Myelin labeled with mercuric chloride. Asymmetric localization of phosphatidylethanolamine plasmalogen

We have recorded modified X-ray diffraction patterns to 15 Å spacing from sciatic nerves treated with mercuric chloride (HgCl₂) at concentrations of 0.5 to 32 mm in water or in saline. The observed changes in repeat period and in the intensities of the low-order reflections indicate closer packing of membranes at their cytoplasmic surfaces after treatments with HgCl₂. In addition, HgCl₂ at 0.25 mm or more prevents swelling in water at the extracellular boundaries. By comparing the distinctive diffraction patterns from nerves treated under different conditions with HgCl₂, we have interpreted the changes in intensities of the higher order X-ray reflections and have calculated electron density profiles of the modified membranes. The most striking difference between membrane profiles before and after treatment with HgCl₂ is the large increase in electron density in the region of the lipid headgroup peak in the cytoplasmic half of the bilayer. The magnitude and location of this increase suggests labeling of myelin lipid. To examine this possibility, we analyzed the lipids from mercury-treated sciatic nerves.Thin-layer chromatography of lipids extracted from nerves treated with HgCl₂ shows a marked decrease of phosphatidylethanolamine, which exists in myelin primarily as plasmalogen. At the same time, a new spot identified as lysophosphatidylethanolamine appears. An identical result was obtained by treating extracted lipids with HgCl₂, suggesting that the same sites of interaction are present in the intact membrane as in the dispersed lipids. Previous studies on plasmalogens indicate that mercury adds to the β-carbon of the α,β-unsaturated ether group to produce a lyso-lipid and an aldehyde with bound mercury (Norton, 1959). From a correlation of our X-ray structural analysis and the chemical studies, we conclude that phosphatidylethanolamine plasmalogen is preferentially localized in the cytoplasmic half of the myelin membrane bilayer.     

X-ray diffraction study of myelin structure in immature and mutant mice

X-ray diffraction patterns were obtained from freshly dissected central and peripheral nerves of quaking, myelin synthesis deficiency ($\text{msd}$), and trembler mutants, as well as immature and adult normal mice. The patterns were compared with respect to strength of myelin diffraction, background scatter level, repeat period, and intensity and linewidth of Bragg reflections. The deficiency of myelin in optic nerves was found to be (in decreasing severity): quaking > immature > trembler ? normal adult; and in sciatic nerves: $\text{trembler > immature > quaking}\text{msd ?}\text{normal}$ adult. Repeat periods about 3 Å less than that for normal adult sciatic myelin were detected in corresponding nerves from immature, quaking, and trembler mice. In some trembler sciatic nerves a second phase having a 190–200 Å period and accounting for about 60% of the total ordered myelin was also evident. Comparison of electron density profiles of membrane units calculated from the repeat periods and diffracted intensities for sciatic myelins indicate structural differences at the molecular level. The main findings are: (1) quaking myelin shows a significant elevation of density in the external protein-water layer between membrane bilayers; (2) the membrane bilayer of immature myelin is ≈ 2 Å thinner than that for normal adult; (3) the membrane bilayer of the more compact phase in trembler myelin is ≈ 5 Å thinner than for normal; and (4) the difference in repeat periods for the two phases present in some of the trembler nerves can be accounted for predominantly by distinct membrane bilayer separations at the external boundary.     

Membrane structure in isolated and intact myelins.

The biochemical composition of myelin and the topology of its constituent lipids and proteins are typically studied using membranes that have been isolated from whole, intact tissue using procedures involving hypotonic shock and sucrose density gradient centrifugation. To what extent, however, are the structure and intermembrane interactions of isolated myelin similar to those of intact myelin? We have previously reported that intact and isolated myelins do not always show identical myelin periods, indicating a difference in membrane-membrane interactions. The present study addresses the possibility that this is due to altered membrane structure. Because x-ray scattering from isolated myelin sometimes consists of overlapping Bragg reflections or is continuous, we developed nonlinear least squares procedures for analyzing the total intensity distribution after film scaling, background subtraction, and Lorentz correction. We calculated electron density profiles of isolated myelin for comparison with membrane profiles from intact myelin. The change in the width of the extracellular space and the relative invariance of the cytoplasmic space as a function of pH and ionic strength that we previously found for intact nerve was largely paralleled by isolated myelin. There were two exceptions: isolated CNS myelin was resistant to swelling under all conditions, and isolated PNS myelin in hypotonic saline showed indefinite swelling at the extracellular apposition. However, electron density profiles of isolated myelins, calculated to 30 A resolution, did not show any major change in structure compared with intact myelin that could account for the differences in interactions.     

