Contrast variation studies of clathrin coated vesicles by small-angle neutron scattering

Structural information on clathrin coated vesicles has been obtained by small angle neutron scattering using contrast variation. A characteristic peak in the neutron scattering profile, which is apparent in 75 % D₂O, as well as in H₂O, disappears when contrast matching the protein component of the coated vesicles in 42% D₂O. Neutron, as well as dynamic, light scattering give a coated vesicle size of about 900 Å in H₂O and D₂O, but for neutron scattering the diameter decreases when matching out the protein coat of the clathrin coated vesicles. From the match point for the clathrin coated vesicles it is demonstrated that the clathrin cages do contain internal membrane. The mass of 34 MD and composition of 75% protein and 25% lipid found from the analysis of the small-angle scattering data are both in good agreement with the values reported in the literature. Electron microscopy gives an average outer diameter of 880 Å for the coated vesicles and an average diameter of 460 Å for the vesicle itself. Offprint requests to: Correspondence to: R. Bauer     

Time-resolved angular dependent measurement of triggered light scattering changes in biological suspensions

We describe a photometer for time-resolved measurements of small changes in light scattering suited for suspensions of biological material. The time resolution is 35 μs, the amplitude resolution for bovine rot outer segments is typically ΔI/I = 5 · 10^?4 at a scattering angle of ? = 20°. The use of the apparatus is demonstrated by recording the near infrared scattering of bovine rod outer segments after excitation with flashes of green light.Semiconductor detector arrays are arranged centrosymmetrically around a hemispherical cuvette. The optical characteristics of a hemispherical cuvette and the resulting geometry of cuvette and detection are discussed.Calculations of optimal signal transfer and noise of the detectors led to the following arrangement for each scattering angle: pairs of parallel connected photodiodes are fed into several current-to-voltage converters, whose output voltages are summed up by a summing amplifier.For the test of the device so-called N signals of fresh and liquid N₂-frozen and thawed ROS samples were measured at four scattering angles simultaneously. A strong angular dependence (difference scattering curve) of the relative light scattering change is seen for fresh ROS which is transformed into a flat curve by freezing and thawing. It is concluded that the competence of the fresh sample to extend the light-induced local events — presumably rhodopsin conformational changes — into the gross-structural range is terminated by freezing.     

Depolarized light-scattering studies of bilayer structures in phospholipid vesicles

Depolarized light scattering has been used to investigate the hydrocarbon chain packing of phospholipids in vesicles below the phase transition and ordering of their chains above the phase transition. The chain packing and ordering have been demonstrated for vesicles of l-α-dipalmitoylphosphatidylethanolamine and some phosphatidylcholines of different hydrocarbon chain lenghts. Anisotropy ratios for phospholipid vesicles could be determined by measuring depolarization ratios for several vesicle sizes at low concentrations of the lipids. The following results were obtained. Hydrocarbon chains of l-α-dimyristoyl and distearoylphosphatidylcholines below their phase transitions pack at tilting angles in good agreement with X-ray diffraction data. On the other hand, hydrocarbon chains of dipalmitoylphosphatidylethanolamine pack perpendicular to the bilayer surface. Values of the averaged order parameter for dimyristoyl, dipalmitoyl and distearoylphosphatidylcholines at 2.5°C above their phase transition are all the same and the value for dipalmitoylphosphatidylcholine is in agreement with results from ²H-NMR experiments. The value of the order parameter for dipalmitoylphosphatidylethanolamine is slightly larger than that for dipalmitoylphosphatidylcholine.     

Temperature dependence of optical properties of aqueous dispersions of phosphatidylcholine

Aqueous dispersions of dipalmitoyl phosphatidyl choline exhibit a sharp decrease in turbidity at the crystal-liquid phase transition temperature of 41°C. The intensity of light scattered at 45°, 90°, and 135° also undergoes a sharp drop at the same temperature. Similarly, the refractive index of such dispersions decreases abruptly with the phase transition. Employing the relationship between light scattering intensity and specific refractive increment, it can be shown that about one half of the change in absorbancy and scattering are accounted for by the change in refractive index. The change in refractive index can be entirely accounted for by the known expansion and corresponding decrease in density of the bilayer. That part of the observed change in scattering and turbidity which is not accounted for by the observed change in average refractive index is apparently due to a decrease in the anisotropy of the bilayer during the melting process. Calculations based on a model which, although oversimplified, is consistent with the known thinning of the bilayer during the melting process, give quantitative agreement with experimental results. Below the phase transition temperature other changes in optical properties are observed; near 32°C, the light scattering envelope changes and the turbidity of dispersions drops markedly. The average refractive index remains constant in this region. For this and other reasons, it is postulated that these pre-transition changes indicate an aggregation-disaggregation phenomenon.     

