This MBP splice isoform also has a plethora of post-translational modifications including phosphorylation, deimination, methylation, and deamidation, that reduce its overall net charge and alter its protein and lipid associations within oligodendrocytes (OLGs). It was originally thought that MBP was simply a structural component of myelin; however, additional investigations have demonstrated that MBP is multi-functional, having numerous protein-protein interactions with Ca²⁺-calmodulin, actin, tubulin, and proteins with SH3-domains, and it can tether these proteins to a lipid membrane in vitro. Here, we have examined cytoskeletal interactions of classic 18.5-kDa MBP, in vivo, using early developmental N19-OLGs transfected with fluorescently-tagged MBP, actin, tubulin, and zonula occludens 1 (ZO-1). We show that MBP redistributes to distinct ‘membrane-ruffled’ regions of the plasma membrane where it co-localizes with actin and tubulin, and with the SH3-domain-containing proteins cortactin and ZO-1, when stimulated with PMA, a potent activator of the protein kinase C pathway. Moreover, using phospho-specific antibody staining, we show an increase in phosphorylated Thr98 MBP (human sequence numbering) in membrane-ruffled OLGs. Previously, Thr98 phosphorylation of MBP has been shown to affect its conformation, interactions with other proteins, and tethering of other proteins to the membrane in vitro. Here, MBP and actin were also co-localized in new focal adhesion contacts induced by IGF-1 stimulation in cells grown on laminin-2. This study supports a role for classic MBP isoforms in cytoskeletal and other protein-protein interactions during membrane and cytoskeletal remodeling in OLGs.     

The Lateral Membrane Organization and Dynamics of Myelin Proteins PLP and MBP Are Dictated by Distinct Galactolipids and the Extracellular Matrix

In the central nervous system, lipid-protein interactions are pivotal for myelin maintenance, as these interactions regulate protein transport to the myelin membrane as well as the molecular organization within the sheath. To improve our understanding of the fundamental properties of myelin, we focused here on the lateral membrane organization and dynamics of peripheral membrane protein 18.5-kDa myelin basic protein (MBP) and transmembrane protein proteolipid protein (PLP) as a function of the typical myelin lipids galactosylceramide (GalC), and sulfatide, and exogenous factors such as the extracellular matrix proteins laminin-2 and fibronectin, employing an oligodendrocyte cell line, selectively expressing the desired galactolipids. The dynamics of MBP were monitored by z-scan point fluorescence correlation spectroscopy (FCS) and raster image correlation spectroscopy (RICS), while PLP dynamics in living cells were investigated by circular scanning FCS. The data revealed that on an inert substrate the diffusion rate of 18.5-kDa MBP increased in GalC-expressing cells, while the diffusion coefficient of PLP was decreased in sulfatide-containing cells. Similarly, when cells were grown on myelination-promoting laminin-2, the lateral diffusion coefficient of PLP was decreased in sulfatide-containing cells. In contrast, PLP''s diffusion rate increased substantially when these cells were grown on myelination-inhibiting fibronectin. Additional biochemical analyses revealed that the observed differences in lateral diffusion coefficients of both proteins can be explained by differences in their biophysical, i.e., galactolipid environment, specifically with regard to their association with lipid rafts. Given the persistence of pathological fibronectin aggregates in multiple sclerosis lesions, this fundamental insight into the nature and dynamics of lipid-protein interactions will be instrumental in developing myelin regenerative strategies.     

