Neutron diffraction from cannabinoids in phospholipid membranes

Neutron diffraction measurements have been utilized to study the effects of delta 9-tetrahydrocannabinol (delta 9-THC) and delta 8-tetrahydrocannabinol (delta 8-THC) incorporated in phospholipid membranes of dipalmitoylphosphatidylcholine (DPPC). Low-angle diffraction indicated that these cannabinoids induce increases in interlamellar spacing similar to those produced by cholesterol. Wide-angle diffraction indicated significant differences in how the intralamellar structure is affected by the inclusion of either cannabinoids or cholesterol. Similar weight percentages of cholesterol and cannabinoids in membranes yielded different thermal analysis profiles but the profiles for membranes with either delta 8 or delta 9-THC were similar. Since the neutron diffraction results for inclusions of delta 8 and delta 9-THC were also similar, this suggests that the difference in psychoactivity of delta 8 and delta 9-THC is probably due to interactions with membrane proteins rather than with phospholipids.     

X-ray diffraction from paclitaxel-loaded zwitterionic and cationic model membranes

We studied the incorporation of the hydrophobic anticancer drug paclitaxel (PXL), into a variety of lipid matrices by X-ray diffraction (XRD) measurements. Liposome suspensions from cationic and zwitterionic lipids, containing different molar fractions of paclitaxel were made and deposited on planar glass substrates. After drying at controlled relative humidity, aligned multilayer stacks were obtained. The structure perpendicular to the substrate plane was investigated by X-ray diffraction measurements. Bragg peaks to several orders were detected, indicative of well-ordered multilamellar lipid layers. The drug induced a modification of the bilayer spacing, which was the characteristic for a given type of lipid matrix. With an excess of the drug, Bragg peaks of drug crystals could be observed. The results provide insight into the solubility of paclitaxel in the different lipid membranes. A structural model of the organization of the drug in the membrane was discussed.     

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.     

Study of the structure of model and biological membranes by neutron diffraction

A model multilamellar structure of dipalmitoyl lecithin was studied with neutron time-of-flight diffractometer. The acquired data permit determination of structure factors and calculation of the bilayer density profile.     

Location and effect of procaine on lecithin/cholesterol membranes using X-ray diffraction methods

X-ray diffraction studies were made on lecithin/cholesterol multilayers with very high water content and containing the local anaesthetic procaine. Narrow-angle diffraction experiments show that the procaine molecules are located with the uncharged aromatic amine group approx. 10 Å from the centre of the bilayer. The polar tertiary amine group of these molecules is almost certainly located in the polar head-group region of the membrane. Wide-angle diffraction experiments show that the incorporation of procaine molecules into such lipid membranes produces an approx. 30% increase in the spread of acyl chain separation, although the average spacing between the chains is slightly reduced.     

X-ray diffraction studies of outer membranes of Salmonella typhimurium.

X-ray diffraction studies were carried out on the outer membranes of various strains of Salmonella typhimurium. Ten distinct diffraction peaks which seem to be caused by protein assemblies were observed for most strains. Three small-angle reflections were used to determine an average structure of the protein assembly in the outer membrane of mutant HN202. An electron density distribution of the averaged assembly was obtained by means of the Fourier-Bessel transform. It has a diameter of about 100A, in agreement with the results of electron microscope observations (Smit, Kamio, and Nikaido (1975) J. Bacteriol. 124, 942--958), and exhibits a low electron density region at its center, suggesting the presence of a pore, as predicted on the basis of transmembrane transport experiments (Nakae (1976) J. Biol. Chem. 251, 2176--2178).     

X-ray diffraction and calorimetric study of N-lignoceryl sphingomyelin membranes.