Light scattering and turbidity measurements on lipid vesicles

The dynamic behaviour of model membranes in the form of sonicated liposomes in excess water was studied by means of 90 °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 change in the refractive index of the lipid and irreversible anomalous changes were explained on the basis of fusion of smaller aggregated vesicles.     

Osmotic swelling of unilamellar vesicles by the stopped-flow light scattering method. Elastic properties of vesicles

Unilamellar vesicles of l-α-dimyristoylphophatidylcholine have been prepared by the ether injection technique. Gel filtration on Sephacryl S1000 was used to obtain fractions of narrow polydispersity, of radius from 300 to 600 Å. Dynamic light scattering was used to determine the change in size of these vesicles in response to an osmotic pressure drop, and its dependence on vesicle size. The amplitude of swelling (ΔR/R) is linearly proportional to the osmotic pressure difference across the bilayer. We have determined the elastic area stretching modulus using a theory of membrane elasticity: it depends on the vesicle radius in the range of size studied. Vesicles having radius smaller than 400 Å show little or no swelling.     

Author index

Using dynamic light scattering we have been able to determine precisely the hydrodynamic radius of l-α-dimyristoylphosphatidylcholine (DMPC) vesicles as a function of temperature. We have detected a sharp, thermally reversible change in the vesicle radius at a phase transition temperature 24°C, corresponding to an approximate 11% increase in surface are. In the range 10–20°C, the change in radius is less than 1%.     

Optical characterization of liposomes by right angle light scattering and turbidity measurement

Liposomes have frequently been used as models of biomembranes or vehicles for drug delivery. However, the systematic characterization of lipid vesicles by right angle light scattering and turbidity has not been carried out despite the usefulness of such studies for size estimation. In this study, liposomes of various sizes were prepared by sonication and extrusion. The mean cumulant radii of the vesicles were determined by dynamic light scattering. The lamellarities were estimated based on fluorescence quenching of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)dipalmitoyl-L-alpha-phosph ati dylethanolamine by sodium dithionite. Right angle light scattering intensity and optical density at 436 nm per unit lipid concentration were measured as a function of vesicle radius. With a vesicle radius < or =100 nm, the optical parameters could be well explained by the Rayleigh-Gans-Debye theory in which the liposomes were modeled as homogeneous spheres with mean refractive indices determined by the volume fractions of lipids in vesicles.     

Determination of solute reflection coefficients in kidney brush-border membrane vesicles by light scattering: influence of the refractive index

Solute reflection coefficients sigma of cell membrane vesicles or liposomes are commonly determined by comparison of the water flow induced by a gradient of the studied solute and that of a reference molecule using light scattering techniques. However, variations in scattered light which are mainly related to change in vesicle volume are also influenced by the refractive index of the surrounding medium. Therefore comparing kinetics of vesicle shrinkage induced by hyperosmotic solutions which have different refractive indexes might lead to an under or over estimation of sigma. We determined sigma NaCl in rat kidney brush-border membrane vesicles by two different approaches using mannitol, a poorly permeant molecule, as reference. (1) The refractive index of the hyperosmotic NaCl solution was adjusted to that of mannitol by addition of polyvinyl pyrrolidone (Mr 40,000), without a significant increase in osmolality. Thereby the change in scattered light intensity induced by osmotic vesicle shrinkage due to gradients of NaCl and mannitol were comparable and led to a sigma NaCl value close to one instead of the previously published value of 0.53. (2) The reflection coefficient was calculated from the lifetime of vesicle shrinkage which is not refractive index-dependent. Again sigma NaCl was not different from one. These results suggest that the water proteic pathways found in the luminal membrane of proximal tubules are not shared by salts.     

Characterization of the size distribution of unilamellar vesicles by gel filtration, quasi-elastic light scattering and electron microscopy

The size and size distribution of unilamellar phospholipid vesicles present in unsonicated phosphatidic acid and mixed phosphatidic acid/phosphatidylcholine dispersions were determined by gel filtration, quasi-elastic light scattering and freeze-fracture electron microscopy. The vesiculation in these dispersions was induced by a transient increase in pH as described previously (Hauser, H. and Gains, N. (1982) Proc. Natl. Acad. Sci. USA 79, 1683–1687). The resulting phospholipid dispersions are heterogeneous consisting of small unilamellar vesicles (average radius r < 50 nm) and large unilamellar vesicles (average r ranging from about 50 to 500 nm). The smallest vesicles with r = 11 ± 2 nm are observed with dispersions of pure phosphatidic acid, the population of these vesicles amounting to about 80% of the total lipid. With increasing phosphatidylcholine content the radius of the small unilamellar vesicles increases and at the same time the population of small unilamellar vesicles decreases. The average radius of small unilamellar vesicles present in phosphatidic acid/phosphatidylcholine dispersions (mole ratio, 1:1) is 17.5 ± 2 nm, the population of these vesicles amounting to about 70% of the total lipid. By a combination of gel filtration, quasi-elastic light scattering and freeze-fracture electron microscopy it was possible to characterize the large unilamellar vesicles. This population is heterogeneous with its mean radius also increasing with increasing phosphatidylcholine content. After separating the large unilamellar vesicles from small unilamellar vesicles on Sepharose 4B it can be shown by quasi-elastic light scattering that in pure phosphatidic acid dispersions 80–90% of the large unilamellar vesicle population consist of vesicles with a mean radius of 170 nm. In mixed phosphatidic acid/phosphatidylcholine dispersions this radius increases to about 265 nm as the phosphatidylcholine content is raised to 90 mol%.     

