THE ULTRASTRUCTURE OF LIPID-DEPLETED ROD PHOTORECEPTOR MEMBRANES

The structure of lipid-depleted retinal rod photoreceptor membranes was studied by means of electron microscopy. Aldehyde-fixed retinas were exhaustively extracted with acetone, chloroform-methanol, and acidified chloroform-methanol. The effect of prefixation on the extractability of lipids was evaluated by means of thin-layer chromatography and fatty acid analysis. Prefixation with glutaraldehyde rendered 38% of the phospholipids unextractable, while only 7% were unextractable after formaldehyde fixation. Embedding the retina in a lipid-retaining, polymerizable glutaraldehyde-urea mixture allows a comparison of the interaction of OsO₄ with lipid-depleted membranes and rod disk membranes which contain all their lipids. A decrease in electron density and a deterioration of membrane fine structure in lipid-depleted tissue are correlated with the extent of lipid extraction. These observations are indicative of the role of the lipid bilayer in the ultrastructural visualization of membrane structure with OsO₄. Negatively stained thin sections of extracted tissue reveal substructures in the lipid-depleted rod membranes. These substructures are probably the opsin molecules which are the major protein component of retinal rod photoreceptor membranes.     

Three-dimensional structure of deoxycholate-treated purple membrane at 6 Å resolution and molecular averaging of three crystal forms of bacteriorhodopsin

The three-dimensional structure of the deoxycholate-treated form of purple membrane has been determined to a resolution of about 6 Å. Using low temperature electron diffraction data, room temperature electron microscope images and improved methods of data analysis, higher resolution has been reached than was obtained using native membranes of the same size. Statistical analysis of the data shows that the new map is considerably better than earlier maps. The map indicates the probable sites for the lipid molecules that remain in the deoxycholate-treated membranes; some of these sites differ from those suggested by the projection map of Glaeser et al. (1985). Comparison of the bacteriorhodopsin structures now determined independently from three crystal forms shows that the monomer structure is independent of the detailed contacts with lipid molecules. The average of the three structures gives a picture with very little noise showing seven similar rod-like features which are clearly best interpreted as -helices; there is no indication that part of the structure is -sheet as suggested by Jap et al. (1983). Phases from the averaged structure at 6 Å resolution will enable better refinement of the parameters that will be required in the analysis of higher resolution images from tilted specimens needed to extend the projection map at 3.5 Å resolution (Henderson et al. 1986) to produce a three-dimensional atomic resolution map.     

Structural analysis of T4 DNA helix destabilizing protein (gp32 I) crystal by electron microscopy

Low dose electron diffraction and imaging techniques have been applied to the study of the crystalline structure of gp32*I, a DNA helix destabilizing protein derived from bacteriophage T4 gene 32 protein. A quantitative analysis of intensities from electron diffraction patterns from tilted, multilayered gp32*I crystal has provided the unit cell thickness of the crystal. The three-dimensional phases indicate that the space group P2(1)2(1)2. By taking into account the unit cell volume and the solvent content in the crystal, it was deduced that there is one gp32*I molecule in each asymmetric unit. A projected density map of unstained, glucose-embedded gp32*I crystal was synthesized with amplitudes from electron diffraction intensities and phases from electron images with reflections out to 7.6 A. Because of the similarity in the scattering density between glucose and protein, this projected map cannot be interpreted with certainty. A low resolution three-dimensional reconstruction shows that the protein molecule is about 90 A long and about 20 A in diameter. Because the dimer is formed around a dyad axis, the protein molecules comprising it must be arranged head-to-head. This dimeric arrangement of the proteins in the unit cell may be implicated as one of the conformational states of this protein in solution.     

