Microcalorimetric studies of the heats of solution of bovine myelin basic protein

Heats of solution for myelin basic protein have been determined using microcalorimetry. All aqueous systems studied yielded negative heats of solution; in contrast, trifluoroethanol produced a small positive heat of solution, while reaction with dimethyl sulfoxide was strikingly exothermic. The heat of interaction for native myelin basic protein with 8 M urea at pH 4.0, 29 degrees C, was found to be -79 +/- 16 kcal/mol. The significance of these results in terms of the protein's structural organization is discussed.     

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.     

Immunocytochemical localization studies of myelin basic protein

The location of myelin encephalitogenic or basic protein (BP) in peripheral nervous system (PNS) and central nervous system (CNS) was investigated by immunofluorescence and horseradish peroxidase (HRP) immunocytochemistry. BP or cross-reacting material could be clearly localized to myelin by immunofluorescence and light microscope HRP immunocytochemistry. Fine structural studies proved to be much more difficult, especially in the CNS, due to problems in tissue fixation and penetration of reagents. Sequential fixation in aldehyde followed by ethanol or methanol provided the best conditions for ultrastructural indirect immunocytochemical studies. In PNS tissue, anti-BP was localized exclusively to the intraperiod line of myelin. Because of limitations in technique, the localization of BP in CNS myelin could not be unequivocally determined. In both PNS and CNS tissue, no anti-BP binding to nonmyelin cellular or membranous elements was detected.     

Resolution and solution behavior of crosslinked myelin basic protein

The use of chemical crosslinking methodologies for the study of the solution structure and folding of the myelin basic protein required the development of a specific protocol for separating the various reaction products. Myelin basic protein treated with the crosslinking reagent dithiobis(succinimidylpropionate) was subjected to analysis by urea-SDS polyacrylamide gel electrophoresis. This permitted the identification of dimer and higher oligomeric crosslinked products. The dissociating conditions of this method precluded the dimerization of the basic protein observed in systems with SDS and without urea. Similar samples analyzed by gel filtration-fast protein liquid chromatography exhibited a complex elution pattern in contrast to the protein not reacted with the crosslinker. The electrophoretic analysis of the different eluted fractions revealed that at least three monomeric forms of modified myelin basic protein had been separated by gel filtration.     

Phosphorylation on basic amino acids in myelin basic protein.

Isolated rat brain myelin when incubated with γ³²P labelled ATP yields proteins bearing acid labile, base stable phosphoryl groups. Phosphorylated myelin basic protein can be isolated and degraded with trypsin and pronase to yield principally phosphoarginine and phosphohistidine. Only a very small amount of phosphorerine survives the base treatment used in the isolation procedure.     

Effects of acyl chain length on the conformation of myelin basic protein bound to lysolipid micelles.

The interactions of myelin basic protein with micelles of lysophosphatidylcholine detergents of different acyl chain lengths were investigated by circular dichroism (CD), small-angle X-ray scattering, Fourier transform infrared spectroscopy (FT-IR), and 1H, 13C and 31P nuclear magnetic resonance spectroscopy (NMR). Circular dichroic, FT-IR, and 1H NMR measurements indicated that the conformational changes induced in the protein molecules by association with micelles depended on the acyl chain length of the detergents. Size is one of the physical properties of micelles which is a function of the length of the acyl chains. The radii of gyration of detergent micelles in complexes with the protein measured by small-angle X-ray scattering indicated that the average size of the micelles was a quadratic function of the acyl chain length. The dependence of the protein conformational changes on micelle size was used to ascertain the order in which different protein segments associate with the detergents. Several procedures were employed to change the fluidity of micelles formed with detergents of given acyl chain lengths. The conformational changes observed on the MBP molecule by varying the micelle properties without changing the length of the chain, suggested that the changes depended on the size and fluidity of the micelles.     