Structural states of myelin observed by x-ray diffraction and freeze- fracture electron microscopy

Coordinated freeze-fracture electron microscopy and x-ray diffraction were used to visualize the morphological relation between compacted and native period membrane arrays in myelinated nerves treated with dimethylsulfoxide (DMSO). Comparison of x-ray diffraction at room temperature and at low temperature was used as a critical measure of the extent of structural preservation. Our x-ray diffraction patterns show that in the presence of cryoprotective agents, it is possible to preserve with only small changes the myelin structure which exists at room temperature. These changes include a slight increase in packing disorder of the membrane, a small, negative thermal expansion of the membrane unit, and some reorganization in the cytoplasmic half of the bilayer. The freeze-fracture electron microscopy clearly demonstrates continuity of compact and native period phases in DMSO-treated myelin. Finally, the use of freezing to trap the transient, intermediate structure during a structural transition in glycerol is demonstrated.     

Reversible changes in myelin structure and electrical activity during anesthesia in vivo

X-ray diffraction patterns have been recorded from sciatic nerve myelin by means of dynamic X-ray diffraction either from frogs, during the early stages of anesthesia in vivo induced by n-pentane inhalation, and from frog and rat sciatic nerves isolated immediately after the animal was anesthetized. This approach has enabled to resolve minor changes in myelin structure that occur during anesthesia which were found to be similar in frogs and mammals. The X-ray patterns show a reversible slight decrease in intensity of the even reflections during anesthesia. The electron density profiles from myelin of anesthetized and recovered nerves revealed that the unit membrane structure is practically identical in both circumstances. However, during anesthesia myelin membrane pairs move toward the cytoplasmic side becoming more closely packed by 1.6 A. Physiological activity was estimated during the recovery process: compound action potential recovered its maximal amplitude before myelin recovered its native structure. On the contrary, the conduction velocity seemed to be closely related to the structural recovery. This work provides evidence that early stages of anesthesia by n-pentane in vivo does not change membrane bilayer structure but perturbs the surface interactions between adjacent membrane pairs.     

Effects of ZnCl2 on membrane interactions in myelin of normal and shiverer mice

X-ray diffraction was used to record the effects of metal cations on the structure of peripheral nerve myelin. Acidic saline (pH 5.0) either with or without added metal cations caused myelin to swell by 10-20 A from its native period of 178 A. The X-ray patterns usually showed broad reflections, and higher orders were either weak or unobserved. With added ZnCl2, however, the swollen myelin gave diffraction patterns that retained sharp reflections to approx. 15 A spacing. Alkaline saline (pH 9.7) containing ZnCl2 produced a reduction of the myelin period by approx. 5 A which was at least twice as much as that produced by other metals. To examine the underlying chemical basis for these unique interactions of Zn2+ with myelin, we carried out parallel X-ray experiments on sciatic nerve from the shiverer mutant mouse, which lacks the major myelin basic proteins. Shiverer myelin responded like normal myelin to ZnCl2 in acidic saline; however, in alkaline saline shiverer myelin showed broadened X-ray reflections which indicated disordering of the regularity of the membrane arrays, and additional reflections were recorded which indicated lipid phase separation. This breakdown may come about by the binding of Zn2+ to negatively-charged lipids which could be more exposed due to the absence of myelin basic proteins. Electron density profiles were calculated on the assumption that, except for changes in their packing, the myelin membranes were minimally altered in structure. For both normal and shiverer myelins, treatments under acidic conditions resulted in swelling at the extracellular apposition and a slight narrowing of the cytoplasmic space. This swelling is likely due to adsorption of protons and divalent cations. Interaction between Zn2+ and myelin P0 glycoprotein could preserve an ordered arrangement of the apposed membrane surfaces. Alkaline saline containing ZnCl2 produced compaction at the cytoplasmic apposition in both normal and shiverer myelins possibly through interactions with a portion of P0 glycoprotein which extends into the cytoplasmic space between membranes.     

