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1.
We present a new method for the determination of structural parameters in biological membranes. Recording the continuous scattering of heavy-atom labeled membranes and applying elementary Fourier methods we obtain the scattering of the heavy-atom distribution alone. The details of this distribution are explored by developing a simple model and testing for cases relevant to biological membranes. We find that the intensity distribution is highly sensitive to many key parameters. The increased signal from heavy-atom labeling and the use of an improved x-ray system make it possible to record patterns from dilute membrane suspensions. Thus determination of these parameters is possible in the same environment where many membrane biochemical studies are performed. Application of the method is made to a model lipid bilayer membrane, dipalmitoyl phosphatidylcholine by labeling with UO2++ ions. We determine the precise distance between UO2++ layers on either side of the membrane as well as the width of the label on each side. This determination permits estimation of phosphate separation across single labeled bilayers in an aqueous suspension. 相似文献
2.
Differential polarization imaging. V. Numerical aperture effects and the contribution of preferential scattering and absorption to the circular dichroism images.
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Chiral objects which scatter and absorb preferentially left versus right circularly polarized light give rise to bright-field circular dichroism (CD) images containing contributions from both these two phenomena. These contributions are separated and characterized mathematically, and the effect of the dimensions of the chiral object on their relative magnitude is discussed. CD images of the long-range chiral organization of the thylakoid membranes in chloroplasts are obtained at two different wavelengths to illustrate the diverse wavelength dependence of the preferential absorption and scattering contributions to the images. The bright field CD images not only depend on the magnitude and sign of the preferential scattering and preferential absorption contributions, but also on the numerical aperture of the lens used. This dependence is obtained formally and a method to extract the angle dependent preferential scattering contributions to the images is presented. The validity of this method is confirmed experimentally. 相似文献
3.
W Lesslauer 《Biochimica et biophysica acta》1976,436(1):25-37
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. 相似文献
4.
A method for structural analysis of biological membranes using neutron scattering from suspensions is described and applied to photosynthetic membranes from bacteria. The variation of scattering density across the membrane is analysed using small-angle scattering and contrast variation with H2O/2H2O mixtures. Effects due to membrane curvature and scattering density variation in the plane of the membrane are evaluated. Thickness parameters (D) are obtained from the small-angle scattering data, which are the one-dimensional analogues of radii of gyration. The formalism of contrast variation is used to describe the change of intensity and thickness parameter with H2O/2H2O mixture. The results are expressed in terms of a thickness parameter at infinite contrast, which is directly related to the physical thickness of the membrane, and a measure of the variation of the scattering density across the membrane, produced, for example, by the higher scattering densities of the polar surfaces relative to a hydrocarbon interior of the membrane. Asymmetry in the membrane scattering density is also evaluated.The results for photosynthetic membranes demonstrate a lipid hydrocarbon core in the membrane. About two-thirds of the protein is closely associated with the lipid layer, and no substantial amounts of protein project more than short distances from the lipid layer. There is a contribution to the variation in scattering density across the membrane that cannot be attributed to lipid, and may involve scattering density heterogeneity within the protein, giving a high proportion of hydrophobic protein segments at the interior of the membrane that have lower scattering densities than the hydrophilic segments at the surfaces of the membrane. The membrane scattering density is not markedly asymmetric. Several alternative structures previously proposed for photosynthetic membranes are incompatible with these results. 相似文献
5.
Effects of physiologically relevant pressures of helium on the structure of cholesterol-containing lipid bilayers. A neutron diffraction study.
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We have used neutron diffraction to study the effects of helium gas (1-210 atm) on the structure of a lipid bilayer model of neuronal plasma membranes. We have recorded diffraction patterns from hydrated multilayers of dimyristoyl lecithin and 40% (molar) cholesterol to a resolution of approximately 6.5 A and have calculated scattering amplitude density distributions as a function of pressure. We find that there are no significant changes in the scattering density profiles at 95% confidence over the range of pressures investigated, suggesting that the physiological effects of high helium pressure are unlikely to be a consequence of changes in the structures of the lipid bilayer portions of membranes. 相似文献
6.
