Four-time 7Li stimulated-echo spectroscopy for the study of dynamic heterogeneities: Application to lithium borate glass.

To study the nature of the nonexponential ionic hopping in solids a pulse sequence was developed that yields four-time stimulated-echo functions of previously inaccessible spin-3/2-nuclei such as 7Li. It exploits combined Zeeman and octupolar order as longitudinal carrier state. Higher-order correlation functions were successfully generated for natural-abundance and isotopically-enriched lithium diborate glasses. Four-time 7Li measurements are presented and compared with two-time correlation functions. The results are discussed with reference to approaches devised to quantify the degree of nonexponentiality in glass forming systems and evidence for the occurrence of dynamic heterogeneities and dynamic exchange were found. Additional experiments using the 6Li species illustrate the challenge posed by subensemble selection when the dipolar interactions are not very much smaller than the quadrupolar ones.

Orientational order in the choline headgroup of sphingomyelin: A 14N-NMR study.

An aqueous dispersion of fully hydrated bovine sphingomyelin was studied using 14N-NMR spectroscopy. Spectra were obtained as a function of temperature over the range 15-80 degrees C, in both the liquid crystal and gel phases. In the liquid crystal phase, powder pattern lineshapes were obtained, whose quadrupolar splitting slowly decreases with increasing temperature. The spectra are increasingly broadened as the temperature is lowered through the phase transition into the gel phase. The linewidths and the second moments of these spectra indicate that the onset of a broad phase transition occurs at approx. 35 degrees C, in agreement with previous calorimetric and 31 P-NMR measurements. There is no evidence from the lineshapes for an hexagonal phase in this system, and this conclusion is supported by X-ray diffraction measurements carried out on aqueous dispersions of sphingomyelin in both phases. Assuming that the static nitrogen quadrupole coupling constant is the same for both sphingomyelin and dipalmitoyl-L-alpha-phosphatidylcholine (DPPC), the decrease observed in the quadrupolar splitting of sphingomyelin compared to that of DPPC indicates that the orientational order of the choline headgroup in liquid crystalline sphingomyelin is not the same as that of its counterpart in DPPC. Preliminary relaxation time measurements of T1 and T2 are presented which suggest that there are also dynamic differences between sphingomyelin and DPPC in the choline headgroup.

A comparison of the headgroup conformation and dynamics in synthetic analogs of dipalmitoylphosphatidylcholine.

14N-NMR spectra and relaxation times for dipalmitoylphosphatidylcholine and three analogs were obtained in both the liquid crystal and gel phases. The analogs either changed the PO-4 to N+ (CH3)3 distance (P-N) within the headgroup by increasing the number of CH2 groups from two in the phosphocholine headgroup (PN-2) to six in the phospho-(N',N',N'-trimethyl)hexanolamine headgroup (PN-6), or replaced the ester linkages to the hydrocarbon chains with either linkages. 31P-NMR spectra were obtained for the four samples in the liquid-crystal phase. (1) The 14N- and 31P-NMR spectra and 14N relaxation times all indicate that increasing the P-N distance within the headgroup causes changes in both the average orientation of the C-N bond and its dynamics. (2) The 14N-NMR spectra provide evidence for a change in orientational order of the headgroup as a result of changing the linkage to the acyl chains. On the other hand, the relaxation time measurements indicate that the molecular motion for the headgroup is independent of the type of linkage. (3) The thermal behaviour of the four samples is clearly reflected in the 14N-NMR spectra. The second moments of the spectra show distinct changes at each of the phase transitions. (4) The 14N-NMR spectra show that the average conformation of the headgroups is not significantly altered by the main phase transition. For the PN-2 samples, T2e, the decay of the quadrupolar echo, decreases discontinuously in the P beta, phase, which is evidence for a possible exchange process between two molecular states within this phase.

Topography of tetrahydrocannabinol in model membranes using neutron diffraction.