Two-dimensional structure of plant light-harvesting complex at 3.7 A [corrected] resolution by electron crystallography

The structure of the light-harvesting chlorophyll a/b-protein complex has been determined at 3.7 A resolution in projection by electron diffraction, electron microscopy and image analysis. Diffraction patterns and high-resolution spotscan images of two-dimensional crystals stabilized with tannin were recorded at low temperature. Phases of structure factors were obtained directly by image processing, after correction of the images for lattice distortions, defocus and beam tilt. Amplitudes were measured by electron diffraction. The projection map shows the detailed structure of the trimeric complex, suggesting the positions of two domains of potential structural and functional homology, of one membrane-spanning alpha-helix approximately perpendicular to the membrane plane and of several tightly bound lipid molecules.     

Structure of CaATPase: electron microscopy of frozen-hydrated crystals at 6 A resolution in projection

Thin, three-dimensional crystals of CaATPase have been studied at high resolution by electron crystallography. These crystals were grown by adding purified CaATPase to appropriate concentrations of lipid, detergent and calcium. A thin film of crystals was then rapidly frozen and maintained in the frozen-hydrated state during electron microscopy. The resulting electron diffraction patterns extend to 4.1 A resolution and images contain phase data to 6 A resolution. By combining Fourier amplitudes from electron diffraction patterns with phases from images, a density map has been calculated in projection. Comparison of this map from unstained crystals with a previously determined map from negatively stained crystals reveals distinct contributions from intramembranous and extramembranous protein domains. On the basis of this distinction and of the packing of molecules in the crystal, we have proposed a specific arrangement for the ten alpha-helices that have been suggested as spanning the bilayer.     

Lipid chain dynamics in stratum corneum studied by spin label electron paramagnetic resonance

The lipid chain motions in stratum corneum (SC) membranes have been studied through electron paramagnetic resonance (EPR) spectroscopy of stearic acid spin-labeled at the 5th, 12th and 16th carbon atom positions of the acyl chain. Lipids have been extracted from SC with a series of chloroform/methanol mixtures, in order to compare the molecular dynamics and the thermotropic behavior in intact SC, lipid-depleted SC (containing covalently bound lipids of the corneocyte envelope) and dispersion of extracted SC lipids. The segmental motion of 5- and 12-doxylstearic acid (5- and 12-DSA) and the rotational correlation time of 16-doxylstearic acid (16-DSA) showed that the envelope lipids are more rigid and the extracted lipids are more fluid than the lipids of the intact SC over the range of temperature measured. The lower fluidity observed for the corneocyte envelope, that may be caused mainly due to lipid-protein interactions, suggests a major contribution of this lipid domain to the barrier function of SC. Changes in the activation energy for reorientational diffusion of the 16-DSA spin label showed apparent phase transitions around 54 degrees C, for the three SC samples. Some lipid reorganization may occur in SC above 54 degrees C, in agreement with results reported from studies with several other techniques. This reorganization is sensitive to the presence of the extractable intercellular lipids, being different in the lipid-depleted sample as compared to native SC and lipid dispersion. The results contribute to the understanding of alkyl chain packing and mobility in the SC membranes, which are involved in the mechanisms that control the permeability of different compounds through skin, suggesting an important involvement of the envelope in the skin barrier.     

Low dose electron microscopy of the crotoxin complex thin crystal

The crotoxin complex from Crotalus d. terrificus rattlesnake venom was crystallized in the form of thin platelets. These crystals were prepared by the glucose embedding technique and examined by low dose electron microscopy. Electron diffraction patterns and images have been recorded to 2.2 and 4.5 A, respectively. By a combination of electron and X-ray diffraction techniques, the space group of this crystal was determined to be P4(2)22 with eight crotoxin complex molecules in one unit cell with dimensions of 38.8 A x 38.8 A x 256.8 A. The Patterson maps and the symmetry reliability factors calculated from the electron diffraction intensities clearly showed the existence of three types of electron diffraction patterns in different crystals. The phases in the computer-calculated transform of the low dose images also show the variation in symmetry among crystals. These phenomena are explained by the presence of crystals consisting of one-half, three-quarter and one unit cell in thickness. The interpretation of the computer reconstructed two-dimensional density map was limited, partly because of the similarity in density between the protein and the embedding glucose and partly because of the non-uniqueness in relating projected structure to the three-dimensional structure.     