NaCl reflection coefficients in proximal tubule apical and basolateral membrane vesicles. Measurement by induced osmosis and solvent drag. 总被引:4,自引:2,他引:2
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Two independent methods, induced osmosis and solvent drag, were used to determine the reflection coefficients for NaCl (sigma NaCl) in brush border and basolateral membrane vesicles isolated from rabbit proximal tubule. In the induced osmosis method, vesicles loaded with sucrose were subjected to varying inward NaCl gradients in a stopped-flow apparatus. sigma NaCl was determined from the osmolality of the NaCl solution required to cause no initial osmotic water flux as measured by light scattering (null point). By this method sigma NaCl was greater than 0.92 for both apical and basolateral membranes with best estimates of 1.0. sigma NaCl was determined by the solvent drag method using the Cl-sensitive fluorescent indicator, 6-methoxy-N-[3-sulfopropyl]quinolinium (SPQ), to detect the drag of Cl into vesicles by inward osmotic water movement caused by an outward osmotic gradient. sigma NaCl was determined by comparing experimental data with theoretical curves generated using the coupled flux equations of Kedem and Katchalsky. By this method we found that sigma NaCl was greater than 0.96 for apical and greater than 0.98 for basolateral membrane vesicles, with best estimates of 1.0 for both membranes. These results demonstrate that sigma NaCl for proximal tubule apical and basolateral membranes are near unity. Taken together with previous results, these data suggest that proximal tubule water channels are long narrow pores that exclude NaCl. 相似文献
7.
Definition of lipid membrane structural parameters from neutronographic experiments with the help of the strip function model. 总被引:3,自引:0,他引:3
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Neutron diffraction is an effective method for investigating model and biological membranes. Yet, to obtain accurate structural information it is necessary to use deuterium labels and much time is needed to acquire experimental data as there are a large number of diffraction reflections to register. This paper offers a way to define the hydrophobic boundary position in lipid membranes with high accuracy and for this purpose it is sufficient to take into consideration three structural factors. The method is based on modeling the density of the neutron diffraction amplitude rho(x) in the direction of the bilayer plane normal by means of a strip function, but it also takes into consideration the fact that the multiplication of the strip function amplitude rho i by the step width zi-zi-1 makes the sum of neutron scattering amplitudes of the atoms included in the step region. On the basis of the analysis of a large number of experimental data for different membranes, the effectiveness of this method in the determination of the position of hydrophilic/hydrophobic boundary is demonstrated, including the case of various rho(x) modifications in the region of polar heads and also the different phase states of membranes. However, it is shown in the present paper that the strip function model is not an adequate instrument for the determination of other structural parameters of membranes. 相似文献
8.
Structure determination of asymmetric membrane profiles using an iterative Fourier method.
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An iterative Fourier method is applied to solving and refining the electron density profile projected into the line perpendicular to a membrane surface. Solutions to the continuous X-ray scattering pattern derived from swelling of multilayer systems or from membrane dispersions can be obtained by this technique. The method deals directly with the observed structure factors and does not rely on deconvolution of the Patterson function. We used this method previously to derive the electron density profile for acetylcholine receptor membranes (Ross et al., 1977). The present paper is an analysis of the theoretical basis for the procedure. In addition, the technique is tested on artificially generated continuous-scattering data, on the data for frog sciatic nerve myelin derived from swelling experiments by Worthington and McIntosh (1974), and on the data for purple membrane (Blaurock and Stoeckenius, 1971). Although the method applies to asymmetric membranes, the special case of centrosymmetric profiles is also shown to be solvable by the same technique. The limitations of the method and the boundary conditions that limit the degeneracy of the solution are analyzed. 相似文献
9.
The ATP-induced quenching of chlorophyll fluorescence in chloroplasts of higher plants is shown to be inhibited when the mobility of the protein complexes into the thylakoid membranes is reduced. Its occurrence also requires the presence of LHC complexes and the ability of the membranes to unstack. These observations, in addition to a slight increase of charge density of the surface—as indicated by 9-aminoacridine fluorescence and high salt-induced chlorophyll fluorescence studies—and partial unstacking of the membranes—as monitored by digitonin method and 540 nm light scattering changes—after phosphorylation, suggest that the ATP-induced quenching of chlorophyll fluorescence could reflect some lateral redistribution of membrane proteins in the lipid matrix of the thylakoids. 相似文献
10.
Small angle neutron scattering (SANS) was used to investigate interaction forces between hemoglobin (Hb) molecules contained within human red cells. The scattering separately attributable to cell membranes and intracellular Hb was identified. A series of D2O-H2O contrast variation measurements were made in order to establish conditions for which scattering from the cell membrane is minimized (approximately 15% D2O). Measurements then were performed to examine changes in intermolecular Hb interactions occurring when the cells are contracted or swollen by varying the ionic strength of the suspension buffer. The scattering cross-sections were fitted to structure factors computed by a mean spherical approximation, and molecular parameters thereby extracted. Oxygenation studies on normal cells were performed, and results contrasted with those of similar studies of erythrocytes obtained from sickle cell disease patients. 相似文献
11.