Small-angle neutron scattering was used to determine the intralamellar location of (-)-delta 8-tetrahydrocannabinol (delta 8-THC) in hydrated dipalmitoylphosphatidylcholine (DPPC) bilayers. Nuclear scattering density profiles were calculated from measurements on deuterium and non-deuterium-labelled inclusions (8.3% (w/w)) of delta 8-THC in DPPC multilayer samples. By comparing pairs of such nuclear density profiles, the locations of the deuterium labels were determined. Present results on the topography of delta 8-THC in membranes are compared with earlier X-ray measurements using iodine labelling. Whereas the position of the phenolic hydroxy group is similar in both types of measurement, a difference is found in the conformation of the terminal methyl groups of the cannabinoid side-chains. The X-ray measurements on dimyristoylphosphatidylcholine (DMPC) indicated that the iodine-labelled cannabinoid side-chains assume an all-trans orientation with the terminal iodine atom pointing inward into the membrane away from the tricyclic region while the neutron measurements indicate that the terminal CH3 group of delta 8-THC aligns itself at the level of the tricyclic ring system implying that the side chain exists in a more compact conformation perpendicular to the DPPC hydrocarbons. A Gaussian function analysis of the data indicates that the delta 8-THC molecule is significantly delocalized in the DPPC membrane in the liquid crystal phase. The mean location of delta 8-THC suggests that the active site on a membrane-embedded receptor protein will lie near the polar interface at the base of the phospholipid headgroups.

Orientational order of unsaturated lipids in the membranes of Acholeplasma laidlawii as observed by 2H-NMR.

Oleic acid specifically deuterated at fifteen different positions along the chain, including the double bond, was biosynthetically incorporated into the membrane lipids of the microorganism Acholeplasma laidlawii B. A detailed study of the dynamic conformation of these chains was carried out using deuterium nuclear magnetic resonance. The deuterium spectra fourteen different samples were recorded as a function of temperature over the range 0-41 degrees C. Spectra were obtained down to -52 degrees C for the sample enriched with oleic acid deuterated at the C-12' position. Above 20 degrees C, where the lipids are in the liquid crystal phase, a single quadrupolar powder pattern was observed for each C2H2 segment, except for the C-2' position which gave rise to a three-component spectrum characteristic for this position in both model and biological membranes. Simulation of this spectrum indicates that there are two conformations of the lipid molecule in the region of the C-2' segment of the sn-2 chain. The orientationa fluctuations of the fatty acid chain segments in the A. laidlawii membranes are described by the deuterium order parameters, and a striking similarity is shown to exist between the oleate chain conformation of the A. laidlawii membrane and a phospholipid model membrane. Remarkable similarities are also demonstrated in the A. laidlawii membrane enriched in palmitic and oleic fatty acids when the order parameter profiles are plotted at the same reduced temperature. Below 15 degrees C a second component, due to gel phase lipid, starts to appear in the spectra. This broad gel phase component grows at the expense of the liquid crystal phase component as the temperature is reduced. The spectra indicate that the center of the phase transition is at about -12 degrees C, in good agreement with DSC studies.

Effects of cholesterol on the orientational order of unsaturated lipids in the membranes of acholeplasma laidlawii. A 2H-NMR study.

We have investigated by 2H-NMR the effects of the incorporation of cholesterol on the orientational order of unsaturated lipid acyl chains in the membranes of acholeplasma laidlawii B. This is the only 2H-NMR study to date of the influence of cholesterol in a biological membrane using specifically labelled fatty acids. We observed the characteristic condensing effect of cholesterol on the lipid acyl chain order in the liquid crystalline phase. In terms of the percentage increase in the quadrupolar splittings, the presence of cholesterol has its greatest effect on the methyl end of the labelled oleoyl chains, with a maximum at the C-14 segment. In absolute terms, the perturbation is greatest in the carboxyl end of the chains. The temperature dependence of the 2H spectra for the cholesterol-containing membranes is very similar to that for the cholesterol-free membranes. The broad phase transition of the membrane lipids, which is characteristic for the samples lacking cholesterol, is apparently little affected by the presence of up to 27 mol% cholesterol. In addition, the temperature of onset of the phase transition is not significantly depressed by the presence of cholesterol.

Purpose-designed probes and their applications for dynamic NMR microscopy in an electromagnet.