Calorimetric studies on the interaction of gangliosides with phospholipids and myelin basic protein

Differential scanning calorimetry was employed to investigate the interaction of GM1 gangliosides with phospholipids (phosphatidylethanolamine, phosphatidylserine or phosphatidylcholine). It was found that GM1 is completely miscible with phosphatidylethanolamine; however, the interaction with phosphatidylserine is minimal. Addition of excess Ca2+ to the interaction products of GM1 with phosphatidylcholine or phosphatidylethanolamine did not induce phase separation. The influence of myelin basic protein on the thermotropic behaviour of GM1 was also studied. It was found that basic protein has a very strong perturbing effect on GM1 micelles.     

Effects of the osmolyte trimethylamine-N-oxide on conformation,self-association,and two-dimensional crystallization of myelin basic protein

The osmolyte trimethylamine-N-oxide (TMAO) is a naturally in vivo occurring "chemical chaperone" that has been shown to stabilise the folding of numerous proteins. Myelin basic protein (MBP) is a molecule that has not yet been suitably crystallized either in three dimensions for X-ray crystallography or in two dimensions for electron crystallography. Here, we describe lipid monolayer crystallization experiments of two species of recombinant murine MBP in the presence of TMAO. One protein was unmodified, whereas the other contained six Arg/Lys-->Gln substitutions to mimic the effects of deimination (i.e., the enzymatic modification of Arg to citrulline), which reduces the net positive charge. Planar arrays of both proteins were formed on binary lipid monolayers containing a nickel-chelating lipid and a phosphoinositide. In the presence of TMAO, the diffraction spots of these arrays became sharper and more distinct than in its absence, indicating some improvement of crystallinity. The osmolyte also induced the formation of epitaxial growth of protein arrays, especially with the mutant protein. However, none of these assemblies was sufficiently ordered to extract high-resolution structural information. Circular dichroic spectroscopy showed that MBP gained no increase in ordered secondary structure in the presence of TMAO in bulk solution, whereas it did in the presence of lipids. Dynamic light-scattering experiments confirmed that the MBP preparations were monomodal under the optimal crystallization conditions determined by electron microscopy trials. The salt and osmolyte concentrations used were shown to result in a largely unassociated population of MBP. The amino acid composition of MBP overwhelmingly favours a disordered state, and a neural-network-based scheme predicted large segments that would be unlikely to adopt a regular conformation. Thus, this protein has an inherently disordered nature, which mitigates strongly against its crystallization for high-resolution structure determination.     

D-aspartic acid in purified myelin and myelin basic protein

The presence of the biologically uncommon D-isomer of aspartic acid in the white matter of human brains has been reported previously from this laboratory (1). We now report that the level of D-aspartate in human brains is higher in purified myelin than in white matter and is even higher in the myelin basic protein fraction. There also appears to be a difference in the level of D-aspartate found in human brain as compared to bovine brain, possibly a species or age-related difference.     

NMR studies of myelin basic protein. X. Conformation of a determinant encephalitogenic in the rabbit

Residues 67 to 75 in myelin basic protein from several species comprise the sequence Thr-His-Tyr-Gly-Ser-Leu-Pro-Gln-Lys that acts as an encephalitogenic determinant in the rabbit. Proton magnetic resonance spectra of human, bovine and porcine proteins display nuclear Overhauser effects between the delta-CH of Tyr-69 and the delta-CH3 of Leu-72, which indicate reverse-turn conformations about the Gly-Ser residues. This effect occurs also in physiological saline solution at pH 6.0 but in dimethylsulfoxide solution the nuclear Overhauser effect disappears. Circular dichroism indicates that the protein when bound to ganglioside micelles acquires 30-40% alpha-helical conformation, but the reverse turn still persists in the sequence of the rabbit encephalitogen. These results suggest that the encephalitogenic region of the protein remains at the aqueous interface of the micelles.     

Insulin-sensitive myelin basic protein phosphorylation on tyrosine residues.

