Structure of photosynthetic membranes of Euglena using X-ray diffraction

Novel X-ray diffraction results of membranes from chloroplasts of Euglena are presented, together with freeze-etch images obtained concurrently. Conditions were found for sharp lamellar reflections corresponding to ordered stacking of thylakoids. The periodicity measured by diffraction agrees well with that observed by microscopy. Intensities of diffraction were analysed in order to calculate the electron density distributions across the membranes. Some arguments in favour of the preferred phases of the reflection are given. The distributions indicate firstly the presence of 25 Å-wide regions where the hydrocarbon chains of the membrane lipids are concentrated. This result is discussed in terms of structural models for the chloroplast membrane. Comparison with results of freeze-etching indicates where in the density distribution are the regons inside and outside the membrane sacs. Secondly, the density distributions show maxima on the outside of the membranes only, corresponding possibly to an asymmetrical distribution of lipids.     

Structure of sarcoplasmic reticulum membranes at low resolution (17A)

Vesicles of fragmented sarcoplasmic reticulum membranes have been prepared and centrifuged into a multilayered form suitable for analysis by X-ray diffraction. X-ray diffraction has been recorded from a regular stacking of flattened vesicles in the presence of excess fluid. Discrete orders of a lamellar repeat distance ranging from 220 to 270 Å have been recorded. The diffraction data extend out to a minimum Bragg spacing of 33 Å. An electron density profile at a resolution of 17 Å has been derived using direct methods of structure analysis. The membrane has a bilayer construction (similar to nerve myelin and retina at low resolution) but the profile is markedly asymmetrical. The protein molecules are predominantly on the inside of the vesicle. A striking resemblance between the disc membranes in retina and the sarcoplasmic reticulum membranes has been noted and is described. X-ray diffraction has been recorded from the protein molecules in the surface of the sarcoplasmic reticulum membrane. The protein molecules are not in an ordered array but appear to have a liquid-like ordering. The observation that vesicles can be prepared in a suitable form for X-ray analysis has importance for membrane research for many different membranes form vesicles and it follows that these membranes can now be profitably studied by X-ray diffraction using a similar method.     

Gap junction structures: V. Structural chemistry inferred from X-ray diffraction measurements on sucrose accessibility and trypsin susceptibility

X-ray diffraction patterns have been recorded from partially oriented specimens of gap junctions isolated from mouse liver and suspended in sucrose solutions of different concentration and thus of different electron density. Analysis of these diffraction patterns has shown that sucrose is excluded from the 6-fold rotation axis of the junction lattice for a length of about 100 Å. This indicates that the aqueous channel of the junctions is in the closed, high resistance state in these preparations. Mapping of the sucrose-accessible space in the junction indicates that the cross-sectional area of the channel entrance on the cytoplasmic side of the membrane could be up to five times larger than the area of the transmembrane channel. Sucrose does not penetrate more than 20 Å into the membrane along the channel. Apparently the aqueous channel, 8 to 10 Å in radius for most of its length, is narrowed or blocked by a small feature about 50 Å from the center of the gap. Very close interactions exist between the gap junction protein and the lipid polar head groups on the cytoplasmic surface of the membrane. In this region, the protein intercalates between the polar head groups. These results suggest that the gap junction protein may have a functional two-domain structure. One domain, with a molecular weight of about 15,000, spans one bilayer and half of the gap and is contained largely within a radius of 25 Å from the 6-fold axis. The second domain is smaller and occupies the cytoplasmic surface of the gap junction membrane. Trypsin digestion removes about 4000 M_rmr from the cytoplasmic surface domain of the junction protein. Most of the material susceptible to trypsin digestion is located more than 28 å from the 6-fold axis.     

The structure of the purple membrane from Halobacterium hallobium: analysis of the X-ray diffraction pattern.