Preliminary X-ray crystallographic data on phospholipase C from Bacillus cereus.

Low-angle X-ray diffraction shows that, despite the well-defined regular axially projected structure, there is no long-range lateral order in the packing of molecules in native (undried) or dried elastoidin spicules from the fin rays of the spurhound Squalus acanthias. The equatorial intensity distribution of the X-ray diffraction pattern from native elastoidin indicates a molecular diameter of 1.1 nm and a packing fraction for the structure projected on to a plane perpendicular to the spicule (fibril) axis of 0.31 (the value for tendon is much higher at around 0.6). Density measurements support this interpretation. When the spicule dries the packing fraction increases to 0.43 but there is still no long-range order in the structure. The X-ray diffraction patterns provide no convincing evidence for any microfibrils or subfibrils in elastoidin. Gel electrophoresis shows that the three chains in the elastoidin molecule are identical. The low packing fraction for collagen molecules in elastoidin explains the difference in appearance between electron micrographs of negatively stained elastoidin and tendon collagen. In elastoidin, but not in tendon collagen, an appreciable proportion of the stain is able to penetrate between the collagen molecules.     

On the effect of lysophosphatidylcholine, platelet activating factor and other surfactants on calcium permeability in sarcoplasmic reticulum vesicles.

The effect of low concentrations of lysophosphatidylcholine (LPC), platelet-activating factor (PAF) and other surfactants (Triton X-100, C12E8, sodium dodecyl sulfate, sodium cholate and sodium deoxycholate) on membrane permeability of native sarcoplasmic reticulum vesicles and sarcoplasmic reticulum lipid vesicles, has been studied. Triton X-100, C12E8, sodium dodecyl sulfate, sodium cholate and sodium deoxycholate were all able to permeabilize membranes at concentrations of surfactants below their critical micellar concentration (CMC) in both lipid and native vesicles, being the K0.5 of calcium release from native vesicles lower than that from lipid vesicles. The values of these K0.5 were well correlated with the corresponding CMC values for each type of membrane. However, both LPC and PAF behaved in a different way since, although they induced permeabilization of the native vesicles at values of K0.5 close to their CMC, their K0.5 values for permeabilizing vesicles, prepared by using lipids extracted from sarcoplasmic reticulum, were much higher than their corresponding CMC.     

A 9 A two-dimensional projected structure of cholera toxin B-subunit-GM1 complexes determined by electron crystallography.

Highly ordered two-dimensional crystals of cholera toxin B-subunit pentamers have been grown by specific interaction with planar lipid films containing monosialoganglioside GM1. Electron diffractograms of frozen-hydrated crystals show diffraction peaks extending to beyond 4 A, while electron images diffract to 8 A. A two-dimensional projected structure of cholera toxin B-subunit-GM1 complex has been calculated at 9 A resolution by combining electron diffraction and image data. Crystals present an approximate pgg projection symmetry, with unit cell dimensions a = 119(+/- 1) A, b = 123(+/- 1) A, gamma = 90 degrees. Each pentameric assembly presents two concentric rings of electron scattering density, separated by an area of lower density. The outer and inner rings are centered at 25 A and and 11 A from the pentamer centre, respectively. The apparent projected density of the outer ring is larger than that of the inner ring. We propose that the outer and inner density rings correspond respectively to the peripheral beta-sheet arrangement and the central alpha-helix barrel, recently identified in the crystal structure of the heat-labile enterotoxin from Escherichia coli.     

Direct determination of crystallographic phases for diffraction data from phospholipid multilamellar arrays.

Direct determination of crystallographic phases based on probabilistic of sigma 1 and sigma 2 "triplet" structure invariants has been found to be an effective technique for structure analysis with lamellar x-ray or electron diffraction intensity data from phospholipids. In many cases, nearly all phase values are determined, permitting a structure density (electron density for x-ray diffraction; electrostatic potential for electron diffraction) map to be calculated, which is directly interpretable in terms of known bilayer lipid structure. The major source of error is found to be due to the distortion of observed electron diffraction intensity data by incoherent multiple scattering, which can significantly affect the appearance of the electrostatic potential map, but not the success of the phase determination, as long as the observed Patterson function can be interpreted.     

