X-ray diffraction from oriented outer mitochondrial membranes: Detection of in-plane subunit structure

X-ray diffraction patterns from ultracentrifugally oriented specimens of plant outer mitochondrial membranes show five distinct maxima in the equatorial direction. These diffraction maxima arise from in-plane subunits whose dimensions are consistent with those of the features (“pits”) seen in electron micrographs of the membranes in negative stain.     

X-ray diffraction from oriented outer mitochondrial membranes. Detection of in-plane subunit structure.

X-ray diffraction patterns from ultracentrifugally oriented specimens of plant outer mitochondrial membranes show five distinct maxima in the equatorial direction. These diffraction maxima arise from in-plane subunits whose dimensions are consistent with those of the features ("pits") seen in electron micrographs of the membranes in negative stain.     

Comparative X-ray studies on the interaction of carotenoids with a model phosphatidylcholine membrane

The interaction of structurally different carotenoids with a membrane molecular model was examined by X-ray diffraction. The selected compounds were beta-carotene, lycopene, lutein, violaxanthin, zeaxanthin, and additionally carotane, a fully saturated derivative of beta-carotene. They present similarities and differences in their rigidity, the presence of terminal ionone rings and hydroxy and epoxy groups bound to the rings. The membrane models were multibilayers of dipalmitoylphosphatidylcholine (DPPC), chosen for this investigation because the 3 nm thickness of the hydrophobic core of its bilayer coincides with the thickness of the hydrophobic core of thylakoid membranes and the length of the carotenoid molecules. Results indicate that the six compounds induced different types and degrees of structural perturbations to DPPC bilayers in aqueous media. They were interpreted in terms of the molecular characteristics of DPPC and the carotenoids. Lycopene and violaxanthin induced the highest structural damage to the acyl chain and polar headgroup regions of DPPC bilayers, respectively.     

On the structure of agglutinated sheep red blood cell membranes

Specimens of isolated sheep red blood cell membranes are prepared by an agglutination technique in which membranes are stacked in regular arrays. X-ray diffraction patterns are recorded from such specimens which show meridional and equatorial diffraction phenomena. The meridional reflections correspond to single lamellar repeat periods of 160–186 Å. It is concluded that two asymmetric membranes are contained inthe elementary period. Lipid phases with preferentialyl oriented hydrocarbon chains are part of the membrane structure. The stacking of membranes is also demonstrated in the electron microscope. The X-ray scattering curve of intracellular hemoglobin of intact sheep red blood cells is recorded to a spacing of about 8 Å^?1. The broad diffraction rings of this scattering curve are replaced by a series of rather sharp rings, when the red blood cells are agglutinated and placed in a hypertonic medium. Both the presence of a functioning membrane and the agglutination appear to be essential for the full expression of this phenomenon.     

On the structure of agglutinated sheep red blood cell membranes.

Specimens of isolated sheep red blood cell membranes are prepared by an agglutination technique in which membranes are stacked in regular arrays. X-ray diffraction patterns are recorded from such specimens which show meridional and equatorial diffraction phenomena. The meridional reflections correspond to single lamellar repeat periods of 160-186 A. It is concluded that two asymmetric membranes are contained in the elementary period. Lipid phases with preferentially oriented hydrocarbon chains are part of the membrane structure. The stacking of the membranes is also demonstrated in the electron microscope. The X-ray scattering curve of intracellular hemoglobin of intact sheep red blood cells is recorded to a spacing of about 8 A-1. The broad diffraction rings of this scattering curve are replaced by a series of rather sharp rings, when the red blood cells are agglutinated and placed in a hypertonic medium. Both the presence of a functioning membrane and the agglutination appear to be essential for the full expression of this phenomenon.     

Interpretation of the absorption and circular dichroic spectra of oriented purple membrane films.

The absorption and circular dichroic (CD) spectra of purple membrane films in which the plane of the membranes is oriented perpendicular to the incident beam are compared with the solution spectra. This enables one to relate structural features of the purple membrane to a coordinate system as defined by a normal to the membrane plane and two mutually perpendicular in-plane axes. The film and solution absorption spectra were similar except for a relative depression in the 200 - 225-nm region of the film spectrum. However, the CD spectra showed significant differences in the visible region, where the biphasic band in the solution spectrum was replaced by a single positive band at 555 nm in the film spectrum and in the far ultraviolet region, where the 208-nm band was deleted from the film spectra of the native and regenerated membranes. Moreover, a small shoulder occurred at 208 nm in the film spectrum of the bleached membrane. The near ultraviolet spectra also showed differences, whereas the 317-nm band remained essentially the same for both spectra. Based on excitonic interpretations of the visible and far ultraviolet spectra the following conclusions were reached: (a) a relatively strong in-plane monomeric interaction occurs between te retinyl chromophore and apoprotein; (b) the helical axes of the native and regenerated membrane proteins are oriented primarily normal to the membrane plane; and (c) the helical axes of the bleached membrane proteins are tilted more in-plane than the axes of the native or regenerated membrane. Additional conclusions were that an interaction occurs between an in-plane magnetic dipole moment of the retinyl chromophore and probably an in-plane electric dipole moment of a nearby aromatic amino acid(s), and that although the membrane is anisotropic with respect to coupling between electric and magnetic moments of the aromatic amino acids, the transition dipole moments of the aromatic amino acids are not preferentially oriented in either direction.     