An X-ray diffraction analysis of oriented specimens of the purple membrane from Halobacterium halobium shows that the protein and lipid components are packed in a P3 hexagonal lattice, with one protein molecule per asymmetric unit. The structure is made up of a single layer of the protein molecules, oriented vectorially in the same direction across the membrane.The presence of strong diffraction peaks equatorially centred at 10 Å, and axially at 5 Å and 1.5 Å, show that the protein molecules, which make up most of the mass of the membrane, are composed to a considerable extent of α-helices, 25 to 35 Å long, arranged roughly perpendicular to the plane of the membrane to form superhelical groupings of the “coiled-coil” type.The surface of the membrane is flat, with no bumps or dimples large enough to affect the X-ray pattern when the electron density of the suspending medium is altered. The phospholipids may be less exactly positioned in the lattice than the protein, since the presence of uranyl acetate, which is expected to co-ordinate with the acidic phosphate groups, produces intensity changes only at low resolution.     

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.     

Two-dimensional lattices of porin diffract to 6 Å resolution

Porin (the product of gene ompF) is an integral membrane protein (M_r 36 500) of the outer membrane of Escherichia coli (strain B^E). The protein has been purified to homogeneity and reconstituted in dimyristoyl-lecithin. Oriented specimen on a flat surface yielded X-ray diffraction pattern, originating from the two-dimensional protein lattice, to a resolution reaching 6 Å. Although these powder rings are broad compared to corresponding diffraction patterns from purple membranes of Halobacterium halobium, porin is the first reconstituted integral membrane protein which shows diffraction to this resolution.     

The reaction center profile structure derived from neutron diffraction

Both reaction center protein from the photosynthetic bacteria Rhodopseudomonas sphaeroides and egg phosphatidylcholine can be deuterium labelled; the reaction center protein can be incorporated into the phosphatidylcholine bilayers forming a homogeneous population of unilamellar vesicles. The lipid profile and the reaction center profile within these reconstituted membrane profiles were directly determined to 32 Å resolution using lamellar neutron diffraction from oriented membrane multilayers containing either deuterated or protonated reaction centers, and either deuterated or protonated phosphatidylcholine. The 32 Å resolution reaction center profile shows that the protein spans the membranes, and has an asymmetric mass distribution along the perpendicular to the membrane plane. These results were combined with previously described X-ray diffraction results in order to extend the resolution of the derived reaction center profile to 9 Å.     

Electron density levels of sarcoplasmic reticulum membranes

Low-angle X-ray diffraction has been recorded from oriented preparations of sacroplasmic reticulum membranes in fluid media containing glycerol solutions in different concentrations. Discrete diffraction orders of a lamellar repeat distance ranging from 200 Å to 250 Å have been recorded. Fourier synthesis at a resolution of 17 Å for 0, 10, 20, and 30% glycerol-treated sarcoplasmic reticulum membranes are described. An electron density scale in electrons/$\text{A}\text{?}{}^3$ for these Fourier syntheses has been determined. The question of the correctness of our asymmetric electron density profile for the sarcoplasmic reticulum membrane is critically examined. A study is made on the choice of phases and on the method used to process the X-ray intensities.     

Molecular structure determination by electron microscopy of unstained crystalline specimens.

The projected structures of two unstained periodic biological specimens, the purple membrane and catalase, have been determined by electron microscopy to resolutions of 7 Å and 9 Å, respectively. Glucose was used to facilitate their in vacuo preservation and extremely low electron doses were applied to avoid their destruction.The information on which the projections are based was extracted from defocussed bright-field micrographs and electron diffraction patterns. Fourier analysis of the micrograph data provided the phases of the Fourier components of the structures; measurement of the electron diffraction patterns provided the amplitudes.Large regions of the micrographs (3000 to 10,000 unit cells) were required for each analysis because of the inherently low image contrast (<1%) and the statistical noise due to the low electron dose.Our methods appear to be limited in resolution only by the performance of the microscope at the unusually low magnifications which were necessary. Resolutions close to 3 Å should ultimately be possible.     

Bacteriorhodospin: a trans-membrane pump containing alpha-helix.