Differential scanning calorimetry and x-ray diffraction have been used to investigate hydrated multibilayers of N-lignoceryl sphingomyelin (C24:0-SM) in the hydration range 0-75 wt % H2O. Anhydrous C24:0-SM exhibits a single endothermic transition at 81.3 degrees C (delta H = 3.6 kcal/mol). At low hydration (12.1 wt % H2O), three different endothermic transitions are observed: low-temperature transition (T1) at 39.4 degrees C (transition enthalpy (delta H1) = 2.8 kcal/mol), intermediate-temperature transition (T2) at 45.5 degrees C, and high-temperature transition (T3) at 51.3 degrees C (combined transition enthalpy (delta H2 + 3) = 5.03 kcal/mol). On increasing hydration, all three transition temperatures of C24:0-SM decrease slightly to reach limiting values of 36.7 degrees C (T1), 44.4 degrees C (T2), and 48.4 degrees C (T3) at approximately 20 wt % H2O. At 22 degrees C (below T1), x-ray diffraction of C24:0-SM at different hydration levels shows two wide-angle reflections, a sharp one at 1/4.2 A-1 and a more diffuse one at 1/4.0 A-1 together with lamellar reflections corresponding to bilayer periodicities increasing from d = 65.4 A to a limiting value of 71.1 A. Electron density profiles show a constant bilayer thickness dp-p approximately 50 A. In contrast, at 40 degrees C (between T1 and T2) a single sharp wide-angle reflection at approximately 1/4.2 A-1 is observed. The lamellar reflections correspond to a larger bilayer periodicity (increasing from d = 69.3-80.2 A) and there is some increase in dp-p (52-56 A) with hydration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reexamining the egg-box model in calcium-alginate gels with X-ray diffraction

The structure of the Ca--alginate junction zones was investigated with X-ray scattering on gels prepared with different methods. Fiber diffraction reveals the popular egg-box model may not be the only possible structure for the junction zones. The (001) reflection, which should be extinguished due to 2/1 helical conformation in the egg-box model, was observed. This was further confirmed by the measurements on Ca--alginate gel beads prepared at different pH where large pieces were formed through a relatively slow process, which leads to a higher crystallinity and a more perfect ordering. The results suggest a 3/1 helical conformation is more proper for Ca--alginate gels formed slowly. This does not exclude the possibility for the 2/1 helical conformation in fast gelatinized Ca--alginate in which the 2/1 helix is a metastable form. Comparing the X-ray scattering results of the fresh, dehydrated, and rehydrated gels, a reversible aggregation of junction zones is found during dehydration and rehydration. The different stabilities of initial bonds and bonds formed during drying are speculated to be the contribution of MG block or short G blocks in the junction zones.     

Amphipathic interactions of cannabinoids with membranes. A comparison between delta 8-THC and its O-methyl analog using differential scanning calorimetry, X-ray diffraction and solid state 2H-NMR.

The effects of (-)-delta 8-tetrahydrocannabinol (delta 8-THC) and its biologically inactive O-methyl ether analog on model phospholipid membranes were studied using a combination of differential scanning calorimetry (DSC), small angle X-ray diffraction and solid state 2H-NMR. The focus of this work is on the amphipathic interactions of cannabinoids with membranes and the role of the free phenolic hydroxyl group which is the only structural difference between these two cannabinoids. Identically prepared aqueous multilamellar dispersions of phosphatidylcholines in the absence and presence of cannabinoids were used. The DSC thermograms and X-ray diffraction patterns of these preparations allowed us to detect the strikingly different manners in which these two cannabinoids affect the thermotropic properties and the thickness of the bilayer. In order study the effects of the cannabinoids on different regions of the bilayer, we used solid state 2H-NMR with four sets of model membranes from dipalmitoylphosphatidylcholine deuterated in different sites, viz., the choline trimethylammonium head group, or one of the following three groups in the acyl chains; the 2'-methylene, 7'-methylene, 16'-methyl groups. Analysis of quadrupolar splittings indicated that delta 8-THC resides near the bilayer interface and the inactive analog sinks deeper towards the hydrophobic region. The temperature dependence of the solid state 2H-NMR spectra showed that, during the bilayer phase transition, the disordering of the choline head groups is a separate event from the melting of the acyl chains, and that amphipathic interactions between delta 8-THC and the membrane separate these two events further apart in temperature. The inactive analog lacks the ability to induce such a perturbation.     

X-ray diffraction studies of lipid phase transitions in cholesterol-rich membranes at sub-zero temperatures

In X-ray diffraction studies of hydrated (greater than 60%) cholesterol/dioleoylphosphatidylcholine mixtures the lipid packing band showed an abrupt transition from liquid crystal-type to gel-type position and definition at a temperature which decreased progressively to almost -50 degrees C as the proportion of cholesterol was increased to a saturation level of about 50 mol%. Plots of transition temperature against composition (mol% cholesterol) and of peak position against composition provided evidence of a significant change in phospholipid configuration at about 20 mol% cholesterol. However, the data overall suggested a uniform dispersion of the cholesterol molecules in the phospholipid bilayer at all concentrations up to the saturation point. Parallel studies of hydrated lipid extract of erythrocyte membranes and of several cholesterol-rich membrane preparations showed a similar overall change from liquid crystal-type packing at +20 degrees C to a gel-type packing at -30 degrees C to -40 degrees C but without displaying a defined transition temperature.     

