Solubilization of PNS myelin membrane proteins by detergents.

We have applied 25 different detergents to the PNS myelin membrane. The extracts produced were analyzed regarding the total amount of protein solubilized, and which myelin glycoproteins were solubilized. The degree of protein extraction was correlated with the critical micelle concentration. For some detergents the protein extraction ability depends clearly on critical micelle concentration. These detergents are more potent if critical micelle concentration is small. The other type of extraction is independent on the critical micelle concentration, but extractability of proteins is low. The best detergents for solubilization of myelin proteins are neutral of the alkyl chain length n =9-11, as follows: dodecyl-beta-D-maltopyranoside, decyl-beta-D-maltopyranoside, n-dodecylsulfobetaine. We expect that these detergents will also be suitable for crystallization of P0 and PASII/PMP22 glycoproteins.

Purification of P0 myelin glycoprotein by a Cu2+-immobilized metal affinity chromatography.

P0 is an abundant myelin glycoprotein of peripheral nerves of vertebrates. Various point mutations of this protein are responsible for hereditary neuropathies. In this paper we described purification of P0 glycoprotein using SDS and a metal chelate affinity chromatography. Purified myelin fraction from bovine spinal roots in 0.5% SDS, 0.5 M NaCl, 50 mM Tris-HCl, pH 7.4 is filtered and applied directly to the Cu2+-immobilized affinity chromatography column, equilibrated with the same buffer. After eluting a void volume (or pass through) fraction, P0 protein was eluted by the same buffer but without salt. To remove contamination from the eluent, further purification is continued on a Concanavalin-A coupled agarose column. We purify within two days, 30 mg of P0 protein of apparent molecular weight 27 kDa. The method can be used to purify recombinant or mutated P0 protein found in severe pathologies.

Ups and downs of protein crystallization: studies of protein crystals by high-performance capillary electrophoresis.

High-performance capillary electrophoresis is a high-technology micro-separation method. Short run time, full automation and minute amounts of sample make it a very attractive technique. In this report we describe studies of protein crystals by capillary electrophoresis. We show how high-performance capillary electrophoresis can be used effectively for rapid evaluation and examination of the protein solution used for crystallization, the protein crystals (solubilized) and surrounding mother liquor. With coated capillaries, the runs were reproducible and disturbing effects, such as electroendosmosis and interaction of the proteins with the capillary wall, were suppressed efficiently. We recommend this new technique as a powerful and routine companion to protein crystallography.

Tetrameric assembly of full-sequence protein zero myelin glycoprotein by synchrotron x-ray scattering.

Highly purified myelin P0 glycoprotein was solubilized to 1-8 mg/ml in 0.1% sodium dodecyl sulfate (SDS), and the solution structure of the P0 assembly was studied using synchrotron x-ray scattering. The full-length P0, which was isolated from bovine intradural roots, included both the extracellular and cytoplasmic domains of the molecule. At the higher concentrations (4, 6, and 8 mg/ml, respectively), an x-ray intensity maximum was observed at 316 A, 245 A, and 240 A Bragg spacing. Because the position of this intensity depended on P0 concentration, it is most likely due to interparticle interference. By contrast, the position of a second intensity maximum, which was at approximately 40 A Bragg spacing, was invariant with P0 concentration. This latter intensity was accounted for by monodispersed, 80 A-diameter particles that are composed of eight, approximately 30 A-diameter spheres. Chemical parameters suggest that the 80 A particles correspond to the size of a tetramer of P0 molecules. Therefore, the approximately 30 A spheres would correspond to the sizes of the extracellular and cytoplasmic domains for each of the P0 monomers. The invariance of the second intensity maximum with P0 concentration indicates that the structure of the 80 A-diameter, tetrameric particles is unaltered. According to the liquid model for interparticle interference from charged spheres, the 80 A-diameter particle has 10 negative surface charges which likely arise from negatively charged SDS molecules bound to the transmembrane domain of P0. This binding, however, apparently does not alter the tetrameric assembly of P0, suggesting that intermolecular interactions involving extracellular domains and cytoplasmic domains likely stabilize this assembly. Some of our results have been published in abstract form (Inouye, H., H. Tsuruta, D. A. Kirschner, J. Sedzik, and K. Uyemura. Abstracts of the 4th International School and Symposium on Synchrotron Radiation in Natural Science, June 15-20, 1998. Ustron-Jaszowiec, Poland. p. 31).

Purification of PASII/PMP22--an extremely hydrophobic glycoprotein of PNS myelin membrane.