The probable arrangement of the bacteriorhodopsin molecules in the purple membrane of Halobacterium halobium is in clusters of three, with a 3-fold axis at the centre of each cluster; the axis is at right angles to the plane of the membrane. The proposed arrangement and the results of model calculations together indicate that each protein molecule spans the entire thickness of the membrane. An earlier proposal for the structure had the protein molecules in two layers, and it was symmetric in projection onto the profile-axis. This model is now rejected since it would be difficult to account for the recently discovered function of pumping protons. There remains a discrepancy in that the calculated number of protein molecules in the unit-cell is 3.4 compared to the three expected.The X-ray diffraction patterns from dispersions of the lipids extracted from the red and purple membranes of H. halobium are described.Model calculations are reported, which are based on the bilayer profile calculated for the extracted lipids and on two simple profiles for the protein. The calculations favour a structure for the purple membrane having the lipid molecules in two layers, as in a bilayer, although there may be more of the lipid on one side of the membrane than on the other. Assuming bilayer structure, the diffraction nearest the centre of the oriented pattern suggests that the lipid molecules may be located mainly in a few discrete regions, roughly 20 Å across, between the protein molecules. An uninterrupted monolayer of the lipid on one surface of a sheet of the protein molecules gives poor agreement with the observed profile-diffraction.The X-ray diffraction pattern from the oriented membranes suggested α-helix in the bacteriorhodopsin, and this has been confirmed by recording a 1.5 Å-reflection oriented on the profile-axis. There appear to be at least two segments of α-helix, which are somewhat inclined to one another, and the two may be packed together. Prominent diffraction on the in-plane axis near 10 Å is consistent with the segments lying more or less perpendicular to the plane of the membrane.     

Erratum

Escherichia coli cells (unsaturated fatty acid auxotroph) have been adapted to grow on branched-chain fatty acids. Membrane vesicles were isolated from cells grown on a mixture of branched-chain fatty acids isolated from the lipids of Bacillus subtilis (E. coli (B. subtilis) membranes) and on a pure synthetic anti-isononadecanoic acid (E. coli (aC19) membranes).We have shown, using wide-angle X-ray diffraction and differential scanning calorimetry, that the ordered state of the lipids is perturbed in the case of E. coli (aC19) membranes. The perturbation leads to the presence of a large wide-angle X-ray diffraction at 4.25–4.3 Å, as opposed to the presence of a sharp 4.2 Å reflection in unperturbed systems.We have shown, using freeze-fracture electron microscopy, that a protein segregation exists in the case of E. coli (aC19) membranes (at low temperature the integral membrane proteins aggregate in the membrane domains containing the disordered lipids); we do not observe such segregation in the case of E. coli (B. subtilis) membranes. We conclude that in cases where the branching of the fatty acids introduces a perturbation of the lipid order, the integral membrane proteins can still be accommodated in membrane domains containing the ‘perturbed’ ordered lipids.Finally, we have determined the rate of β-galactoside transport in E. coli (aC19) and E. coli (B. subtilis) membranes as a function of temperature. We have shown that, in both cases, the Arrhenius representations display an increased slope in the region of the disorder-to-order transition. We conclude that such an increased slope may have different origins. In the case of E. coli (aC19) membranes, it is the result of the aggregation of the β-galactoside carriers together with other integral membrane proteins which may lead to the inactivation of the carriers; in the case of E. coli (B. subtilis) membranes, it is the result of the partial immobilisation of the carriers embedded in a lipid environment, of which the fluidity, despite the perturbation of its lipid order, is still much less than that associated with lipids in a totally disordered state.     

A 12 Å resolution X-ray diffraction study of the profile structure of isolated bovine retinal rod outer segment disk membranes

Electron density profiles of disk membranes isolated from bovine retinal rod outer segments have been determined to 12 Å resolution by analysis of the X-ray diffraction from oriented multilayers, in the absence of lipid phase separation. Data were collected on both film and a two-dimensional TV-detector; both detectors yielded identical patterns consisting of relatively sharp lamellar reflections of small mosaic spread. The unit cell repeat was reversibly varied over the range of 143 to 183 Å. The diffraction patterns changed dramatically at 150 Å; consequently, the low (less than 150 Å) and high (greater than 150 Å) periodicity data were independently analyzed via a swelling algorithm. The high periodicity data yielded two statistically equivalent phase choices corresponding to two symmetric, but different membrane profiles. The low periodicity data yielded essentially one, characteristically asymmetric profile. These profiles have been modeled with regard to the separate profiles of rhodopsin, lipid and water, subject to the known composition of the isolated disk membranes.     