Conformational choreography of a molecular switch region in myelin basic protein--molecular dynamics shows induced folding and secondary structure type conversion upon threonyl phosphorylation in both aqueous and membrane-associated environments

The 18.5 kDa isoform of myelin basic protein is essential to maintaining the close apposition of myelin membranes in central nervous system myelin, but its intrinsic disorder (conformational dependence on environment), a variety of post-translational modifications, and a diversity of protein ligands (e.g., actin and tubulin) all indicate it to be multifunctional. We have performed molecular dynamics simulations of a conserved central segment of 18.5 kDa myelin basic protein (residues Glu80-Gly103, murine sequence numbering) in aqueous and membrane-associated environments to ascertain the stability of constituent secondary structure elements (α-helix from Glu80-Val91 and extended poly-proline type II from Thr92-Gly103) and the effects of phosphorylation of residues Thr92 and Thr95, individually and together. In aqueous solution, all four forms of the peptide bent in the middle to form a hydrophobic cluster. The phosphorylated variants were stabilized further by electrostatic interactions and formation of β-structures, in agreement with previous spectroscopic data. In simulations performed with the peptide in association with a dimyristoylphosphatidylcholine bilayer, the amphipathic α-helical segment remained stable and membrane-associated, although the degree of penetration was less in the phosphorylated variants, and the tilt of the α-helix with respect to the plane of the membrane also changed significantly with the modifications. The extended segment adjacent to this α-helix represents a putative SH3-ligand and remained exposed to the cytoplasm (and thus accessible to binding partners). The results of these simulations demonstrate how this segment of the protein can act as a molecular switch: an amphipathic α-helical segment of the protein is membrane-associated and presents a subsequent proline-rich segment to the cytoplasm for interaction with other proteins. Phosphorylation of threonyl residues alters the degree of membrane penetration of the α-helix and the accessibility of the proline-rich ligand and can stabilize a β-bend. A bend in this region of 18.5 kDa myelin basic protein suggests that the N- and C-termini of the proteins can interact with different leaflets of the myelin membrane and explain how a single protein can bring them close together.     

Structured functional domains of myelin basic protein: cross talk between actin polymerization and Ca(2+)-dependent calmodulin interaction

The 18.5-kDa myelin basic protein (MBP), the most abundant isoform in human adult myelin, is a multifunctional, intrinsically disordered protein that maintains compact assembly of the sheath. Solution NMR spectroscopy and a hydrophobic moment analysis of MBP's amino-acid sequence have previously revealed three regions with high propensity to form strongly amphipathic α-helices. These regions, located in the central, N- and C-terminal parts of the protein, have been shown to play a role in the interactions of MBP with cytoskeletal proteins, Src homology 3-domain-containing proteins, Ca(2+)-activated calmodulin (Ca(2+)-CaM), and myelin-mimetic membrane bilayers. Here, we have further characterized the structure-function relationship of these three domains. We constructed three recombinant peptides derived from the 18.5-kDa murine MBP: (A22-K56), (S72-S107), and (S133-S159) (which are denoted α1, α2, and α3, respectively). We used a variety of biophysical methods (circular dichroism spectroscopy, isothermal titration calorimetry, transmission electron microscopy, fluorimetry, and solution NMR spectroscopy and chemical shift index analysis) to characterize the interactions of these peptides with actin and Ca(2+)-CaM. Our results show that all three peptides can adopt α-helical structure inherently even in aqueous solution. Both α1- and α3-peptides showed strong binding with Ca(2+)-CaM, and both adopted an α-helical conformation upon interaction, but the binding of the α3-peptide appeared to be more dynamic. Only the α1-peptide exhibited actin polymerization and bundling activity, and the addition of Ca(2+)-CaM resulted in depolymerization of actin that had been polymerized by α1. The results of this study proved that there is an N-terminal binding domain in MBP for Ca(2+)-CaM (in addition to the primary site located in the C-terminus), and that it is sufficient for CaM-induced actin depolymerization. These three domains of MBP represent molecular recognition fragments with multiple roles in both membrane- and protein-association.     