B A Wallace 《Biophysical journal》1986,49(1):295-306
Gramicidin A, a hydrophobic linear polypeptide, forms channels in phospholipid membranes that are specific for monovalent cations. Nuclear Magnetic Resonance (NMR) spectroscopy provided the first direct physical evidence that the channel conformation in membranes is an amino terminal-to-amino terminal helical dimer, and circular dichroism (CD) spectroscopy has shown the sensitivity of its conformation to different environments and the structural consequences of ion binding. The three-dimensional structure of a gramicidin/cesium complex has been determined by x-ray diffraction of single crystals using single wavelength anomalous scattering for phasing. The left-handed double helix in this crystal form corresponds to one of the intermediates in the process of folding and insertion into membranes. Co-crystals of gramicidin and lipid that appear to have gramicidin in their membrane channel conformation have also been formed and are presently under investigation. Hence, we have used a combination of spectroscopic and diffraction techniques to examine the conformation and functionally-related structural features of gramicidin A. 相似文献
12.
S S Vogel L V Chernomordik J Zimmerberg 《The Journal of biological chemistry》1992,267(36):25640-25643
We studied calcium-triggered fusion of sea urchin egg secretory granules to test whether membrane bound fusion proteins are required in both fusing membranes. Using both light scattering assays and video microscopy, we found that native granules fused to granules that had been inactivated with either trypsin or N-ethylmaleimide. Granules also fused with liposomes prepared from lipids extracted from egg cortices and with liposomes made from synthetic phospholipids and cholesterol. Granule-liposome fusion required no cytoplasmic proteins and was inhibited by N-ethylmaleimide. Thus, membrane fusion of exocytotic granules can be promoted by proteins residing on only one of the two membranes. 相似文献
13.
W. Lesslauer 《生物化学与生物物理学报:生物膜》1976,436(1):25-37
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. 相似文献
14.
Erythrocyte membrane interactions with insulin fibrils (amyloid) have been investigated using centrifugation, fluorescence spectroscopy, light scattering, and flow cytometric techniques. The results indicate that insulin fibrils are having moderate affinity to erythrocyte membrane. However, analysis of the apparent dissociation constants of human erythrocyte membranes (leaky and resealed vesicles) with amyloid insulin reveal that the insulin binding is drastically reduced on attaining the fibrillar state compared with native insulin. To understand the role of insulin receptors on erythrocytes binding to amyloid, we have studied the interaction of biotinylated forms of denatured and amyloidic insulin with erythrocytes. FITC-streptavidin was used as a counter staining in flow cytometry measurements. We found that insulin fibrils bind 10 times more with erythrocyte membranes than with amylin and denatured insulin. 相似文献
15.
Intensities of x-ray scattering from a series of fragmented rabbit muscle sarcoplasmic reticulum (SR) samples have been measured over the range x = 0.05 to s = 0.25. By varying the relative concentrations of lipid and protein (chiefly the Mg++-dependent, Ca++- stimulated ATPase) in the membranes of this series, and by employing methods of analysis appropriate to the scattering from binary liquid mixtures, we have identified the separable contributions of protein and lipid, and the protein-lipid interaction contributions to the total scattering profiles. The shape of the protein term is consistent with scattering from a cylindrical ATPase particle 142 A in length and 35 A in diameter. These data imply that the dominant ATPase species is monomeric. The protein-lipid interaction term has been analyzed by a novel treatment based on a determination of the pair correlation function between the electrons of the protein molecule with the electrons of the lipid bilayer in terms of the asymmetry of the transbilayer disposition of the protein. Applied to our results, the analysis indicates a fully asymmetric disposition of ATPase, in which one end of the molecule is contiguous with either the lumenal or cytoplasmic surface of the bilayer. 相似文献
16.