The electromagnet provides a favorable environment for certain applications of NMR microscopy. These include plant imaging experiments and measurements of slow molecular diffusion, where high magnetic field gradients for the pulsed gradient spin echo (PGSE) technique are required. In this paper, two probes designed specifically for these two applications are described. In the first case, the open space within the probe has been maximized in order to incorporate environmental support systems for the plant, while in the second the smallest possible PGSE gradient coil former has been used to maximize the gradient strength. Examples are given of Dynamic NMR Microscopy experiments on a castor bean stem and on poly(ethylene oxide)/water solutions under shear thinning conditions.

A dielectric analysis of liquid and glassy solid glucose/water solutions.

Dielectric relaxation data covering a temperature range from above room temperature to below the glass transition for 40% (w/w) and 75% (w/w) glucose/water solutions in the frequency range between 5 and 13 MHz are presented. These data are used to obtain correlation times for the dielectric relaxation in the viscous liquid and the glass and are compared with correlation times determined from deuterium nuclear spin relaxation times [J. Chem. Phys., 110 (1999) 3472-3483]. The two sets of results have the same temperature dependence, but differ in magnitude by a factor of 3, implying that the relaxation is a small-step rotational diffusion. Both the structural relaxation (alpha process) and the slow beta process are present. In the 40% glucose/water sample, there is a dielectric relaxation attributable to the ice that forms at low temperature. It is shown that the reciprocal of the viscosity, the correlation time derived from the dielectric relaxation, and the dc conductivity have a similar dependence on temperature.

Molecular dynamics simulations and 2H NMR study of the GalCer/DPPG lipid bilayer.

Molecular dynamics simulations were performed on a two-component lipid bilayer system in the liquid crystalline phase at constant pressure and constant temperature. The lipid bilayers were composed of a mixture of neutral galactosylceramide (GalCer) and charged dipalmitoylphosphatidylglycerol (DPPG) lipid molecules. Two lipid bilayer systems were prepared with GalCer:DPPG ratio 9:1 (10%-DPPG system) and 3:1 (25%-DPPG system). The 10%-DPPG system represents a collapsed state lipid bilayer, with a narrow water space between the bilayers, and the 25%-DPPG system represents an expanded state with a fluid space of approximately 10 nm. The number of lipid molecules used in each simulation was 1024, and the length of the production run simulation was 10 ns. The simulations were validated by comparing the results with experimental data for several important aspects of the bilayer structure and dynamics. Deuterium order parameters obtained from (2)H NMR experiments for DPPG chains are in a very good agreement with those obtained from molecular dynamics simulations. The surface area per GalCer lipid molecule was estimated to be 0.608 +/- 0.011 nm(2). From the simulated electron density profiles, the bilayer thickness defined as the distance between the phosphorus peaks across the bilayer was calculated to be 4.21 nm. Both simulation systems revealed a tendency for cooperative bilayer undulations, as expected in the liquid crystalline phase. The interaction of water with the GalCer and DPPG oxygen atoms results in a strong water ordering in a spherical hydration shell and the formation of hydrogen bonds (H-bonds). Each GalCer lipid molecule makes 8.6 +/- 0.1 H-bonds with the surrounding water, whereas each DPPG lipid molecule makes 8.3 +/- 0.1 H-bonds. The number of water molecules per GalCer or DPPG in the hydration shell was estimated to be 10-11 from an analysis of the radial distribution functions. The formation of the intermolecular hydrogen bonds was observed between hydroxyl groups from the opposing GalCer sugar headgroups, giving an energy of adhesion in the range between -1.0 and -3.4 erg/cm(2). We suggest that this value is the contribution of the hydrogen-bond component to the net adhesion energy between GalCer bilayers in the liquid crystalline phase.

Silver ion dynamics in silver borate glasses: spectra and multiple-time correlation functions from 109Ag-NMR.

The motion of silver ions in (AgI)x-(Ag2O-B2O3)1-x glasses with AgI concentrations of x=0.5 and 0.7 was studied using 109Ag-NMR. The NMR spectra were analyzed in terms of a superposition of two different contributions. These are associated with Ag ions, which hop on vastly different time scales. The existence of dynamic heterogeneity, i.e. the existence of slow and of fast contributions to the hopping dynamics was directly demonstrated using a four-time stimulated-echo experiment. The results are compatible with an intrinsically exponential response. From measurements of the spectra and of two-time correlation functions a Gaussian distribution of energy barriers, hindering the ionic motion, could be mapped out.