A New Form of Myelin Basic Protein Found in Human Brain

Human myelin basic protein was subjected to ion-exchange chromatography at high pH to separate the differently charged components. Polyacrylamide gel electrophoretic patterns of the fractions showed that the less basic fractions 3, 4, and 5 contained significant amounts of a protein somewhat smaller than the more common 18.5-kDa form. Fraction 3 consisted of approximately equal amounts of this smaller polypeptide and component 3, the 18.5-kDa form found in other mammalian myelin basic protein preparations. The two proteins in fraction 3 were separated by fast protein liquid chromatography. Both have blocked N termini and identical C termini (-Met-Ala-Arg-Arg). When the tryptic digests of the two proteins were fractionated by HPLC, the elution profiles were similar, except that four peaks found in the chromatogram of the larger protein were missing from the chromatogram of the smaller one. In addition, an extra peak was found in the elution pattern of the latter chromatogram. Amino acid analysis of the individual tryptic peptides indicated that the smaller protein lacked residues 106-116 (-Gly-Arg-Gly-Leu-Ser-Leu-Ser-Arg-Phe-Ser-Trp-). The deleted portion corresponds exactly to the amino acid sequence encoded by exon 5 of the mouse basic protein gene. This new form of myelin basic protein has a molecular weight of 17,200, calculated from its amino acid composition.     

Myelin management by the 18.5‐kDa and 21.5‐kDa classic myelin basic protein isoforms

The classic myelin basic protein (MBP) splice isoforms range in nominal molecular mass from 14 to 21.5 kDa, and arise from the gene in the oligodendrocyte lineage (Golli) in maturing oligodendrocytes. The 18.5‐kDa isoform that predominates in adult myelin adheres the cytosolic surfaces of oligodendrocyte membranes together, and forms a two‐dimensional molecular sieve restricting protein diffusion into compact myelin. However, this protein has additional roles including cytoskeletal assembly and membrane extension, binding to SH3‐domains, participation in Fyn‐mediated signaling pathways, sequestration of phosphoinositides, and maintenance of calcium homeostasis. Of the diverse post‐translational modifications of this isoform, phosphorylation is the most dynamic, and modulates 18.5‐kDa MBP's protein‐membrane and protein‐protein interactions, indicative of a rich repertoire of functions. In developing and mature myelin, phosphorylation can result in microdomain or even nuclear targeting of the protein, supporting the conclusion that 18.5‐kDa MBP has significant roles beyond membrane adhesion. The full‐length, early‐developmental 21.5‐kDa splice isoform is predominantly karyophilic due to a non‐traditional P‐Y nuclear localization signal, with effects such as promotion of oligodendrocyte proliferation. We discuss in vitro and recent in vivo evidence for multifunctionality of these classic basic proteins of myelin, and argue for a systematic evaluation of the temporal and spatial distributions of these protein isoforms, and their modified variants, during oligodendrocyte differentiation.     

Myelin basic protein binds microtubules to a membrane surface and to actin filaments in vitro: effect of phosphorylation and deimination

Myelin basic protein (MBP) is a multifunctional protein involved in maintaining the stability and integrity of the myelin sheath by a variety of interactions with membranes and other proteins. It assembles actin filaments and microtubules, can bind actin filaments and SH3-domains to a membrane surface, and may be able to tether them to the oligodendrocyte membrane and participate in signal transduction in oligodendrocytes/myelin. In the present study, we have shown that the 18.5 kDa MBP isoform can also bind microtubules to lipid vesicles in vitro. Phosphorylation of MBP at Thr94 and Thr97 (bovine sequence) by MAPK, and deimination of MBP (using a pseudo-deiminated recombinant form), had little detectable effect on its ability to polymerize and bundle microtubules, in contrast to the effect of these modifications on MBP-mediated assembly of actin. However, these modifications dramatically decreased the ability of MBP to tether microtubules to lipid vesicles. MBP and its phosphorylated and pseudo-deiminated variants were also able to bind microtubules to actin filaments. These results suggest that MBP may be able to tether microtubules to the cytoplasmic surface of the oligodendrocyte membrane, and that this binding can be regulated by post-translational modifications to MBP. We further show that MBP appears to be co-localized with actin filaments and microtubules in cultured oligodendrocytes, and also at the interface between actin filaments at the leading edge of membrane processes and microtubules behind them. Thus, MBP may also cross-link microtubules to actin filaments in vivo.     

