Through the application of static and high resolution solid state NMR techniques we are observing detailed structural and dynamic information pertaining to acetylcholine and its covalent analogue bromoacetylcholine in the ligand binding site on the nicotinic acetylcholine receptor. Using high resolution cross polarization magic angle spinning experiments (CP-MAS) we have been able to assign resonances from bound acetylcholine. This approach has enabled us to determine detailed dynamic information about acetylcholine in the binding site. A change in electrostatic environment, observed through the perturbation in chemical shift (-1.6ppm) upon binding has been shown to be similar to that which has been simulated for homologous ligands bound to the acetylcholinesterase whose binding site is proposed to have homology to that of the nAcChoR. Using homonuclear recoupling techniques we are currently investigating the structure of carbon-13 enriched acetylcholine bound to the receptor with the goal of elucidating the bound conformation. Currently the geometries within the crystalline ligand have been obtained and are in good agreement with the values expected from the crystal structure. Through the covalent attachment of deuterated (N(CD3)3-bromoacetylcholine we have been able to carry out dynamic studies of the covalent ligand in the binding site. Through the application of isopotential spinning techniques we have been able to obtain aligned nAcChoR membranes as determined by 31P NMR. Using such membranes labelled with N(CD3)3 bromoacetylcholine we are defining the orientation of the N(CD3)3 group within the ligand to the macroscopic orientation of the nAcChoR membranes.
Sedimentation in non-Newtonian suspending fluids such as polymer solutions is of great industrial interest because of its relevance to separation processes, composite materials and foods. However, relatively little work has been devoted to the study of sedimentation of concentrated suspensions in such media. There is experimental evidence that flow-induced structures arise when particles are suspended in non-Newtonian media [1], and one would expect such phenomena to affect sedimentation strongly. It has been demonstrated recently that NMRI can provide a useful means for studying sedimentation in Newtonian fluids [2], and we are applying these ideas to examine the qualitatively different phenomena that occur in rheologically complex fluids. Experiments were performed with a horizontal bore 0.6T GE CSI imaging spectrometer. Concentration profiles were obtained with a modified 1-D Multiple Spin Echo sequence to obtain, after processing, pure intensity profiles. 2-D imaging was performed with a standard spin-warp imaging sequence. In this work, we examined the sedimentation of concentrated, noncolloidal suspensions in the viscous-dominated flow regime. Our results show evidence, not reported before, of non-Newtonian effects in the sedimentation behavior caused by the elastic properties of the suspending fluid. The study of the NMR concentration profiles during the sedimentation process, and in particular the dynamics of the supernatant/suspension interface, reveal qualitatively new behavior: the speed of the sedimenting front is not constant in time. Imaging experiments show structure development (aggregates) in the suspension during the sedimentation. We found a significant tendency toward alignment of the structures in the flow direction, and clear signs of dynamic aggregation/segregation of the structures during the sedimentation process. 1.- J. Michele, R. Ptzol, and R. Donis, "Alignment and Aggregation Effects in Suspensions of Spheres in Non-Newtonian Media". Rheol. Acta, 16, 317-321 (1977). 2.- M.K. Cheung, R.L. Powell, and M.J. McCarthy, "Sedimentation of Noncolloidal Bidisperse Suspensions", AIChE J., 42, 1: 271-276, 1996
One of the most important NMR techniques for the study of liquid crystals is the removal of homonuclear dipolar couplings. Since the introduction of the multiple-pulse WAHUHA sequence, many dipolar-decoupling sequences have been proposed. Recently, a method based on z-rotational decoupling has been published, and is abbreviated as MSHOT-3 for Magic Sandwich High Order Truncation [N. C. Nielsen and co-workers, J. Chem. Phys. 106, 7571(1997); Chem. Phys. Lett. 273, 297 (1997)]. It gives very good results for CRAMPS experiments. On the other hand, we have found that it does not offer much advantage over MREV-8 for the removal of dipolar couplings in the proton NMR of liquid crystals, for which the proton-proton dipolar couplings are larger than those in solids with magic-angle spinning. The effect of removing proton dipolar couplings for the 13C NMR of liquid crystal solutions has also been examined for MSHOT-3 and other sequences, including MREV-8, frequency-switched Lee-Goldburg, and BLEW-48. The best results was obtained for the BLEW-48 sequence, but the result of MSHOT-3 was disappointing. The BLEW-48 sequence has also been applied to the technique of proton-detected local field spectroscopy, and the results will be presented.