Rat brain plasma membranes were solubilized in detergent and a glycoprotein-enriched fraction was obtained by lectin affinity chromatography. This glycoprotein fraction contained insulin receptors, as well as protein kinases capable of phosphorylating some exogenously added substrates such as MAP2 (microtubule associated protein 2) and MBP (myelin basic protein), but not ribosomal protein S6. Phosphoamino acid analysis of MAP2 and MBP showed that phosphotyrosine residues, as well as phosphoserine/phosphotheronine residues, were present in both proteins under basal conditions. Whereas the addition of insulin to the rat brain membrane glycoprotein fraction in vitro had no effect on MAP2 phosphorylation, MBP phosphorylation was stimulated 2.7-fold in response to insulin. This phenomenon was dose-dependent, with half-maximal stimulation of MBP phosphorylation observed with 2 nM insulin. Phosphoamino acid analysis of MBP indicated that insulin stimulated the phosphorylation of tyrosine residues nearly three-fold, whereas the phosphorylation of serine or threonine residues was not increased. These results identify MBP as a substrate for the rat brain insulin receptor tyrosine-specific protein kinase in vitro.     

Basis of microheterogeneity of myelin basic protein.

The basic protein of bovine central nervous system myelin contains a single polypeptide chain of 170 amino acids. Multiple components of basic protein have been observed on disc gel electrophoresis and ion exchange chromatography at alkaline pH, but the basis of the microheterogeneity has not been established. In the present study myelin basic protein from bovine spinal cord was chromatographed on carboxymethylcellulose at pH 10.4 in glycine buffer/2 M urea. Three major peaks were obtained, identified as components 4, 5, and 6 in the oder of their elution from the column by a linear salt gradient. The amino acid compositions of tryptic peptides from components 4 and 6 were identical and the COOH-terminal sequence, Ala-Arg-Arg, was intact for all three components. Component 4 was found to differ from component 6 by partial phosphorylation of threonine 98 and serine 165. This modification was estimated to account for 50% of component 4. Component 5 differed from component 6 by partial deamidation of glutamine residues 103 and 147, which accounted for 80% of this component. These modified glutamine residues were also present in component 4 and constituted another 15% of this component. It was considered that component 6 was the native, unmodified species of basic protein and that component 4 differed by a net negative charge of 2, and component 5 by a net negative charge of.1 as a result of these modifications. The nonrandom nature of the modifications suggested the involvement of specific enzymes.     

Phosphoprotein phosphatases for myelin basic protein in myelin and cytosol fractions of brain.

Phosphoprotein phosphatase (phosphoprotein phosphohydrolase EC 3.1.3.16) activity for myelin basic protein was found to be present in the myelin fraction of rat brain. The enzyme activity was in a latent form and solubilized by 0.2% Triton X-100 treatment with about 50% increase of activity. The cytosol fraction from bovine brain also had phosphoprotein phosphatase activity for myelin basic protein, which was resolved into at least two peaks of activity on DEAE-cellulose column chromatography. Myelin basic protein was the best substrate for both the solubilized myelin fraction and the cytosol enzymes among the substrate proteins tested. The Km values of the solubilized myelin fraction were 4.2 muM for myelin basic protein, 7.4 muM for arginine-rich histone, 8.0 muM for histone mixture and 14.3 muM for protamine, respectively.     

Mechanism of interaction of myelin basic protein and S-100 protein: metal binding and fluorescence studies.

Abstract— It has been reported that myelin basic protein (MBP) forms a specific complex with S-100 protein in the presence of either Ca²⁺ or Mn²⁺, as detected by Immunoelectrophoresis. We have now studied the binding of Ca²⁺ and Mn²⁺ to these two proteins. We find that MBP binds 1 mol of Mn²⁺/mol of protein, and this binding produces an increment in its fluorescence, indicating a conformational change. Ca²⁺ does not bind to MBP nor does it affect the fluorescence of MBP. S-100 protein, as has been reported, binds about 10 mol of Ca²⁺/mol and this binding produces a conformational change. S-100 protein also has 25 binding sites for Mn²⁺, but this binding does not alter fluorescence and does not appear to affect conformation. Competitive binding experiments demonstrate that the binding sites of S-100 protein for Ca²⁺ and Mn²⁺ are independent. The alteration of electrophoretic migration in gels of S-100 protein produced by Ca²⁺ and of MBP produced by Mn²⁺ are in accord with the observations based on fluorescence. Mn²⁺ does not affect the electrophoretic mobility of S-100. These results indicate that the formation of the complex between MBP and S-100 protein in the presence of either Ca²⁺ or Mn²⁺ is due to the conformational change induced by these ions in S-100 protein, MBP, or both.