Cryoelectron microscopy of protein-lipid complexes of human myelin basic protein charge isomers differing in degree of citrullination

Myelin basic protein (MBP) is considered to be essential for the maintenance of stability of the myelin sheath. Reduction in cationicity of MBP, especially due to conversion of positively charged arginine residues to uncharged citrulline (Cit), has been found to be associated with multiple sclerosis (MS). Here, the interactions of an anionic phosphatidylserine/monosialoganglioside-G(M1) (4:1, w:w) lipid monolayer with 18.5-kDa MBP preparations from age-matched adult humans without MS (no Cit residues), with chronic MS (6 Cit), and with acute Marburg-type MS (18 Cit) were studied by transmission and ultralow dose scanning transmission electron microscopy under cryogenic conditions. Immunogold labeling and single particle electron crystallography were used to define the nature of the complexes visualized. These electron microscopical analyses showed that the three different MBP charge isomers all formed uniformly sized and regularly shaped protein-lipid complexes with G(M1), probably as hexamers, but exhibited differential association with and organization of the lipid. The least cationic Marburg MBP-Cit(18) formed the most open protein-lipid complex. The data show a disturbance in lipid-MBP interactions at the ultrastructural level that is related to degree of citrullination, and which may be involved in myelin degeneration in multiple sclerosis.     

Characterization of Myelin Basic Protein Charge Microheterogeneity in Developing Mouse Brain and in the Transgenic Shiverer Mutant

Abstract: Myelin basic protein (MBP) is a highly heterogeneous family of membrane proteins consisting of several isoforms resulting from alternative splicing and charge isomers arising from posttranslational modifications. Although well characterized in the bovine and human species, those in the mouse are not. With the availability of a number of transgenic and knockout mice, the need to understand the chemical nature of the MBPs has become very important. To isolate and characterize the MBP species in murine brain, two methods were adapted for use with the small amounts of MBP available from mice. The first was a scaled-down version of the preparative CM-52 chromatographic system commonly used to isolate MBP charge isomers; the second was an alkaline-urea slab gel technique that required five times less material than the conventional tube gel system and, from these gels, western blots were readily obtained. Murine MBP was resolved into two populations of charge isomers: the 18.5- and 14-kDa isoforms. Isolation and characterization of these charge isomers or components permitted us to assign possible posttranslational modifications to some of them. Component 1 (C-1), the most cationic isomer, had a molecular weight of 14,140.38 ± 0.79. C-2 consisted of two 14-kDa species, 14,136.37 ± 0.74 and 14,204.45 ± 0.70. Two variants, 14,215.57 ± 0.94 and 18,413.57 ± 0.76, constituted C-3. C-4, C-5, and C-8 (the least cationic isomer) each consisted of both 14- and 18.5-kDa isoforms. During myelinogenesis, the 18.5-kDa isoform appeared first (day 4); the 14-kDa isoform appeared at day 16 and subsequently became the dominant isoform. The transgenic shiverer mutant synthesized mainly the 18.5-kDa isoform, but none of the 14-kDa isoform, similar to the 4-day-old mouse. We concluded that the trangenic shiverer was able to initiate myelinogenesis with the 18.5-kDa isoform, but was unable to complete myelinogenesis because of the absence of the 14-kDa isoform.     

髓鞘碱性蛋白对不同头部基团的髓磷脂质单层膜影响的热力学特性

髓鞘碱性蛋白(myelin basic protein,MBP)是中枢神经系统(central nervous system,CNS)髓鞘成熟期的主要蛋白质之一.研究资料表明,MBP与变态反应性脑脊髓炎(allergic encephalomyelitis,EAE)、多发性硬化等多种神经疾病有关,是反映中枢神经系统有无...     