Dietary fats are hydrolyzed by water-soluble lipases acting on the surface of aggregates formed by acylglycerols, phospholipids, and bile salts (BS). We are studying the aggregate structure of chemical mixtures of BS, monoglycerides (MG), and fatty acids (FA) in order to model the later stages of fat digestion and rationalize the nutritional therapies used to treat digestive dysfunction. The two-dimensional NMR methods used previously for steroids have been extended in order to assign all 1H and 13C resonances in C8-based model digestive mixtures. Paramagnetic (Mn++) broadening of the 1H and 13C spectra has been used to reveal aqueous-assessible functional groups, and 2D nuclear Overhauser enhancements have identified proton pairs that are located close in space. With the addition of dynamic (quasielastic) laser light scattering (QLS), the shape of the model digestive aggregates has been deduced from the dependence of scattered light intensity on hydrodynamic radius Rh. Based on the experimental results from NMR and QLS, a prolate ellipsoid model is proposed to describe molecular organization in BS/MG/FA aggregates.
X. Tang1, Y. Cheng1, S. Peled1, W. E. Maas2, D. G. Cory1 1. Department of Nuclear Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 2. Bruker Analytical Systems, Inc., 15 Fortune Drive, Billerica, MA 01821 A fundamental limitation for NMR studies of heterogeneous samples is that the local variation of the bulk susceptibility introduces background gradients. These background gradients lead to excess line broadening in static samples. As is well known from Garroway's work[1], MAS averages out variations in the local field introduced by changes in the isotropic bulk susceptibility. Here we demonstrate that MAS may be used to avoid the commonly observed signal attenuation seen for spins diffusing in local gradients during echo experiments. The application of this method permits accurate measurements of diffusion behavior in heterogeneous samples, including studies of excised tissue and cultured cells. 1. A. N. Garroway, J. Magn. Reson. 49, 168 (1982).
We have extended the utility of NMR as a technique to probe porous media microstructure over length scales of ~ 100 - 2000 microns by using the spin 1/2 noble gases (3He and 129Xe) imbibed into the system's pore space. There are two primary advantages to this technique, relative to the existing liquid-saturated NMR method [1]: (i) gas diffusion coefficients are orders of magnitude larger than for liquids, hence noble gases can diffuse over distances much greater than a pore size before spin de-coherence; and (ii) noble gases interact much less strongly with grain surfaces than do most liquid molecules, allowing inter-pore noble gas diffusion without spin de-polarization. We used noble gas NMR to determine the restricted diffusion (or tortuosity) of randomly packed bead samples, measuring the time-dependent noble gas diffusion coefficient D(t) using pulsed-field- gradient NMR techniques modified for application in heterogenous systems. We studied uniform size bead samples, with the bead diameter in different samples ranging from 100 microns to 4 mm, and found that the time-dependent noble gas diffusion coefficient decreases with increasing diffusion time, reaching an asymptote of ~ 0.64 of the free gas diffusion coefficient, which is consistent with numerical calculations [2] and previous NMR measurements in random bead packs saturated with water [1]. We are currently applying the imbibed-noble gas NMR technique to other porous media (e.g. reservoir rocks), and investigating the imaging of pore spaces using laser-polarized noble gas [3]. 1. L. L. Latour et al., J. Magn. Reson., A 101, 342 (1993). 2. P.N. Sen, et al., Phys. Rev., B 49, 49 (1994). 3. Y.-Q. Song, et. al., J. Magn. Reson., A 115, 127 (1995).
Unusual phospholipids which are of potential interest in the field of protein immobilisation and reconstruction are examined using 2H and 31P NMR techniques. The phospholipids differ from other model membranes by the introduction of fatty acids containing acetylenic groups and by the usage of unconventional head groups. Dynamic NMR methods - comprising lineshape, relaxation and 2D exchange NMR investigations - have been applied to evaluate the molecular features of these systems over a large temperature range. Moreover, the composition of the samples has been varied by the addition of further components like cholesterol, etc. It is found that the motional behaviour of such systems is rather complex exhibiting various intra- and intermolecular contributions. The conformational and overall reorientational motions were examined by 2H NMR investigations of selectively deuterated compounds (deuterated fatty acids); 31P NMR studies were used to determine the head group dynamics and overall motions including lateral diffusion processes. The analysis of the available experimental data could provide a comprehensive picture about the dynamical properties of this new class of phospholipids. The same is true for the structural behaviour (conformational order, chain order) which also has been studied by variable temperature FT IR methods. Finally, we will report on recent results from13C NMR studies performed on these model membranes.
Lipophilic compounds play an important role in all sciences. In environmental studies, the binding and permeation of hydrophobic compounds, such as organochlorines and fungicidal amines, is directly linked to their toxicity. Pharmacologically, a variety of hydrophobic local anesthetics exhibit a correlation between increased membrane partitioning and effect. Other studies of hydrophobic molecular interactions with membranes include amine drugs and antibacterial peptides. In the present study, we have observed changes in the 1H NMR spectrum of a suspension of large unilamellar vesicles (LUVs) upon the addition of the lipophilic anion, tetraphenylborate (TPB). In the absence of an ion carrier, the addition of TPB to an LUV suspension causes a time dependent change (k=(7.60.3)x10-4 s-1) in the chemical shift of the -N(CH3)3 resonance which may be used to calculate a permeability coefficient (P=(1.00.3)x10-11m s-1). This permeability is more representative of the rate of Na+ transport, since it limits the rate at which TPB may traverse the bilayer. In the presence of a Na+ carrier (monensin), the chemical shift time dependence is removed and the addition of TPB now causes a concentration dependent shift of the EPC resonances. From the concentration dependence of the chemical shift, the affinity of TPB for the EPC choline headgroup may be quantified. Effects on the binding affinity by membrane cholesterol content and salt concentrations in the intra- and extravesicular solutions will be discussed. These studies may provide an NMR method of obtaining permeability coefficients and binding constants of similar lipophilic molecules without the need for fluorescent or paramagnetic probes.