Sizes of two amino acyl-tRNA synthetase complexes from E. coli with their cognate tRNAs.

The complexes of valyl-tRNA synthetase with tRNA_I^Val and arginyl-tRNA synthetase with tRNA_II^Arg from $\text{E. coli}$ were studied by light scattering measurements and analytical ultracentrifugation of concentrations as low as 40 μg/ml. The molecular weights determined from these studies were 260,000 ± 2,000 for the valyl-tRNA synthetase·tRNA complex, and 310,000 ± 1,500 for the arginyl-tRNA synthetase·tRNA complex at pH 7.1. The stoichiometry for the complexes are apparently 2:1 for valyl-tRNA synthetase and tRNA and 4:1 in the case of the arginyl-tRNA synthetase and tRNA. From the angular dependence of the scattered intensity a radius of gyration of 54.5 Å for the complex between valyl-tRNA synthetase and tRNA was found, whereas for the other complex a value of 59.1 Å was found.     

Bilayer undulation dynamics in unilamellar phospholipid vesicles: Effect of temperature,cholesterol and trehalose

We report a combined dynamic light scattering (DLS) and neutron spin-echo (NSE) study on the local bilayer undulation dynamics of phospholipid vesicles composed of 1,2-dimyristoyl-glycero-3-phosphatidylcholine (DMPC) under the influence of temperature and the additives cholesterol and trehalose. The additives affect vesicle size and self-diffusion. Mechanical properties of the membrane and corresponding bilayer undulations are tuned by changing lipid headgroup or acyl chain properties through temperature or composition. On the local length scale, changes at the lipid headgroup influence the bilayer bending rigidity κ less than changes at the lipid acyl chain: We observe a bilayer softening around the main phase transition temperature T_m of the single lipid system, and stiffening when more cholesterol is added, in concordance with literature. Surprisingly, no effect on the mechanical properties of the vesicles is observed upon the addition of trehalose.     

Hexose and amino acid transport by chicken embryo fibroblasts infected with temperature-sensitive mutant of rous sarcoma virus: Comparison of transport properties of whole cells and membrane vesicles

The effect of transformation on hexose and amino acid transport has been studied using whole cells and membrane vesicles of chicken embryo fibroblasts infected with the temperature-sensitive mutant of the Rous sarcoma virus, TS-68. In whole cells, TS-68-infected chicken embryo fibroblasts cultured at the permissive temperature (37°C) had a 2-fold higher rate of 2-deoxy-d-glucose uptake than the same cells cultured at the non-permissive temperature (41°C). However, both the non-transformed and transformed cells had comparable rates of α-aminoisobutyric acid transport. Membrane vesicles, isolated from TS-68-infected chicken embryo fibroblasts cultured at 41°C or 37°C, displayed carrier-mediated, intravesicular uptake of d-glucose and α-aminoisobutyric acid. Membrane vesicles from TS-68-infected chicken embryo fibroblasts cultured at 37°C had an approx. 50% greater initial rate of stereospecific hexose uptake than the membrane vesicles from fibroblasts cultured at 41°C. The two types of membrane vesicle had similar uptake rates of α-aminoisobutyric acid. The results of hexose and amino acid uptake by the membrane vesicles correlated well with those observed with the whole cells. K_m values for stereospecific d-glucose uptake by the membrane vesicles from TS-68-infected chicken embryo fibroblasts cultured at 41 and 37°C were similar, but the V value was greater for the membrane vesicles from TS-68-infected cells cultured at 37°C. Cytochalasin B competitively inhibited stereospecific hexose uptake in both types of membrane vesicle. These findings suggest that the membrane vesicles retained many of the features of hexose and amino acid transport observed in whole cells, and that the increased rate of hexose transport seen in the virallytransformed chicken embryo fibroblasts was due to an increase in the number or availability of hexose carriers.     