Shiverer and Normal Peripheral Myelin Compared: Basic Protein Localization, Membrane Interactions, and Lipid Composition

We have correlated membrane structure and interactions in shiverer sciatic nerve myelin with its biochemical composition. Analysis of x-ray diffraction data from shiverer myelin swollen in water substantiates our previous localization of an electron density deficit in the cytoplasmic half of the membrane. The density loss correlates with the absence of the major myelin basic proteins and indicates that in normal myelin, the basic protein is localized to the cytoplasmic apposition. As in normal peripheral myelin, hypotonic swelling in the shiverer membrane arrays occurs in the extracellular space between membranes; the cytoplasmic surfaces remain closely apposed notwithstanding the absence of basic protein from this region. Surprisingly, we found that the interaction at the extracellular apposition of shiverer membranes is altered. The extracellular space swells to a greater extent than normal when nerves are incubated in distilled water, treated at a reduced ionic strength of 0.06 in the range of pH 4-9, or treated at constant pH (4 or 7) in the range of ionic strengths 0.02-0.20. To examine the biochemical basis of this difference in swelling, we compared the lipid composition of shiverer and normal myelin. We find that sulfatides, hydroxycerebroside, and phosphatidylcholine are 20-30% higher than normal; nonhydroxycerebroside and sphingomyelin are 15-20% lower than normal; and ethanolamine phosphatides, phosphatidylserine, and cholesterol show little or no change. A higher concentration of negatively charged sulfatides at the extracellular surface likely contributes to an increased electrostatic repulsion and greater swelling in shiverer. The cytoplasmic surfaces of the apposed membranes of normal and shiverer myelins did not swell apart appreciably in the pH and ionic strength ranges expected to produce electrostatic repulsion. This stability, then, clearly does not depend on basic protein. We propose that P0 glycoprotein molecules form the stable link between apposed cytoplasmic membrane surfaces in peripheral myelin.     

LOCATING THE MAJOR GLYCOPROTEIN (Po PROTEIN) IN THE X-RAY PROFILE OF FROG SCIATIC-NERVE MYELIN

Some detailed X-ray profiles have been put forward for frog sciatic-nerve myelin in the past 8 y. In this paper we interpret one of these. Much of the myelin protein is located in the extracellular half of the membrane. On the basis of a chemical analysis and other recently published data indicating the sidedness of some myelin proteins, we suggest that the major glycoprotein (P_o protein) is inserted into the extracellular half of the myelin bilayer and that it extends into the extracellular space. Our interpretation is consistent with known physical properties of the P_o protein. Other myelin proteins have not yet been located. Electron densities and average hydrophobicities of some myelin proteins have been calculated, as well as electron densities and scattering powers of some myelin lipids.     

Myelin Membrane Structure and Composition Correlated: A Phylogenetic Study

We have correlated myelin membrane structure with biochemical composition in the CNS and PNS of a phylogenetic series of animals, including elasmobranchs, teleosts, amphibians, and mammals. X-ray diffraction patterns were recorded from freshly dissected, unfixed tissue and used to determine the thicknesses of the liquid bilayer and the widths of the spaces between membranes at their cytoplasmic and extracellular appositions. The lipid and protein compositions of myelinated tissue from selected animals were determined by TLC and sodium dodecyl sulfate-polyacrylamide gel electrophoresis/immunoblotting, respectively. We found that (1) there were considerable differences in lipid (particularly glycolipid) composition, but no apparent phylogenetic trends; (2) the lipid composition did not seem to affect either the bilayer thickness, which was relatively constant, or the membrane separation; (3) the CNS of elasmobranch and teleost and the PNS of all four classes contained polypeptides that were recognized by antibodies against myelin P0 glycoprotein; (4) antibodies against proteolipid protein (PLP) were recognized only by amphibian and mammalian CNS; (5) wide extracellular spaces (ranging from 36 to 48 A) always correlated with the presence of P0-immunoreactive protein; (6) the narrowest extracellular spaces (approximately 31 A) were observed only in PLP-containing myelin; (7) the cytoplasmic space in PLP-containing myelin (approximately 31 A) averaged approximately 5 A less than that in P0-containing myelin; (8) even narrower cytoplasmic spaces (approximately 24 A) were measured when both P0 and 11-13-kilodalton basic protein were detected; (9) proteins immunoreactive to antibodies against myelin P2 basic protein were present in elasmobranch and teleost CNS and/or PNS, and in mammalian PNS, but not in amphibian tissues; and (10) among mammalian PNS myelins, the major difference in structure was a variation in membrane separation at the cytoplasmic apposition. These findings demonstrate which features of myelin structure have remained constant and which have become specifically altered as myelin composition changed during evolutionary development.     