Location of the carboxyl terminus of bacteriorhodopsin in purple membrane.

Purple membrane samples have been prepared by trypsin digestion to have either 10 or 21 residues removed from the carboxyl terminus of the proteins. Electron diffraction of single membranes and x-ray diffraction of unoriented membrane pellets have been carried out on both these specimens and on native purple membranes. the main conclusion from this work is that the carboxyl terminus is almost entirely disordered, being free to take up many positions, and that its removal does not affect the packing in the crystal. The low resolution x-ray diffraction difference map may also suggest the approximate location of the carboxyl terminus.     

Fine structural studies on the digestion of chloroplasts in the rumen ciliate Entodinium caudatum

The rumen ciliate Entodinium caudatum engulfed both native and glutaraldehyde-fixed chloroplasts, which were then usually found in vesicles located in the protozoal endoplasm. The native chloroplasts lost their characteristic morphological appearance in less than 5 min. The grana stacks disappeared and the thylakoid membranes were altered and appeared as many single rings or as concentric rings of membranous material resembling myelin figures. The membranes were shown to be of chloroplast rather than protozoal origin.     

Retinal location in purple membrane of Halobacterium halobium: a neutron diffraction study of membranes labelled in vivo with deuterated retinal

Purple membranes were prepared by growing Halobacterium halobium in a medium containing nicotine (which inhibits biosynthesis of retinal) and the oxidation products of fully deuterated beta-carotene. This allowed the in vivo incorporation of deuterated retinal into the membranes. The labelled membranes were crystalline and isomorphous with native membrane as determined by X-ray diffraction, and their optical absorption spectra were very similar. Neutron diffraction data for the two dimensional in-plane lattice from labelled and native membranes were analysed by difference Fourier and direct methods to 8.6 A resolution. The difference Fourier shows the retinal to be located in the centre of the bacteriorhodopsin molecule. The best fit to the data was obtained with the projection of retinal as a 10 A long rod forming an angle of -40 degrees +/- 10 degrees with the x axis centred at x = -0.19 +/- 0.02, y = -0.35 +/- 0.02 in fractional unit cell coordinates. The main peak in the difference Fourier map is at x = -0.17, y = -0.33.     

Electron microscopic studies of carboxysomes of Thiobacillus neapolitanus

The outer part of the carboxysomes of Thiobacillus neapolitanus was examined by electron microscopy using negatively stained, cryo-treated, frozen hydrated and freeze dried specimens. From stereo-micrographs of freeze dried and fixated carboxysomes the three dimensional structure of the carboxysomes was elucidated. The carboxysomes always appear as hexagonal bodies, which possess twelve pentameric planes. This indicates that carboxysomes have the form of a pentagonal dodecahedron. Inside the carboxysomes the ribulose-1,5-bisphosphate carboxylase molecules are arranged in rows and concentric rings. Negatively stained and cryo-treated carboxysomes do not differ significantly in size. The mean size of these carboxysomes is 117.3±6.9 nm (n=782)     

A ring-shaped structure with a crown formed by streptolysin O on the erythrocyte membrane.

Streptolysin O (SLO) is a membrane-damaging toxin produced by most strains of group A beta-hemolytic streptococci. We performed ultrastructural analysis of SLO-derived lesions on erythrocyte membranes by examining electron micrographs of negatively stained preparations. SLO formed numerous arc- and ring-shaped structures with or without holes on membranes. Rings formed on intact cell membranes had an inner diameter of ca. 24 nm and had distinct borders of ca. 4.9 nm in width, but the diameter of rings varied from 24 to 30 nm on membranes of erythrocyte ghosts. Image analysis of electron micrographs demonstrated that each ring was composed of an inner and an outer layer. Each layer contained an array of 22 to 24 SLO molecules. On the top of the ring, we found a characteristic crown that projected from the cell membrane. The crown was separated by an electron-dense layer from the basal part of the ring that was embedded in the lipid bilayer of the erythrocyte membrane. Heights of the three parts, namely, the crown (head), the space (neck), and the basal portion (base), were ca. 3.2, 1.6, and 5.0 nm, respectively, and we postulated that these parts are the constituents of a single SLO molecule. The volumes of SLO molecules in the inner and outer layers were calculated to be 77 and 88 nm3. On the basis of a model of the structure of SLO, we propose some new details of the mechanisms of hemolysis by SLO toxin.     