Conformation of <Emphasis Type="Italic">Thermus thermophilus</Emphasis> ribosomal protein S1 in solution at different ionic strengths

Small-angle x-ray and neutron scattering were used to study the structure of the ribosomal protein S1 (61 kDa) from Thermus thermophilus in solution at low and moderate ionic strength (0 and 100 mM NaCl). The protein was found to be globular in both cases. Modeling of the S1 structure comprising six homologous domains on the basis of the NMR data for one domain showed that the best fit to scattering data was provided by compact domain packing. The calculated gyration radius was 28–29 Å, as typical of globular proteins about 60 kDa. The protein was prone to self-association, forming mainly dimers and trimers at moderate ionic strength and higher compact associates at low ionic strength. Neutron scattering assays in heavy water at 100 mM NaCl revealed markedly elongated associates. The translational diffusion coefficient calculated for S1 at 100 mM NaCl from dynamic light scattering was markedly lower than the one expected for its globular monomer (D _20,w = (2.7 ± 0.1)·10^?7 versus (5.8–6.0)·10^?7 cm² s^?1), confirming protein association under equilibrium conditions.     

High-relaxivity supramolecular aggregates containing peptides and Gd complexes as contrast agents in MRI

Mixed supramolecular aggregates, obtained by assembling together two amphiphilic monomers (C₁₈H₃₇)₂NCO(CH₂)₂CO(AdOO)₅-G-CCK8 (AdOO is 8-amino-3,6-dioxaoctanoic acid, CCK8 is C-terminal octapeptide of cholecystokinin) and (C₁₈H₃₇)₂NCO(CH₂)₂COLys(DTPAGlu)CONH₂ (DTPAGlu is N,N-bis[2-[bis(carboxyethyl)amino]ethyl]-l-glutamic acid), are characterized for their structural parameters by dynamic light scattering and for their relaxometric properties, in the absence and in the presence of 0.9 wt% NaCl. Two different aggregates (micelles and bilayer structures) are present in the absence of NaCl, while only bilayer structures are observed at physiological ionic strength. The presence of NaCl increases the ionic strength, promoting a decrease in the repulsions between the polar heads and among the aggregates in solution, thus supporting the formation of large-curvature aggregates such as bilayer structures like vesicles. In these conditions the closed, vesicular shape and the large size (hydrodynamic radius of about 300 Å) of the aggregates allow a high number of paramagnetic gadolinium complexes and bioactive peptides to be accommodated on the inner and external surfaces . The presence of the salt causes a variation in the structural arrangement of the molecules and a partial rigidification of the assembled Gd(III) complexes on the surface vesicles, reducing their internal motions and giving an approximately 15% higher relaxivity value (r _1p = 21.0 and 18.6 Mm^?1 s^?1 in the presence and in the absence of NaCl, respectively). The vesicles obtained, for the high relaxivity of each gadolidium complex and for the presence of a surface-exposed bioactive peptide, are very promising candidates as target-selective MRI contrast agents.     

Properties of pancreatic zymogen granules studied by quasi-elastic light scattering

Physico-chemical properties of isolated zymogen granules of the mouse pancreas were studied by means of quasi-elastic light scattering. The average diameter of the granules in 0.3 M sucrose was found to be 1.1 ± 0.1 μm from the correlation time of intensity fluctuation of the scattered light. The average diameter altered depending on the osmolality of the medium in a manner that the alteration was smaller than that expected from the van't Hoff relation. Aggregation of the granules induced by the increase of Ca²⁺ concentration or the decrease of pH in the medium was also detected. The aggregation started at a critical level of 1 mM CaCl₂ or at pH 5.4.     

Permeability to ions of bovine retinal disk membrane vesicles in the bleached state

The permeability of the bleached disk membrane of retinal rod outer segments to univalent and divalent ions is studied by light scattering. The membranes are isolated from frozen dark-adapted bovine retinae, swollen into spherical vesicles in a hypotonic medium and bleached in dilute suspension and their size is determined by elastic and quasi-elastic light scatterings. Various electrolytes are then added to the suspending medium in order to examine their osmotic activity relative to the vesicles deformation characteristics. By following the deformation behavior of the membrane vesicles by elastic light scattering in terms of the oblate ellipsoidal shell model, the osmotic activity of a given electrolyte is qualitatively deduced and thereby the permeability of the membrane to the electrolyte is ranked in reference to a chosen standard, i.e., sucrose. By this method, we show that the permeabilities to Na⁺, K⁺, Mg²⁺ and Ca²⁺ are all alike, and those to halides (F^?, Cl^?, Br^?, I^?), nitrate and phosphates (HPO₄^2?/H₂PO₄^?) are similar. Acetate, however, is about 3-times more permeative, while sulfate is less permeative than the other anions by about the same factor. The viability of our method is checked with use of an ionophore, lasolocid (X-537A), by establishing partial recovery from the osmotic deformation through the suppression of the cation osmotic effect. Ion-induced aggregation and pH-dependent size and shape changes are both found to be insignificant.