Protein and Lipid Composition of Radial Component-Enriched CNS Myelin

Abstract: The radial component is a junctional complex that is believed to stabilize the apposition of myelin membranes in the internode of CNS myelin. Based on our previous finding that the radial component of compact myelin retains its structure in tissue treated with the detergent Triton X-100, we have attempted to isolate the junctional complex from spinal cord myelin treated with this detergent. Using 0.5% Triton X-100, our procedures yielded a fraction of isolated myelin that was enriched in well-preserved radial component. This fraction that contained morphologically well-defined radial component was examined by sodium dodecyl sulfate-polyacrylamide gel electropho-resis and immunoblotting, and TLC, and was found to be significantly and consistently enriched in the 21.5-kDa and 17-kDa isoforms of myelin basic protein, and in cerebro-sides, hydroxy sulfatide, and sphingomyelin. In addition, the myelin-associated enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase, tubulin, and actin tended to be resistant to Triton extraction. The fraction of isolated myelin that contained radial component was deficient in proteolipid protein and DM-20, the 18.5-and 14-kDa isoforms of myelin basic proteins, and in the major phospholipids, phosphatidylethanolamine, phosphatidylcholine, and phosphatidylserine. Our data indicate that the radial component can be isolated and that certain myelin and cytoskeletal proteins and lipids are closely associated with it.     

Hydrophobic Regions in Myelin Proteins Characterized Through Analysis of "Hydropathic"Profiles

Computer-generated "hydropathic" profiles were constructed for graphic comparison of the amino acid sequences for P2 protein, 18.5 kilodalton (kDa) myelin basic protein (BP), and myelin proteolipid protein (PLP). Profiles were also obtained for cytochrome b5, a membrane protein known to be capable of reversible association with lipid bilayers and of a size comparable to that of the myelin BPs. Analysis of the PLP sequence produced profiles generally compatible with the suggestions that PLP has three transbilayer and two bilayer intercalating segments. Profiles for P2 and 18.5 kDa BP were found to contain hydrophilic segments separated by relatively short hydrophobic regions. Whereas hydropathic indices in hydrophobic regions of P2, 18.5 kDa BP, and PLP fall in the value ranges recently reported for cores of globular proteins and intrabilayer domains of membrane proteins, hydrophobic sections of P2 and 18.5 kDa BP have hydropathic indices similar to those in the hydrophobic core (transprotein) regions of globular proteins. None of them are comparable to the region of cytochrome b5 known to anchor that protein in its membrane or to the segments of PLP sequence proposed as intrabilayer domains. This comparison suggests that neither BP has structural characteristics compatible with insertion into the hydrocarbon core of the myelin lipid bilayer, a conclusion that is consistent with a recently published study that identified the bilayer penetrating proteins of myelin with a hydrophobic probe. The above findings suggest an enhancement for some details of myelin architecture and a cautious approach to interpreting data for BP intercalation into bilayers.     

Assembly of tubulin by classic myelin basic protein isoforms and regulation by post-translational modification

Myelin basic protein (MBP), a highly cationic protein that maintains the structure of the myelin sheath, associates with tubulin in vivo. The in vitro assembly of tubulin by MBP was examined here using several assays. The unmodified C1 component of 18.5 kDa bovine MBP (bC1) assembled tubulin into microtubules in a dose-dependent manner via filamentous intermediates, and was able simultaneously to promote the formation of microtubule bundles. The critical tubulin concentration in the presence of bC1 was 0.69 +/- 0.05 microM. The effects of post-translational modifications (such as deamidation and phosphorylation) were assayed by comparing the bC1-bC6 components of 18.5 kDa bovine MBP; an increasing level of modification enhanced the ability of MBP to assemble tubulin. The effects of charge reduction via deimination were examined using recombinant murine isoforms emulating the unmodified C1 and deiminated C8 isoforms of 18.5 kDa MBP; both rmC1 and rmC8 exhibited a comparable ability to assemble tubulin. The effects of alternate exon recombination of the classic MBP variants were tested using the recombinant murine 21.5, 17.22, and 14 kDa isoforms. The isoforms containing regions derived from exon II of the classic MBP gene, 21.5 and 17.22 kDa MBP, showed no substantial difference in the extent of tubulin polymerization and bundling when compared to those of 18.5 kDa MBP. The 14 kDa isoform and two terminal deletion mutants of rmC1 were able to induce microtubule polymerization, but not bundling, to the same degree as the longer proteins. Finally, bC1 was shown to disrupt and aggregate planar sheets of crystalline tubulin stabilized by paclitaxel, establishing that these structures are not suitable substrates for the formation of MBP cocrystals.     