PASII/PMP22 protein (mol. wt 22 kDa, pI 8.8) is an abundant and extremely hydrophobic glycoprotein of PNS myelin which is solubilized effectively by SDS. In humans, this protein is involved in hereditary neuropathies. Here we describe a simple method for purification of PASII/PMP22, suitable for crystallization trials. We usually obtained 10-20 mg PASII/PMP22 from 10 g bovine spinal roots. It is notable, that the original protocol was designated for purification of P0 myelin glycoprotein, and purification of PASII/PMP22 is a bonus. Extensive crystallization trials are in progress.

Regression analysis of factorially designed trials--a logical approach to protein crystallization.

The specific composition of the mother liquor that induces the formation of protein crystal is usually quoted as the 'best condition' for crystallization. However, very little is described about how these conditions were determined. These missing and non-reported details would certainly lead to a better understanding of why a specific purified protein does not crystallize, and why crystallization seems to be a totally unpredictable enterprise. In this paper, it is shown that a simple regression model applied to a set of factorially designed experiments (comprising successful and failed experiments) is a useful tool for the analysis of crystallization outcomes. The applications of this approach to the proteins lysozyme and calmodulin yielded results that did not substantially differ from published accounts. When this procedure is applied to myelin basic protein (MBP) and to a MBP and calmodulin complex no crystals formed, which would suggest that neither is easy to crystallize. In summary, factorial design and regression analysis can be an important methodological approach for crystallizing proteins.

Myelin basic protein purified on an ion-exchange continuous polymer bed in the presence of ethylene glycol and salt possesses activity against p-nitrophenyl acetate.

In this paper we describe a fast and mild method based on the use of a unique cation exchanger and buffers containing ethylene glycol and salt for the purification of the myelin basic protein (MBP; MW 18.5 kDa). MBP thus purified hydrolyses catalytically p-nitrophenyl acetate. This esterase activity facilitates not only the purification of MBP but also indicates that probably it is in its native state, i.e. there is a good chance that the purified molecules are structurally and chemically identical. This is a prerequisite to obtain crystals appropriate for x-ray diffraction and other studies.

Myelin vesicles: what we know and what we do not know.

In the following review, we address difficulties that have arisen when attempting to convert the myelin multilayers into vesicles. The emphasis is on CNS myelin of adult mammals although both central nervous system (CNS) and peripheral nervous system (PNS) myelin are considered. The ability to prepare vesicle of myelin membrane has yet not been feasible. We hope to clarify some aspect of this problem and offer some possible approaches. Special attention is paid to myelin swelling phenomena because these indicate ways in which the myelin multilayer can break down. Images of isolated myelin are reviewed with special attention to the ways in which the multilayer actually breaks down. Attempts at reproducing a procedure for vesiculating myelin are summarized, and a critique is given to account for the inability to reproduce the published results. Finally, novel approaches for vesiculating myelin are proposed, which are based on well-characterized swelling phenomena.

DESIGN: a guide to protein crystallization experiments.

Existing guidelines for crystallizing proteins generally require substantial amounts of protein and scarce laboratory resources. To address this problem we have developed the program DESIGN, which, suited to daily laboratory usage, utilizes statistics when screening variables of crystallization. The program DESIGN is described in considerable detail and examples of its outcome and capabilities are shown. In addition, a new scoring scale for crystallization trials is described. It is expected that the use of such an approach may provide additional constraints toward crystallizing the protein, even if the conditions applied are not successful.

Is myelin basic protein crystallizable?

Myelin basic protein (MBP) is the predominant extrinsic protein in both central and peripheral nervous system myelins. It is thought to be involved in the stabilizing interactions between myelin membranes, and it may play an important role in demyelinating diseases such as multiple sclerosis. In spite of the fact that this abundant protein has been known for almost three decades, its three-dimensional crystal structure has not yet been determined. In this study we report on our extensive attempts to crystallize the major 18.5 kDa isoform of MBP. We used MBP having different degrees of purity, ranging from crude MBP (that was acid or salt extracted from isolated myelin), to highest purity single isoform. We used convention strategies in our search for a suitable composition of a crystallization medium. We applied both full and incomplete factorial searches for crystallization conditions. We analyzed the available data on proteins which have previously resisted crystallization, and applied this information to our own experiments. Nevertheless, despite our efforts which included 4600 different conditions, we were unable to induce crystallization of MBP. Previous work on MBP indicates that when it is removed from its native environment in the myelin membrane and put in crystallization media, the protein adopts a random coil conformation and persists as a population of structurally non-identical molecules. This thermodynamically preferred state presumably hinders crystallization, because the most fundamental factor of protein crystallization - homogeneity of tertiary structure--is lacking. We conclude that as long as its random coil flexibility is not suppressed, 18.5 kDa MBP and possibly also its isoforms will remain preeminent examples of proteins that cannot be crystallized.