Myelin x-ray patterns reconciled.

Two different X-ray diffraction patterns have been published for frog sciatic myelin. Here the apparent discrepancy is attributed to different spacings between the myelin membranes in the two experiments. Assuming the single membrane has the same structure in the two cases, some restrictions on the phasing are indicated. Several possible profiles for the single membrane are then considered. A profile derived by assuming a lecithin cholesterol-like bilayer within the membrane accounts for all the published data. Three published profiles also are considered. These are not quite in as good agreement with observation, but they cannot be excluded at present.     

An X-ray diffraction study of model membrane raft structures

Protein sorting and assembly in membrane biogenesis and function involves the creation of ordered domains of lipids known as membrane rafts. The rafts are comprised of all the major classes of lipids, including glycerophospholipids, sphingolipids and sterol. Cholesterol is known to interact with sphingomyelin to form a liquid-ordered bilayer phase. Domains formed by sphingomyelin and cholesterol, however, represent relatively small proportions of the lipids found in membrane rafts and the properties of other raft lipids are not well characterized. We examined the structure of lipid bilayers comprised of aqueous dispersions of ternary mixtures of phosphatidylcholines and sphingomyelins from tissue extracts and cholesterol using synchrotron X-ray powder diffraction methods. Analysis of the Bragg reflections using peak-fitting methods enables the distinction of three coexisting bilayer structures: (a) a quasicrystalline structure comprised of equimolar proportions of phosphatidylcholine and sphingomyelin, (b) a liquid-ordered bilayer of phospholipid and cholesterol, and (c) fluid phospholipid bilayers. The structures have been assigned on the basis of lamellar repeat spacings, relative scattering intensities and bilayer thickness of binary and ternary lipid mixtures of varying composition subjected to thermal scans between 20 and 50 °C. The results suggest that the order created by the quasicrystalline phase may provide an appropriate scaffold for the organization and assembly of raft proteins on both sides of the membrane. Co-existing liquid-ordered structures comprised of phospholipid and cholesterol provides an additional membrane environment for assembly of different raft proteins.     

An X ray diffraction study of frog sciatic nerve myelin in dimethyl sulfoxide.

Reversible structure modification of frog sciatic nerve myelin bathed in Ringer's solution containing dimethyl sulfoxide (DMSO) at a concentration of 33% has been studied by low-angle X-ray diffraction using a linear position-sensitive counter. Fourier images of native myelin layers, derived using low-order reflections measured at various stages of the DMSO treatment, reveal that the bilayer profile of native myelin membrane undergoes a specific asymmetric change prior to the phase transformation: The high-density peak on the extracellular side of the central lipid hydrocarbon layer decreases reversibly as the nerve is permeated by DMSO, while the internal peak and the central layer remain virtually unaltered. The dynamic process by which the contracted phase of myelin is derived from native myelin is speculated on the basis of the observed profile change.     