Membrane-associated components of the bacterial flagellar apparatus

At the position of insertion of the flagellum into the Gram-negative bacterial cell envelope, a specialized membrane differentiation has been observed by electron microscopy. This structure, termed concentric membrane rings, is harboured on the under-side of the outer membrane of Spirillum serpens, and forms a plate-like array of up to seven rings (diameter 90 nm) and an interior supporting collar. The concentric membrane rings are sensitive to proteolytic digestion, but are lysozyme and phospholipase resistant. The structures are disrupted by ionic detergents, yet resistant to the action of non-ionic detergents. A model integrating the basal organelle of the bacterial flagellum and the outer membrane of the cell wall is presented.     

Electron diffraction studies of human erythrocyte membrane and its lipid extracts. Effects of hydration, temperature and hydrolysis.

The organization of lipid molecules in individual human erythrocyte ghost membranes and single bilayers of their total lipid extracts were studied by low-dose electron diffraction in a controlled environment. The highest onset temperature (Ts) at which diffraction rings corresponding to a gel state appeared, were found to be in the range of -2 to -4 degrees C for both the whole ghost membrane and bilayers of its total lipid extracts. The onsets were abolished by dehydration before separated crystallizations of cholesterol and phospholipid occurred. Ts increased as a result of free fatty acids accumulation in membranes after phospholipase A2 treatment or storage.     

Membrane associated components of the bacterial flagellar apparatus

At the position of insertion of the flagellum into the Gram-negative bacterial cell envelope, a specialized membrane differentiation has been observed by electron microscopy. This structure, termed concentric membrane rings, is harboured on the under-side of the outer membrane of Spirillum serpens, and forms a plate-like array of up to seven rings (diameter 90 nm) and an interior supporting collar. The concentric membrane rings are sensitive to proteolytic digestion, but are lysozyme and phospholipase resistant. The structures are disrupted by ionic detergents, yet resistant to the action of non-ionic detergents. A model integrating the basal organelle of the bacterial flagellum and the outer membrane of the cell wall is presented.     

X-ray diffraction and electron microscopy studies of frozen erythrocyte membrane preparations

Well-defined X-ray diffraction patterns have been recorded from erythrocyte membranes in the frozen state. At ?40°C, lamellar periodicities range from 19 to 95 nm depending on the glycerol content (0–40%, respectively). Freeze-fracture electron micrographs of samples frozen in two stages to approximate to the diffraction conditions show ice formation external to membrane stacks. The membrane stacks have periodicities of the same order of magnitude as those obtained by X-ray diffraction.     

Imaging the membrane protein bacteriorhodopsin with the atomic force microscope.

The membrane protein bacteriorhodopsin was imaged in buffer solution at room temperature with the atomic force microscope. Three different substrates were used: mica, silanized glass and lipid bilayers. Single bacteriorhodopsin molecules could be imaged in purple membranes adsorbed to mica. A depression was observed between the bacteriorhodopsin molecules. The two dimensional Fourier transform showed the hexagonal lattice with a lattice constant of 6.21 +/- 0.20 nm which is in agreement with results of electron diffraction experiments. Spots at a resolution of approximately 1.1 nm could be resolved. A protein, cationic ferritin, could be imaged bound to the purple membranes on glass which was silanized with aminopropyltriethoxysilane. This opens the possibility of studying receptor/ligand binding under native conditions. In addition, purple membranes bound to a lipid bilayer were imaged. These images may help in interpreting results of functional studies done with purple membranes adsorbed to black lipid membranes.     

Structure and thermotropic phase behaviour of detergent-resistant membrane raft fractions isolated from human and ruminant erythrocytes