Structure of light-harvesting chlorophyll a/b protein complex from plant photosynthetic membranes at 7 A resolution in projection

The structure of thin three-dimensional crystals of the light-harvesting chlorophyll a/b protein complex, an integral membrane protein from the photosynthetic membrane of chloroplasts, has been determined at 7 A (1 A = 0.1 nm) resolution in projection. The structure analysis was carried out by image processing of low-dose electron micrographs, and electron diffraction of thin three-dimensional crystals preserved in tannin. The three-dimensional crystals appeared to be stacks of two-dimensional crystals having p321 symmetry. Results of the image analysis indicated that the crystals were disordered, due to random translational displacement of stacked layers. This was established by a translation search routine that used the low-resolution projection of a single layer as a reference. The reference map was derived from the symmetrized average of two images that showed features consistent with the projected structure of negatively stained two-dimensional crystals. The phase shift resulting from the displacement of each layer was corrected. Phase shifts were then refined by minimizing the phase residual, bringing all layers to the same phase origin. Refined phases from different images were in agreement and reliable to 7 A resolution. A projection map was generated from the averaged phases and electron diffraction amplitudes. The map showed that the complex was a trimer composed of three protein monomers related by 3-fold symmetry. The projected density within the protein monomer suggested membrane-spanning alpha-helices roughly perpendicular to the crystal plane. The density in the centre and on the periphery of the trimeric complex was lower than that of the protein, indicating that this region contained low-density matter, such as lipids and antenna chlorophylls.     

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.     

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 of a multifunctional protein. Mammalian phosphatidylinositol transfer protein complexed with phosphatidylcholine

Eukaryotic phosphatidylinositol transfer protein is a ubiquitous multifunctional protein that transports phospholipids between membrane surfaces and participates in cellular phospholipid metabolism during signal transduction and vesicular trafficking. The three-dimensional structure of the alpha-isoform of rat phosphatidylinositol transfer protein complexed with one molecule of phosphatidylcholine, one of its physiological ligands, has been determined to 2.2 A resolution by x-ray diffraction techniques. A single beta-sheet and several long alpha-helices define an enclosed internal cavity in which a single molecule of the phospholipid is accommodated with its polar head group in the center of the protein and fatty acyl chains projected toward the surface. Other structural features suggest mechanisms by which cytosolic phosphatidylinositol transfer protein interacts with membranes for lipid exchange and associates with a variety of lipid and protein kinases.     

Protein-Associated Lipid of Bacillus stearothermophilus

The composition and patterns of metabolism of phospholipids isolated as part of a lipid-depleted membrane fragment (LDM fragment) and associated with the membrane adenosine triphosphatase complex have been compared with those of the bulk membrane phospholipid. The bulk lipid was extracted from washed membranes with sodium cholate. The LDM fragments, which contained a portion of the electron transport system and the membrane adenosine triphosphatase complex, were purified by chromatography with Sepharose 6B. The LDM fragment preparations contained 0.10 +/- 0.02 mumol of lipid phosphorus per mg of protein, compared with 0.54 +/- 0.05 mumol of lipid phosphorus per mg of protein for washed membranes. The phospholipid associated with the LDM fragments consisted of 78 +/- 4% cardiolipin, 7 +/- 1% phosphatidylglycerol, and 15 +/- 3% phosphatidylethanolamine. Changes in the total membrane lipid composition (produced by culture conditions) did not alter the phospholipid composition of the LDM fragments. The adenosine triphosphate complex was separated from the other components of the LDM fragments by suspension of the fragments in 1% Triton X-100 and precipitation with antibody specific for the F(1) component of the adenosine triphosphatase complex. The phospholipid isolated with the adenosine triphosphatase complex consisted of 86% cardiolipin, 8% phosphatidylglycerol, and 6% phosphatidylethanolamine. In pulse-chase experiments with (32)P and [2-(3)H]glycerol, the labeling patterns of the phosphatididylglycerol and phosphatidylethanolamine associated with the LDM fragments were different from those of the bulk membrane phosphatidylglycerol and phosphatidylethanolamine. It was concluded that at least a portion of the phospholipid isolated with the LDM fragments was part of a native lipid-protein complex.     