An X-ray diffraction analysis of oriented lipid multilayers containing basic proteins

X-ray diffraction techniques have been used to study the structures of lipid bilayers containing basic proteins. Highly ordered multilayer specimens have been formed by using the Langmuir-Blodgett method in which a solid support is passed through a lipid monolayer held at constant surface pressure at an air/water interface. If the lipid monolayer contains acidic lipids then basic proteins in the aqueous subphase are transferred with the monolayer and incorporated into the multi-membrane stack. X-ray diffraction patterns have been recorded from multilayers of cerebroside sulphate and 40% (molar) cholesterol both with and without polylysine, cytochrome c and the basic protein from central nervous system myelin. Electron density profiles across the membranes have been derived at between 6 A and 12 A resolution. All of the membrane profiles have been placed on an absolute scale of electron density by the isomorphous exchange of cholesterol with a brominated cholesterol analog. The distributions and conformations of the various basic proteins incorporated within the cerebroside sulphate/cholesterol bilayer are very different. Polylysine attaches to the surface of the lipid bilayer as a fully extended chain while cytochrome c maintains its native structure and attaches to the bilayer surface with its short axis approximately perpendicular to the membrane plane. The myelin basic protein associates intimately with the lipid headgroups in the form of an extended molecule, yet its dimension perpendicular to the plane of the membrane of approx. 15 A is consistent with the considerable degree of secondary structure found in solution. In the membrane plane, the myelin basic protein extends to cover an area of about 2500 A2. There is no significant penetration of the protein into the hydrocarbon region of the bilayer or, indeed, beyond the position of the sulphate group of the cerebroside sulphate molecule.     

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.     

Thermotropic and structural evaluation of the interaction of natural sphingomyelins with cholesterol

The structural transitions in aqueous dispersions of egg-sphingomyelin and bovine brain-sphingomyelin and sphingomyelin co-dispersed with different proportions of cholesterol were compared during temperature scans between 20° and 50 °C using small-angle and wide-angle X-ray scattering techniques. The Bragg reflections observed in the small-angle scattering region from pure phospholipids and codispersions of sphingomyelin:cholesterol in molar ratios 80:20 and 50:50 could all be deconvolved using peak fitting methods into two coexisting lamellar structures. Electron density profiles through the unit cell normal to the bilayer plane were calculated to derive bilayer and water layer thicknesses of coexisting structures at 20° and 50 °C. Codispersions of sphingomyelin:cholesterol in a molar ratio 60:40 consisted of an apparently homogeneous bilayer structure designated as liquid-ordered phase. Curve fitting analysis of the wide-angle scattering bands were applied to correlate changes in packing arrangements of hydrocarbon in the hydrophobic domain of the bilayer with changes in enthalpy recorded by differential scanning calorimetry. At 20 °C the wide-angle scattering bands of both pure sphingomyelins and codispersions of sphingomyelin and cholesterol could be deconvolved into two symmetric components. A sharp component located at a d-spacing of 0.42 nm was assigned to a gel phase in which the hydrocarbon chains are oriented perpendicular to the bilayer plane. A broader symmetric band centered at d-spacings in the region of 0.44 nm was assigned as disordered hydrocarbon in dispersions of pure sphingomyelin and as liquid-ordered phase in codispersions of sphingomyelin and cholesterol. It is concluded from the peak fitting analysis that cholesterol is excluded from gel phases of egg and brain sphingomyelins at 20 °C. The gel phases coexist with liquid-ordered phase comprised of egg-sphingomyelin and 27 mol% cholesterol and brain-sphingomyelin and 33 mol% cholesterol, respectively. Correlation of the disappearance of gel phase during heating scans and the enthalpy change recorded by calorimetry in codispersions of sphingomyelin and cholesterol leads to the conclusion that a major contribution to the broadened phase transition endotherm originates from dilution of the cholesterol-rich liquid-ordered phase by mobilization of sphingomyelin from the melting gel phase.     

The partition of cholesterol between ordered and fluid bilayers of phosphatidylcholine: a synchrotron X-ray diffraction study