Three-dimensional reconstruction of bovine intradural spinal root myelin by electron microscope tomography.

Electron microscope tomography was used to reconstruct three-dimensionally the configuration of heavy metal staining in bovine intradural spinal root myelin. Samples were fixed with glutaraldehyde, exposed to osmium tetroxide, embedded, thin sectioned, and finally stained with uranyl-acetate and lead citrate. Reconstructions up to 4.2 nm resolution showed a non-uniform distribution of stain in the planes of individual cytoplasmic appositions (major dense lines). In each reconstructed major dense line the stain was distributed in striated, well-defined structures. Those structures appear to be nearly parallel between neighboring major dense lines. The distribution of stain in the Schmidt-Lanterman cleft did not resemble the distribution of stain in the major dense line; however, weak striations were present. Evidence that the striated structures are not an artifact due to image calculation is discussed.

The three-dimensional structure of P2 myelin protein.

The three-dimensional structure of P2 protein from peripheral nervous system myelin has been determined at 2.7 A resolution by X-ray crystallography. The single isomorphous replacement/anomalous map was interpreted using skeletonized electron density on a computer graphics system. An atomic model was built using fragment fitting. The structure forms a compact 10-stranded up-and-down beta-barrel which encapsulates residual electron density that we interpret as a fatty acid molecule. This beta-barrel shows some similarity to, but is different from, the retinol binding protein family of structures. The relationship of the P2 structure to a family of cytoplasmic, lipid binding proteins is described.

Bovine P2 myelin basic protein crystallizes in three different forms.

P2 protein is a minor component of the myelin membrane. We have crystallized this protein for high-resolution crystallographic study. Three crystal morphologies are available. Two of them are from ammonium sulfate, and one is from polyethyleneglycol (PEG). The unit cell of the most suitable crystals from PEG 4000 has the dimensions a = 91.3 A, b = 99.8 A, c = 56.0 A; is of space group P2(1)2(1)2(1); and contains up to four molecules per asymmetric unit. The limit of resolution is 2.7 A.

Crystallization of P2 myelin protein.

Single crystals of bovine P2 myelin protein have been grown in polyethylene glycol 4000 by the hanging-drop vapor diffusion method. Crystals belonging to space group P2(1)2(1)2(1) with cell dimensions a = 91.8 A, b = 99.5 A, c = 56.5 A (1 A = 0.1 nm). The diffraction pattern extends to better than 2.3 A resolution.

Reconstituted P2/myelin-lipid multilayers.

A complex forms when bovine P2 protein is added to single-bilayer vesicles created by sonicating myelin lipids. The complex was studied by biochemical analysis, freeze-fracture (FF) and thin-section electron microscopy (EM), and by X-ray diffraction. Smaller amounts of P2 cause the vesicles to aggregate and fuse whereas larger amounts (greater than or equal to 4 wt%) cause multilayers to form. Binding saturates at 15 wt% P2. FF EM shows that large, flat multilayers form within 15 min of addition of P2. Only smooth fracture faces are seen, as expected for a peripheral membrane protein. X-ray diffraction shows a constant repeating distance in the multilayers: 86.0 +/- 0.7 A between the centers of bilayers in the range 4 wt% less than or equal to P2/(P2 + lipid) less than or equal to 15 wt%. Assuming a 53 A-thick bilayer, the space between bilayers is 33 A wide. This is a wider space than for myelin basic protein (MBP) (20-25 A wide). The respective widths are consistent with a compact, globular structure for P2 and a flattened shape for MBP. Calculated electron-density profiles of the lipids with and without P2 reveal the protein largely in the interbilayer spaces, with a small part possibly inserted into the lipid headgroup layers. The different proportions of P2 in the sciatic nerve of various species are tentatively correlated with the different average widths observed by X-ray diffraction for the cytoplasmic space (major period line) between bilayers in the respective sciatic myelins.

Resistance to disruption of multilamellar fragments of central nervous system myelin.