Benjamin F. Dickens T. Russel Snow Vickie Green William B. Weglicki 《Molecular and cellular biochemistry》1988,79(1):91-94
Summary The apparent membrane fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene has been reported to be lower in intact erythrocytes than in isolated erythrocyte membranes. Although this difference was once suggested to be caused by the fluidizing effect associated with the loss of erythrocyte proteins during membrane isolation, it is currently thought to be an artifact resulting from intense light scattering properties of intact erythrocytes which overwhelm extrapolation methods of correcting for light scattering. This study confirmed that, at erythrocyte concentrations greater than 107 cells/ml, this difference was caused by intense light scattering; however, at erythrocyte concentrations less than 4.0 × 106 cells/ml, the anisotropy values for erythrocytes and isolated membranes are identical, demonstrating that intense light scattering can be overcome with dilute suspensions of cells. 相似文献
17.
Mg2+-ATP produces a large decrease in near-IR light scattering when added to suspensions of rod outer segments (ROS) when the plasma membranes have been disrupted by a gentle dialysis procedure. When this process is studied by light microscopy with video-enhanced image contrast, the Mg2+-ATP-dependent signal is seen to be associated with the formation of filaments which extend only from those ROS lacking plasma membranes. Both the IR light scattering signal and filament growth are inhibited by vanadate and DCCD but not by colchicine, colcemid or cytochalasins. 相似文献
18.
Hydration dependence of chain dynamics and local diffusion in L-alpha-dipalmitoylphosphtidylcholine multilayers studied by incoherent quasi-elastic neutron scattering. 总被引:2,自引:2,他引:0
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Incoherent quasi-elastic neutron scattering is applied to study the local diffusion and chain dynamics of L-alpha-diplamiotylphosphatidylcholine molecules in oriented model membranes. Different motions are distinguished by changing the hydration of the multilayers as well as by measuring below and above the gel-to-liquid crystalline phase transition. The time range of the utilized time-of-flight spectrometer permits to observe two types of motion to be observed more closely: chain defect motions and the local diffusion of the whole molecule in its solvation cage. Oriented lipid membranes are a useful system for the observation of chain defects, as they can be macroscopically oriented, in contrast to most polymers. As a representative model for a chain defect a kink is chosen and the corresponding scattering functions are derived. The kink motion can explain the entire dynamics seen in the gel phase, and the lifetime of such a defect was found to be 10-15 ps, in good agreement with theoretical predictions. On the other hand the dynamics in the liquid crystalline phase cannot be explained even by a superposition of several kinks and thus requires the consideration of an additional motion: the local diffusion of the molecule in its solvation cage. The size of the solvation cage is increasing with multilayer hydration and reduced temperature. Particularly interesting in view of recent discussions about the origin of the short-range repulsive forces between membranes is the experimental finding of an out-of-plane motion with an amplitude of 1-1.5 A, which cannot be explained by the undulation of the whole membrane. 相似文献
19.
The dynamic behaviour of model membranes in the form of sonicated liposomes in excess water was studied by means of 90 degrees C light scattering and turbidity measurements. Computer calculations based on the Rayleigh-Gans theory of light scattering were used to estimate the average size of lipid vesicles dispersed in water, taking into account the various structures of the vesicles. Normal reversible changes in the scattered light intensity and turbidity with temperature could be accounted for mainly by the changes in the refractive index of the lipid and irreversible anomalous changes were explained on the basis of fusion of smaller aggregated vesicles. 相似文献
20.
《生物化学与生物物理学报:生物膜》2022,1864(9):183944
Fluid lipid bilayers are the building blocks of biological membranes. Although there is a large amount of experimental data using incoherent quasi-elastic neutron scattering (QENS) techniques to study membranes, very little theoretical works have been developed to study the local dynamics of membranes. The main objective of this work is to build a theoretical framework to study and describe the local dynamics of lipids and derive analytical expressions of intermediate scattering functions (ISF) for QENS. As results, we developed the dynamical Matryoshka model which describes the local dynamics of lipid molecules in membrane layers as a nested hierarchical convolution of three motional processes: (i) individual motions described by the vibrational motions of H-atoms; (ii) internal motions including movements of the lipid backbone, head groups and tails, and (iii) molecule movements of the lipid molecule as a whole. The analytical expressions of the ISF associated with these movements are all derived. For use in analyzing the QENS experimental data, we also derived an analytical expression for the aggregate ISF of the Matryoshka model which involves an elastic term plus three inelastic terms of well-separated time scales and whose amplitudes and rates are functions of the lipid motions. And as an illustrative application, we used the aggregated ISF to analyze the experimental QENS data on a lipid sample of multilamellar bilayers of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine). It is clear from this analysis that the dynamical Matryoshka model describes very well the experimental data and allow extracting the dynamical parameters of the studied system. 相似文献