Dynamics of the phosphate group in phospholipid bilayers. A 31P angular dependent nuclear spin relaxation time study.

To understand 31P relaxation processes and hence molecular dynamics in the phospholipid multilayer it is important to measure the dependence of the 31P spin-lattice relaxation time on as many variables as the physical system allows. Such measurements of the 31P spin-lattice relaxation rate have been reported both as a function of Larmor frequency and temperature for egg phosphatidylcholine liposomes (Milburn, M.P., and K.R. Jeffrey. 1987. Biophys. J. 52:791-799). In principle, the spin-lattice relaxation rate in an anisotropic environment such as a bilayer will be a function of the angle between the bilayer normal and the magnetic field. However, the measurement of this angular dependence has not been possible because the rapid (on the time-scale of the spin-lattice relaxation rate) diffusion of the lipid molecules over the curved surface of the liposome average this dependence (Milburn, M.P., and K.R. Jeffrey. 1987. Biophys. J. 52:791-799; Brown, M.F., and J.H. Davis. 1981. Chem. Phys. Lett. 79:431-435). This paper reports the results of the measurement of the 31P spin-lattice relaxation rate as a function of this angle, beta', (the angle between the bilayer normal and the external magnetic field) using samples oriented between glass plates. These measurements were made at high field (145.7 MHz) where the spin-lattice relaxation processes are dominated by the chemical shielding interaction (Milburn, M.P., and K.R. Jeffrey. 1987. Biophys. J. 52:791-799). A model of molecular motion that includes a fast axially symmetric rotation of the phosphate group (tau i approximately 10(-9) s) and a wobble of the head group tilt with respect to this rotation axis has been used to describe both the angular dependence of the spin-lattice relaxation and the spectral anisotropy. Cholesterol is seen to have a negligible effect on the motional properties of the phospholipid phosphate segment as measured by the orientation dependence of the spin-lattice relaxation.

Dynamics of the phosphate group in phospholipid bilayers. A 31P-1H transient Overhauser effect study.

Two recent studies have addressed the question of the dynamics of the phosphate in egg phosphatidylcholine multilayers by measurement and interpretation of 31P NMR spin-lattice relaxation. In the first (Milburn, M. P., and K. R. Jeffrey. 1987. Biophys. J. 52:791-799), the temperature dependences of the two contributions to the 31P relaxation rate, a dipolar interaction of the phosphorus with neighboring protons and a time-dependent anisotropic chemical shielding interaction were separately measured. A further study (Milburn, M. P., and K. R. Jeffrey. 1989. Biophys. J. 56:543-549) incorporated the anisotropic nature of phospholipid motions into the dynamic model of the headgroup motion by measuring the 31P spin-lattice relaxation time in oriented samples as a function of angle between the bilayer normal and the magnetic field. These angular dependent measurements were made at high field so that analysis could by made using the chemical shielding interaction because the 31P-1H dipolar interaction in phospholipid systems is complex and as such poorly understood. Nuclear Overhauser effect (NOE) studies have attempted to identify the important proton species contributing to the 31P-1H dipolar interaction (Yeagle, P. L., W. C. Hutton, C. Huang, and R. B. Martin. 1975. Biochemistry. 15:2121-2124) and despite some controversy in interpretation (Burns, R. A., R. E. Stark, D. A. Vidusek, and M. F. Roberts. 1983. Biochemistry. 22:5084-5090), it was generally agreed that the choline methyl and methylene protons are the major contributors to the 31P-1H NOE. To further understand the nature of the 31P-1H dipolar interaction, we carried out 31P-1H Transient Overhauser effect (TOE) measurements on egg phosphatidylcholine multilayers. Protons from both the lipids and water are important in understanding the TOE measurements in both D20 dispersions and H20 dispersions of egg PC. A quantitative analysis of the TOE has enabled the cross-relaxation rate between the phosphorus and the two proton types to be determined. It is suggested that these TOE experiments are a direct observation of the interaction between the phospholipid phosphate and surrounding water protons. The correlation time describing the relative motion of the phosphate group and the water molecules is on the order of 10- 11 s. The TOE measurements in phospholipid dispersions can be easily understood in terms of a straight forward model of the dipolar interaction and provide complementary information to NOE and T1 measurements.