Structure of molecular aggregates of 1-(3-sn-phosphatidyl)-l-myo-inositol 3,4-bis(phosphate) in water

The molecular organization of 1-(3-sn-phosphatidyl)-l-myo-inositol 3,4-bis-(phosphate)/water systems is investigated over a wide range of lipid concentrations using X-ray diffraction, calorimetry, analytical ultracentrifugation, densitometry and viscometry.At high lipid concentrations, the lipid molecules are found to form a lamellar phase. The repeat distance increases from 60 to 120 Å with increasing water content to 70 wt% and the surface area per lipid molecule increases from 41.7 Ų to a limiting value of 100 Ų.On the other hand, at very low lipid concentrations the molecules are found to form not vesicles but micelles, the total molecular weight of which takes a value of 93 000.This finding revises the prevalent view that lipids containing two (or more) hydrocarbon chains form extended bilayers or vesicles, whereas single chained lipids form micelles. (Tanford, C.(1972) J. Phys. Chem. 76, 3020–3024).     

Structure of polar pili from Pseudomonas aeruginosa strains K and O

The polar pili of Pseudomonas aeruginosa strains K and O are hollow cylinders with 52 Å outer diameter and 12 Å inner diameter. There is a girdle of low electron density (interpreted as due to a local concentration of hydrophobic amino acid side-chains) centred at 31 Å diameter. Similar X-ray diffraction patterns are obtained from oriented fibres of the two types of pili, to a resolution of 7 Å in the equatorial direction and 4 Å in the meridional direction. The two types of pilin protein subunits have a similar molecular weight, and their sequences contain a number of homologous regions. They form a helical array with 4.06 to 4.08 units per turn of a basic helix that has a pitch of 40.8 Å for strain K pili and 41.3 Å for strain O pili at 75% relative humidity. A method is described for distinguishing between very similar diffraction patterns.There is strong intensity at 10 Å near the equator and at 5 Å near the meridian on the diffraction patterns. This intensity distribution is characteristic of α-helical rods running roughly in the direction of the fibre axis. The orientation of these rods was established by the fit between the transform of an α-helical polyalanine model and the strong near-equatorial layer-line.     

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 °C. Non-lamellar phases are not observed in erythrocytes or detergent-resistant membrane preparations heated up to 55 °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.     

X-ray scattering studies of heavy atom bound erythrocyte membrane suspensions

We have observed a periodic intensity variation of scattered x-rays from uranyl labeled erythrocyte membranes. Using unique x-ray scattering methods, we have made these measurements from membrane suspensions in which the vesicles appear, by phase contrast microscopy, to be normal in shape. The periodic intensity variation is not present for membranes labeled on one side only. The frequency of the variation permits calculation of membrane width, which we find to be 55Å.     

Low resolution structure of adenylate kinase

A low resolution model of adenylate kinase has been derived from a 6 Å electron density map. The molecular shape can be described approximately as an oblate ellipsoid with dimensions 40 Å × 40 Å × 30 Å. The molecule is composed of two globular units separated by a 10 Å deep cleft. In contrast to the bigger unit, the smaller globule appears to contain a high amount of α-helical structure. The location of the active centre is discussed.The crystals used for X-ray diffraction analysis belong to one of the enantiomorphic trigonal space groups P3₁21 or P3₂21, with one molecule in the asymmetric unit. The phase determination was based on four isomorphous heavy atom derivatives. Frequent transitions between different crystal forms complicate the analysis.     

the stability in various detergents of transferrin-transferrin receptor complexes from reticulocyte plasma membranes