Fluorescence studies on the interactions of myelin basic protein in electrolyte solutions.

This paper examines the influence of electrolytes on fluorescence spectral properties of the single tryptophanyl residue, Trp-115, within the 18.5-kDa species of myelin basic protein from bovine brain. Steady-state fluorescence spectra and intensities and time-correlated fluorescence lifetimes increased in the presence of increasing concentrations of mono- and divalent electrolytes (Li+, Na+, K+, Mg2+, Ca2+, Cl-, ClO4-, SO4(2-), and PO4(3-)). In all cases, the increases closely paralleled the ionic strength of the bulk aqueous medium and resembled that observed upon immersion of the protein in solutions of urea. This behavior was therefore concluded to reflect changes in the solution conformation of myelin basic protein. Bimolecular quenching of Trp-115 by acrylamide was rapid (10(9) M-1 s-1), approaching the diffusion limitation, and markedly dependent on the viscosity of the bulk aqueous medium. Rotational depolarization of myelin basic protein was rapid (phi less than or equal to 1 ns), occurring at rates exceeding those predicted for a rigid particle of revolution, and markedly dependent on the viscosity of the surrounding medium. Whereas the bimolecular quenching constants were unaltered in the presence of electrolytes, rotational depolarization of myelin basic protein underwent substantial slowing as indicated by the appearance of an additional decay component characterized by a correlation time of 5-10 ns. These studies indicate that Trp-115 of myelin basic protein is readily accessible to the bulk aqueous medium and is associated with a highly mobile segment of the protein. The slowing of rotational depolarization upon immersion of myelin basic protein in electrolyte solutions is consistent with an electrolyte-induced self-association of myelin basic protein molecules and indicates a relationship between the lability of solution conformation on the one hand and the capacity for self-association on the other.     

Solid-state NMR spectroscopy of 18.5 kDa myelin basic protein reconstituted with lipid vesicles: spectroscopic characterisation and spectral assignments of solvent-exposed protein fragments

Myelin basic protein (MBP, 18.5 kDa isoform) is a peripheral membrane protein that is essential for maintaining the structural integrity of the multilamellar myelin sheath of the central nervous system. Reconstitution of the most abundant 18.5 kDa MBP isoform with lipid vesicles yields an aggregated assembly mimicking the protein's natural environment, but which is not amenable to standard solution NMR spectroscopy. On the other hand, the mobility of MBP in such a system is variable, depends on the local strength of the protein-lipid interaction, and in general is of such a time scale that the dipolar interactions are averaged out. Here, we used a combination of solution and solid-state NMR (ssNMR) approaches: J-coupling-driven polarization transfers were combined with magic angle spinning and high-power decoupling to yield high-resolution spectra of the mobile fragments of 18.5 kDa murine MBP in membrane-associated form. To partially circumvent the problem of short transverse relaxation, we implemented three-dimensional constant-time correlation experiments (NCOCX, NCACX, CONCACX, and CAN(CO)CX) that were able to provide interresidue and intraresidue backbone correlations. These experiments resulted in partial spectral assignments for mobile fragments of the protein. Additional nuclear Overhauser effect spectroscopy (NOESY)-based experiments revealed that the mobile fragments were exposed to solvent and were likely located outside the lipid bilayer, or in its hydrophilic portion. Chemical shift index analysis showed that the fragments were largely disordered under these conditions. These combined approaches are applicable to ssNMR investigations of other peripheral membrane proteins reconstituted with lipids.