Dynamics of the phosphate group in phospholipid bilayers. A 31P nuclear relaxation time study.

The spin-lattice relaxation time of the 31P nucleus in the phosphate group of egg yolk phosphatidylcholine multilamellar dispersions has been investigated at four resonant frequencies (38.9, 81.0, 108.9, and 145.7 MHz) in the temperature range from -30 degrees to 60 degrees C. The observed frequency dependence of the relaxation indicates that both dipolar relaxation and relaxation due to anisotropic chemical shielding are significant mechanisms. The experimental data have thus been modeled assuming both mechanisms and the analysis has allowed the contribution of each to the relaxation to be determined along with the correlation time for the molecular reorientation as a function of temperature. Dipolar relaxation was found to dominate at low nuclear magnetic resonance frequencies while at high frequencies the anisotropic chemical shift dominates. The correlation time of the phosphate group is on the order of 10(-9) s at 60 degrees C and increases to approximately 10(-7) s at -30 degrees C. It is observed that the freezing of the buffer which occurs at approximately -8 degrees C has a significant effect on the phosphate group reorientation. This effect of the freezing is to change the activation energy for the phosphate group reorientation from 16.9 KJ/mol above -8 degrees C to 32.5 KJ/mol below -8 degrees C.

14N NMR of lipid bilayers: effects of ions and anesthetics.

The interaction of divalent and trivalent metal cations, ferricyanide, a lipophilic ion (tetraphenylborate), and a local anesthetic (tetracaine) with the phosphocholine head group of egg lecithin was investigated by using wide-line 14N and 31P NMR. Measurements of the 14N quadrupolar splittings in the presence of a variety of perturbing agents demonstrated that the 14N NMR technique can be used to directly monitor ion or anesthetic binding. The 14N quadrupolar splitting (delta nu Q) is a measure of the order parameter of the C beta-N bond segment, and changes in delta nu Q as large as 3.5 kHz were observed. Moreover, a comparison of the changes in the quadrupolar splittings induced by the binding of ions or anesthetics provided a sensitive method of discriminating between these perturbing agents in their ability to alter the orientational order of the C beta-N bond segment of the phosphocholine moiety. Without exception, addition of metal ions or anesthetics always resulted in a decrease of the 14N delta nu Q. This reduction reflects a change in the average orientation or degree of motional averaging at the C beta-N bond segment position. In the case of metal ion binding, the strength of the interaction increased with the charge of the metal ion in the order Ca2+ less than Ln3+, in agreement with a previous 2H NMR study [Akutsu, H., & Seelig, J. (1981) Biochemistry 20, 7366-7373].(ABSTRACT TRUNCATED AT 250 WORDS)

Direct evidence for the partial dehydration of phosphatidylethanolamine bilayers on approaching the hexagonal phase.

X-ray diffraction studies on oriented multilayers of 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE) in the lamellar gel (L beta) and inverted hexagonal (HII) phases at various temperatures (5-50 degrees C) and relative humidities (0-100%) are reported. One-dimensional electron density profiles of the L beta phase bilayers were constructed to a resolution of better than 4 A using direct methods to solve for the phase problem. In addition, the electron density profiles were fitted favorably using a model in which the atomic groups were assumed to be Gaussian distributed [Wiener, M. C., & White, S. H. (1992) Biophys. J. 61, 434-447]. The X-ray data clearly demonstrate that, at 100% relative humidity (RH), POPE samples exist in two distinct L beta phases, differing primarily in the amount of water between the lamellae. As the hexagonal phase transition temperature is approached, 100% RH POPE samples partially dehydrate, releasing approximately 5 water molecules per phospholipid and experiencing on average a 3-A decrease in repeat spacing. The lower temperature hydrated L beta phase POPE electron density distribution resembles that obtained from the L beta phase 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) bilayers and is unlike the partially dehydrated POPE bilayers.