The three-dimensional structure of the regular surface protein of Comamonas acidovorans derived from native outer membranes and reconstituted two-dimensional crystals

The three-dimensional structure of the regular surface protein (p4 symmetry, lattice constant a = b = 10.5 nm) of Comamonas acidovorans has been determined to a resolution of about 1.5 nm by means of electron microscopy and image processing. Three-dimensional reconstructions were performed using native outer membranes and artificial two-dimensional crystals of the surface protein, which was selectively solubilized by deoxycholate and recrystallized on carbon films. The two-fold symmetric morphological complex is composed of two identical monomers which are in tight contact with the outer membrane and presumably anchored to it by a small hydrophobic domain.     

脂对泛醌细胞色素c还原酶的影响

用羟基磷灰石柱亲和层析法制备了高纯度的缺脂泛醌细胞色素c还原酶．脂的缺失使该酶活力丢失,部分细胞色素（约52.8％细胞色素b和82.5％细胞色素c₁）呈现还原状态．将缺脂泛醌细胞色素。还原酶与磷脂重组,可恢复其活性,同时那些呈还原状态的细胞色素也恢复到氧化态.此结果表明如此制备的缺脂泛醌细胞色素c还原酶仍保持着活力所必需的构象状态,细胞色素氧化还原状态随脂缺失的变化反映了脂与蛋白的相互作用.     

Effects of delipidation on proton translocation and ATPase activity in beef heart electron transport particles

Delipidation of beef heart electron transport particles with phospholipase A2 has been examined. When the particles were treated with the lipase and subjected to a low bovine serum albumin wash, ATPase activity was unaffected as was the lipid/protein ratio of the particles. However, energisation by ATP/Mg2+ was abolished. Furthermore, unsaturated but not saturated fatty acids discharged the steady-state ATP-driven membrane potential of control samples. When the phospholipase A2 hydrolysis products were removed, inhibition of energy-linked reactions in the lipid-depleted particles was still observed and was interpreted in terms of non-specific leaks in the vesicle membranes, and 'specific' leaks through impaired H+-ATPase complexes. ATPase activity was less susceptible to delipidation than energisation but was, nevertheless, strongly inhibited at 50 percent lipid depletion. Spin label studies indicated a decrease in the fluidity of particle membranes accompanying delipidation. Moreover, the discontinuity seen in Arrhenius plots of ATPase activity was shifted from 17 degrees C (control) to 22 degrees C at 50 percent phospholipid depletion. The data are consistent with a release of unsaturated fatty acids by phospholipase A2 rendering the transport particles both leakier and the membranes less fluid than controls.     

Altered 5'-nucleotidase specific activity and distribution between two plasma membrane domains of ascites tumor cells with modified lipid composition

1. The cholesterol and phospholipid content of the surface membranes of ascites tumor cells cultivated in lipid-depleted medium was reduced to about 60(70)% of the control, but the relative composition of the individual phospholipids was not altered. 2. Differences in lipid composition were also observed between the two plasma membrane domains isolated from the cells cultured in normal and lipid-depleted medium respectively. 3. The fatty acid spectrum of the lipid-depleted membranes showed a greater fraction of saturated vs unsaturated acids. 4. The membrane lipid fluidity measured by fluorescence polarization was decreased in the modified surface membranes. 5. The 5'-nucleotidase specific activity was drastically reduced (46-66%) in the lipid-deleted membranes, and in addition its distribution between the two vesicle fractions was altered.