Detergent-resistant membrane raft fractions have been prepared from human, goat, and sheep erythrocyte ghosts using Triton X-100. The structure and thermotropic phase behaviour of the fractions have been examined by freeze-fracture electron microscopy and synchrotron X-ray diffraction methods. The raft fractions are found to consist of vesicles and multilamellar structures indicating considerable rearrangement of the original ghost membrane. Few membrane-associated particles typical of freeze-fracture replicas of intact erythrocyte membranes are observed in the fracture planes. Synchrotron X-ray diffraction studies during heating and cooling scans showed that multilamellar structures formed by stacks of raft membranes from all three species have d-spacings of about 6.5 nm. These structures can be distinguished from peaks corresponding to d-spacings of about 5.5 nm, which were assigned to scattering from single bilayer vesicles on the basis of the temperature dependence of their d-spacings compared with the multilamellar arrangements. The spacings obtained from multilamellar stacks and vesicular suspensions of raft membranes were, on average, more than 0.5 nm greater than corresponding arrangements of erythrocyte ghost membranes from which they were derived. The trypsinization of human erythrocyte ghosts results in a small decrease in lamellar d-spacing, but rafts prepared from trypsinized ghosts exhibit an additional lamellar repeat 0.4 nm less than a lamellar repeat coinciding with rafts prepared from untreated ghosts. The trypsinization of sheep erythrocyte ghosts results in the phase separation of two lamellar repeat structures (d=6.00; 5.77 nm), but rafts from trypsinized ghosts produce a diffraction band almost identical to rafts from untreated ghosts. An examination of the structure and thermotropic phase behaviour of the dispersions of total polar lipid extracts of sheep detergent-resistant membrane preparations showed that a reversible phase separation of an inverted hexagonal structure from coexisting lamellar phase takes place upon heating above about 30 degrees C. Non-lamellar phases are not observed in erythrocytes or detergent-resistant membrane preparations heated up to 55 degrees C, suggesting that the lamellar arrangement is imposed on these membrane lipids by interaction with non-lipid components of rafts and/or that the topology of lipids in the erythrocyte membrane survives detergent treatment.     

Orientations of the retinyl and the heme chromophores in the brown membrane of Halobacterium halobium

The orientations of the retinyl and heme chromophores of bacteriorhodopsin and cytochrome b-561 of the brown membrane of Halobacterium halobium have been determined by linear dichroic spectroscopy of oriented brown membrane films. Both chromophores exhibit cylindrical symmetry with respect to the membrane normal. However, the retinyl transition dipole moment is polarized at an angle of 20 to 24 ° with respect to the plane of the membrane while the plane of the heme is oriented nearly perpendicular to the membrane plane. Therefore, the orientation of retinal bound to bacterio-opsin in the brown membrane is approximately the same as in the purple membrane. This is supportive of our previous conclusions that the fine structures of the bacteriorhodopsins of these membranes are very similar in spite of differences in the composition and structure of the two membranes. The orientation of the heme plane of the membrane-bound cytochrome b-561 is very similar to orientations of several membrane-bound heme proteins that are involved in electron transfer processes and may be suggestive of its function in the brown membrane. Analysis of the linear dichroic spectrum over the entire bacteriorhodopsin band using an exciton formalism is in accord with the energy separation of the in-plane and out-of-plane excitonic transitions being less than 5 nm. Since a similar energy separation was reported for the purple membrane, the relative positions of the retinals must be approximately the same in both membranes. A similar analysis of the Soret region, based on the existence of two degenerate mutually perpendicular porphyrin transitions, indicates that the energy separation should be from 5 to 20 nm. However, the smaller value is unlikely for it would imply very large circular dichroic bands not yet encountered in any heme proteins.     

Ultrastructure of the concentric membrane system in Arthrinium aureum

An ultrastructural study was performed on Arthrinium aureum. The fungi were treated with glutaraldehyde and osmium tetroxide fixation. The hypha and conidia has a concentric membrane system which consisted of multiple membranes of a myelinoid appearance, and continued to the conidia and hypha plasma membrane. The fungi were also treated with periodic acid-alkaline bismuth (PABi) staining after glutaraldehyde and osmium tetroxide fixation. PABi positive materials were found on the marginal glycogen granules, the concentric membrane system and the conidia plasma membrane.     

Lipid membrane structure and interactions in dimethyl sulfoxide/water mixtures.

In this paper we have investigated via x-ray diffraction the influence of dimethyl sulfoxide (DMSO), known for its biological and therapeutic properties, on the structure of lipid membranes of dipalmitoylphosphatidylcholine (DPPC) in excess of the solvent (DMSO/water) at mole DMSO fractions XDMSO in (0.1) and under equilibrium conditions. At small XDMSO </= 0.133 the repeat distance d is reduced remarkably, whereas wide-angle x-ray diffraction pattern remains almost unchanged with the increase in XDMSO. It agrees well with previous study (Yu and Quinn, 1995). At 0.133 < XDMSO < 0.3 the repeat period d reduces slowly; however, an orthorombic in-plane lattice of hydrocarbon chains transfers to a disordered quasihexagonal lattice. The increase in XDMSO from 0.3 up to approximately 0.9 leaves d almost unchanged, whereas it leads to less disordered packing of hydrocarbon chains. At XDMSO approximately 0.9, Lbeta' phase transfers into interdigitated phase. The chain-melting phase transition temperature of DPPC membranes increases by several degrees with the increase of DMSO concentration. It points to a strong concentration-dependent solvation of membrane surface by DMSO. Thus DMSO strongly interacts with the membrane surface, probably displacing water and modifying the structure of the lipid bilayer. It appears to determine some of the properties of DMSO as a biologically and therapeutically active substance.