Transferrin-membrane protein complexes were solubilized either with 0.4% sodium dodecyl sulfate (SDS), 1% Triton X-100 or 0.5% sulfobetaine 3-14 from the plasma membranes of rabbit reticulocytes previously labeled with 125I and then incubated with 131-labeled transferrin. When the solubilized membranes were analyzed by gel filtration fractionation, marked variation in the preservation of transferrin-transferrin receptor interaction was noted between the three detergents. After SDS solubilization, more than 80% of the 131I-labeled transferrin remained associated with membrane proteins with apparent molecular weight of the transferrin-receptor complexes of 1400 000 and 240 000. In contrast, after Triton X-100 solubilization only 40% of the transferrin was still complexed to membrane proteins with an apparent molecular weight of the complex of 450 000. Dissociation of transferrin from its receptor was most marked following sulfobetaine solubilization, with less than 30% of the transferrin still complexed. Following gel filtration 131I-labeled transferrin-125I-labeled membrane protein complexes were immunoprecipitated with goat specific anti-rabbit transferrin antibodies. The immunoprecipitates were analyzed under stringent dissociating conditions by two SDS-polyacrylamide gel electrophoretic techniques. In a linear 5-25% polyacrylamide gradient the 125I-labeled receptor obtained after membrane solubilization with all three detergents had an apparent molecular weight of 80 000. In contrast, in a different system using 10% polyacrylamide gel two 125I-labeled receptor components were detected wih apparent molecular weights of 90 000 and 80 000. These results demonstrate that estimates of the molecular weight of the transferrin receptor depended on the conditions of electrophoresis and suggest that the transferrin receptor is partially modified, perhaps by glycosylation.     

Purification and crystallization of a complex between human interferon γ receptor (extracellular domain) and human interferon γ

X-ray diffraction quality crystals have been obtained from a complex between interferon γ and the extracellular domain of its high-affinity cell surface receptor. The crystals were obtained from interferon γ/interferon γ receptor complexes purified by size exclusion chromatography. Diffraction quality crystals required analyzing these complex samples by isoelectric focusing gels to select purified complex fractions devoid of unbound interferon γ. These studies used interferon γ receptor engineered with an eight amino acid N-terminal deletion to eliminate heterogeneity generated due to proteolytic cleavage. In addition, the receptor was expressed in an E. coli secretion cell line which eliminated the need to refold the protein. Hexagonal crystals were grown from 1.6 M ammonium phosphate solutions and belong to a spacegroup of P6₅22 with unit cell dimensions a = 145.9 Å and c = 180.3 Å. These crystals diffract to at least 2.9 Å resolution when exposed to synchrotron radiation. SDS PAGE analysis of the crystals demonstrated that both interferon γ and the receptor were present. Analysis of the x-ray diffraction data revealed that the crystals contain complexes with a stoichiometry of 2:1 receptor: ligand within the crystallographic asymmetric unit and consist of approximately 55% solvent. © 1996 Wiley-Liss, Inc.     

An ordered membrane-cytoskeleton network in squid photoreceptor microvilli

To study the organization of microvilli in the photoreceptor cells of an invertebrate. X-ray diffraction patterns were obtained from aldehyde-fixed squid retinas to a resolution of (40 Å)^?1 and correlated with results from electron microscopy and sodium dodecyl sulphate/polyacrylamide gel electrophoresis. Squid photoreceptor microvilli are packed in extensive hexagonal arrays; in addition each microvillus has a hexagonal substructure. Image reconstruction from thin section electron micrographs shows that the microvilli are linked together with specialized membrane junctions at their neighbour contacts, and phosphotungstic acid-stained sections show a central cytoskeleton connected to the membrane by side-arms.The X-ray patterns also reveal two axial periodicities in the microvilli. A weak and diffuse (50 Å)^?1 band is tentatively assigned to rhodopsin molecules ordered in the plane of the membrane. In addition, an arc at (85 Å)^?1 is attributed to a cytoplasmic or extracellular structure.Sodium dodecyl sulphate/polyacrylamide gel electrophoresis of the isolated microvilli shows that the major component, rhodopsin, comprises about 50% of the total protein. There are two major detergent-insoluble polypeptides with molecular weights of 145,000 and 42,000. The 42,000 component is identified as actin by papain digestion fragment mapping.Cephalopod photoreceptors are highly sensitive to the polarization vector of linearly polarized light. In consequence, the linear rhodopsin chromophores must be aligned relative to the microvillar axes. The membrane junctions and cytoskeleton described here may provide a mechanism for maintaining this rhodopsin alignment.