Single-bilayer vesicles of myelin are desirable for studying myelin development and metabolism. Accordingly, our interest was drawn to a procedure for vesiculating myelin (Steck et al., Biochim, Biophys. Acta 509, 397-408, 1978). We used X-ray diffraction analysis to examine these putative vesicle preparations because much larger amounts of material can be surveyed by this method than by electron microscopy. The sharpness (width) of the rings in the X-ray diffraction pattern varies inversely with the number of bilayers per multilayer structure. We therefore expected to see the diffuse diffraction pattern characteristic of single bilayers. Diffraction patterns were recorded from isolated rat brain myelin before and after the vesiculation procedure. Both patterns showed sharp rings, indicating numerous multilayered structures. Average values ranging from 7 to 10 bilayers per multilayer were calculated in both cases. This procedure did produce a small fraction of single-bilayer structures, which were isolated by differential centrifugation; however, these accounted for only about 1% of the total myelin present. The diffraction pattern of this material showed the diffuse band typical of single-bilayer structures, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated it had the same protein composition as in normal myelin. Similar results were also obtained using either fresh or frozen bovine brain myelin. Variations of the published vesiculation procedure (incubation in 0.1 M NaCl or in buffers containing glycerol; disruption by sonication or use of a Tissumizer) also were not effective in breaking down the multilamellar fragments into thinner structures. The conclude that the multilamellar fragments of isolated CNS myelin resist disruption into single-bilayer structures.

Lipid/myelin basic protein multilayers. A model for the cytoplasmic space in central nervous system myelin.

A multilayered complex forms when a solution of myelin basic protein is added to single-bilayer vesicles formed by sonicating myelin lipids. Vesicles and multilayers have been studied by electron microscopy, biochemical analysis, and X-ray diffraction. Freeze-fracture electron microscopy shows well-separated vesicles before myelin basic protein is added, but afterward there are aggregated, possibly multilayered, vesicles and extensive planar multilayers. The vesicles aggregate and fuse within seconds after the protein is added, and the multilayers form within minutes. No intra-bilayer particles are seen, with or without the protein. Some myelin basic protein, but no lipid, remains in the supernatant after the protein is added and the complex sedimented for X-ray diffraction. A rather variable proportion of the protein is bound. X-ray diffraction patterns show that the vesicles are stable in the absence of myelin basic protein, even under high g-forces. After the protein is added, however, lipid/myelin basic protein multilayers predominate over single-bilayer vesicles. The protein is in every space between lipid bilayers. Thus the vesicles are torn open by strong interaction with myelin basic protein. The inter-bilayer spaces in the multilayers are comparable to the cytoplasmic spaces in central nervous system myelins . The diffraction indicates the same lipid bilayer thickness in vesicles and multilayers, to within 1 A. By comparing electron-density profiles of vesicles and multilayers, most of the myelin basic protein is located in the inter-bilayer space while up to one-third may be inserted between lipid headgroups. When cytochrome c is added in place of myelin basic protein, multilayers also form. In this case the protein is located entirely outside the unchanged bilayer. Comparison of the various profiles emphasizes the close and extensive apposition of myelin basic protein to the lipid bilayer. Numerous bonds may form between myelin basic protein and lipids. Cholesterol may enhance binding by opening gaps between diacyl-lipid headgroups.

The effect of transplacental intoxication with ethylnitrosourea on myelin proteins.

Pregnant mice at the 15th day of gestation were injected with a single intravenous dose of ENU/80 mg/kg of body weight). The protein composition of the myelin fraction isolated by means of differential centrifugation from brains of the offsprings was studied. The results obtained lead to the following conclusions: 1. The myelin protein spectrum of transplacentally intoxicated animals aged 40 days postnatal, shows decreased percentages of the Wolfgram protein and of the low molecular weight basic protein, whereas the proportions of the high molecular weight component of the myelin basic protein as well that of the Agrawal's protein are elevated. 2. The alterations in the profile of myelin proteins obtained from experimental mice aged 70 days postnatal are less severe and consist in a decline of the percentage of Wolfgrams protein and elevation of the Agrawal's protein content. 3. The intraplacental intoxication of mouse fetuses leads to development of animals defective with respect to the protein composition of the central myelin.

Myelin lipids in the brain after transplacental intoxication with ethylnitrosourea.

At day 15 of gestation pregnant mice were injected with a single intravenous dose of ethylnitrosourea (ENU) (80 mg/kg of body weight). The lipid composition of the myelin fraction isolated by means of differential centrifugation from brains of their offsprings was studied. The transplacentally intoxicated mice developed severe changes in the lipid composition of their central myelin sheaths, such as a markedly decreased galactolipid (both cerebroside and sulfatide) as well as plasmalogen contents. The results show, that besides being a well known carcinogenic agent, ENU is capable of affecting the lipid metabolism of the developing central nervous system. The resultant effect is the formation of a chemically defective myelin sheath.