Internodal myelination during development quantitated using X-ray diffraction

Characterizing the formation, accretion, and stability of myelin during development, maturation, and senescence is important for better understanding critical periods in the function of the nervous system in normal growth and following environmental insult or genetic mutation. Although there are numerous studies on the ultrastructural, biochemical, and genetic aspects of myelin development and maturation, few have used X-ray diffraction (XRD), which can rapidly provide unique metrics about internodal myelin based on measurements from whole, unfixed tissue. Besides periodicity (the classic attribute of internodal myelin measured by XRD), other parameters include: relative amount of myelin, membrane dimensions, and packing disorder. To provide a baseline for future experiments on myelin structural integrity, we used XRD to characterize internodal myelin as a function of age (from 5 to 495 days) in the mouse, a species increasingly used for developing transgenic models of human neurological diseases. As expected, the relative amount of myelin increased with age in both PNS and CNS, with the most rapid accumulation occurring in the youngest age group. Changes in rate of myelin accretion yielded three distinct age brackets during which small but significant changes in structural parameters were detected: in PNS, myelin period increased, packing distortion decreased, width of extracellular apposition (EXT) decreased, and widths of cytoplasmic apposition (CYT) and lipid bilayer (LPG) increased; in CNS, myelin period decreased, packing distortion decreased, EXT and CYT decreased, and LPG increased. We propose that the data obtained here can serve as a basis for rapidly detecting abnormal pathologies during myelination.     

An X-ray diffraction study of model membrane raft structures

Protein sorting and assembly in membrane biogenesis and function involves the creation of ordered domains of lipids known as membrane rafts. The rafts are comprised of all the major classes of lipids, including glycerophospholipids, sphingolipids and sterol. Cholesterol is known to interact with sphingomyelin to form a liquid-ordered bilayer phase. Domains formed by sphingomyelin and cholesterol, however, represent relatively small proportions of the lipids found in membrane rafts and the properties of other raft lipids are not well characterized. We examined the structure of lipid bilayers comprised of aqueous dispersions of ternary mixtures of phosphatidylcholines and sphingomyelins from tissue extracts and cholesterol using synchrotron X-ray powder diffraction methods. Analysis of the Bragg reflections using peak-fitting methods enables the distinction of three coexisting bilayer structures: (a) a quasicrystalline structure comprised of equimolar proportions of phosphatidylcholine and sphingomyelin, (b) a liquid-ordered bilayer of phospholipid and cholesterol, and (c) fluid phospholipid bilayers. The structures have been assigned on the basis of lamellar repeat spacings, relative scattering intensities and bilayer thickness of binary and ternary lipid mixtures of varying composition subjected to thermal scans between 20 and 50 °C. The results suggest that the order created by the quasicrystalline phase may provide an appropriate scaffold for the organization and assembly of raft proteins on both sides of the membrane. Co-existing liquid-ordered structures comprised of phospholipid and cholesterol provides an additional membrane environment for assembly of different raft proteins.     

Difficult macromolecular structures determined using X-ray diffraction techniques

Macromolecular crystallography has been, for the last few decades, the main source of structural information of biological macromolecular systems and it is one of the most powerful techniques for the analysis of enzyme mechanisms and macromolecular interactions at the atomic level. In addition, it is also an extremely powerful tool for drug design. Recent technological and methodological developments in macromolecular X-ray crystallography have allowed solving structures that until recently were considered difficult or even impossible, such as structures at atomic or subatomic resolution or large macromolecular complexes and assemblies at low resolution. These developments have also helped to solve the 3D-structure of macromolecules from twin crystals. Recently, this technique complemented with cryo-electron microscopy and neutron crystallography has provided the structure of large macromolecular machines with great precision allowing understanding of the mechanisms of their function.     

X-ray diffraction evidence for the presence of discrete water layers on the surface of membranes

X-ray diffraction spacings in multilayered membranes obtained from frog sciatic nerves were found to increase in discrete steps of approx. 5 A during swelling. These observed jumps in the repeat period suggest that the lipid bilayers exist in distinct states of hydration, and perhaps the swelling occurs by step-wise addition of water layers between the polar head groups. Our analysis and statistical tests of this hypothesis are presented.