The structure and composition of coexisting bilayer phases separated in binary mixtures of dipalmitoylphosphatidylcholine and cholesterol and ternary mixtures of equimolar proportions of dipalmitoyl- and dioleoylphosphatidycholines containing different proportions of cholesterol have been characterized by synchrotron X-ray diffraction methods. The liquid-ordered phase is distinguished from gel and fluid phases by a disordering of the hydrocarbon chains intermediate between the two phases as judged from the wide-angle X-ray scattering profiles. Electron density distribution calculated in coexisting bilayer phases shows that liquid-ordered phase is enriched in dipalmitoylphosphatidylcholine and cholesterol and a higher electron density in the methylene chain region of the bilayer ascribed to the location of the sterol ring of cholesterol. The ratio of the two constituents in the liquid-ordered phase is not constant because the stoichiometry is temperature-dependent as seen by respective changes in bilayer thickness over the range 20 degrees to 36 degrees C where coexisting phases are observed. Three coexisting phases were deconvolved in the ternary mixture at 20 degrees C. From an analysis of the ternary mixtures containing mole fractions of cholesterol from 0.09 to 0.15 it was found that the liquid-crystal and gel phases each contained about 10% of the cholesterol molecules and the liquid-ordered phase was comprised of 30% cholesterol molecules.     

Comparison of the liquid-ordered bilayer phases containing cholesterol or 7-dehydrocholesterol in modeling Smith-Lemli-Opitz syndrome

The phase behavior of egg sphingomyelin (ESM) mixtures with cholesterol or 7-dehydrocholesterol (7-DHC) has been investigated by independent methods: fluorescence microscopy, X-ray diffraction, and electron spin resonance spectroscopy. In giant vesicles, cholesterol-enriched domains appeared as large and clearly delineated domains assigned to a liquid-ordered (L_o) phase. The domains containing 7-DHC were smaller and had more diffuse boundaries. Separation of a gel phase assigned by X-ray examination to pure sphingomyelin domains coexisting with sterol-enriched domains was observed at temperatures less than 38°C in binary mixtures containing 10-mol% sterol. At higher sterol concentrations, the coexistence of liquid-ordered and liquid-disordered phases was evidenced in the temperature range 20°–50°C. Calculated electron density profiles indicated the location of 7-DHC was more loosely defined than cholesterol, which is localized precisely at a particular depth along the bilayer normal. ESR spectra of spin-labeled fatty acid partitioned in the liquid-ordered component showed a similar, high degree of order for both sterols in the center of the bilayer, but it was higher in the coexisting disordered phase for 7-DHC. The differences detected in the models of the lipid membrane matrix are said to initiate the deleterious consequences of the Smith-Lemli-Opitz syndrome.     

Cytoplasmic domain of zebrafish myelin protein zero: adhesive role depends on beta-conformation

Solution spectroscopy studies on the cytoplasmic domain of human myelin protein zero (P0) (hP0-cyt) suggest that H-bonding between beta-strands from apposed molecules is likely responsible for the tight cytoplasmic apposition in compact myelin. As a follow-up to these findings, in the current study we used circular dichroism and x-ray diffraction to analyze the same type of model membranes previously used for hP0-cyt to investigate the molecular mechanism underlying the zebrafish cytoplasmic apposition. This space is significantly narrower in teleosts compared with that in higher vertebrates, and can be accounted for in part by the much shorter cytoplasmic domain in the zebrafish protein (zP0-cyt). Circular dichroism measurements on zP0-cyt showed similar structural characteristics to those of hP0-cyt, i.e., the protein underwent a beta-->alpha structural transition at lipid/protein (L/P) molar ratios >50, and adopted a beta-conformation at lower L/P molar ratios. X-ray diffraction was carried out on lipid vesicle solutions with zP0-cyt before and after dehydration to study the effect of protein on membrane lipid packing. Solution diffraction revealed the electron-density profile of a single membrane bilayer. Diffraction patterns of dried samples suggested a multilamellar structure with the beta-folded P0-cyt located at the intermembrane space. Our findings support the idea that the adhesive role of P0 at the cytoplasmic apposition in compact myelin depends on the cytoplasmic domain of P0 being in the beta-conformation.     

Disorder is characteristic of nerve myelin.

An X-ray diffraction pattern from the myelin in frog sciatic nerve has been obtained using the intense synchrotron radiation from the storage ring, SPEAR. Data with good statistical accuracy are obtained in a few minutes by using a scintillation counter or position-sensitive detector. The same indications for stacking disorder are seen as in previous conventional exposures which required one to two days. Thus, the stacking disorder is characteristic of myelin in a freshly dissected nerve. The present data, obtained with a more nearly monochromatic X-ray beam than in the previous study, remove one of two ambiguities which bear on the possible phasing of the higher order Bragg reflections.