It has recently been shown that dilute liquid crystalline solutions can be used to observe residual dipolar couplings in partially oriented macromolecules, while still maintaining high resolution NMR spectra. The advantages of this technique have recently been demonstrated in high resolution solution NMR spectra of proteins.1 We have used a mixture of DMPC and DHPC, which form bicelles,2 to obtain spectra of partially oriented carbohydrates in D2O. Preliminary results for sucrose show significant differences between one-bond C-H coupling constants, measured in the presence and the absence of bicelles. We will discuss the extraction of structural information from these spectra, as well as the application of this technique to other more complicated carbohydrates. 1. N. Tjandra and A. Bax, Science , 278, 1111 (1997) 2. C. R. Sanders and J. P. Schwonek, Biochem.,31,8898 (1992)
By restricting the focus on the ability of a series of homologous lipids to promote hexagonal (HII) phase formation in a phosphatidylethanolamine (PtdEtn) matrix, we are able to quantitatively analyze the potency of hydrocarbon chains in tail regions to promote membrane curvature (1,2). In order to investigate if the tail group effects can be considered independently, a dilute concentration (1-5 mole %) of a series of phosphatidylethanol (PtdEth) or PtdEtn with variable acyl chain composition [M(14:0), P(16:0), S(18:0), L(18:2)] were incorporated into liposomes of POPtdEtn and the effect on the La\HII transition (TH) was studied by 31P NMR. The potency of PtdEth and PtdEtn species (e.g. DM, DP, DS, MO, OP, SO, OS, DL, SL) to promote the HII phase was measured and shown to coincide with the calculated value within the experimental error ( 0.3oC). Furthermore, two natural phospholipids, EggPtdEth and EggPtdEtn, were also examined. The potency to promote the HII phase for EggPtdEth is -1.10.3oC/mf%, which is in good agreement with the calculated value of -1.10.3oC/mf%. The potency for EggPtdEtn was found as -0.10.3oC/mf%, which is also in agreement with the calculated value of -0.20.3oC/mf%. This investigation provides a quantitative analysis which supports the hypothesis that the effects on membrane curvature by lipid tail regions can be considered independently. Supported by PHS AA07215, AA07463, AA07186. 1. Lee et al. Biochem., 35:3677-3684 (1996). 2. Lee et al. 5th Scientific Meeting of Society for Magnetic Resonance in Medicine (1997).
*Anthony A. Ribeiro, Zhimin Zhou and Christian R. H. Raetz, Duke NMR Center and Departments of Biochemistry and Radiology, Duke University Medical Center, Durham, North Carolina 27710 The Lipid A (endotoxin) moiety of Gram negative bacterial lipopolysaccharides may be a suitable target to address simultaneously issues of antibiotic resistance, outer membrane permeability and Gram- negative sepsis. As these glycolipids are phosphorylated disaccharides of glucosamine derivatized with several long fatty acyl chains, 1D 1H and 31P NMR has previously been used to assess sites of acylation and phosphorylation. The Lipid A of bacterial mutants have further substitutions linked to either 1 or 4' phosphate, such as phosphoethanolamine and 4-amino-4-deoxyarabinose which are associated with polymixin resistance. We have designed and implemented multidimensional 1H, 31P and 13C NMR strategies which elucidate the NMR assignments and unequivocally establish the phosphate and glycosidic linkages of several known and two newly identified Lipid A compounds purified from wild type and mutant bacterial sources.
Modern NMR spectroscopy is a prospective nondestructive analytical tool for investigating the composition and isotope fractionation of natural samples. So far, this technique found no wide application in geochemistry, especially, to dating the fossil samples of geochemical and geological origin. A number of such samples, powdered or sorbed on inorganic oxides, including peats, coals, mammoth and bison bones and meat of different age (from 10 to 40 kY), were investigated without any pretreatment (with except for drying and grinding) by means of multinuclear solid state MAS NMR. Additionally, some samples were extracted by water or various solvents and concentrated by evaporation or were chemically modified (fermentation or enzymatic degradation) and then measured by multinuclear (1H, 13C, 15N, 2D) and 2D high resolution NMR spectroscopy. A range of spectral characteristics, such as aromatic region integrals in 13C and 1H spectra and isotope fractionation (12C/13C and 1H/2H) for peats and extracts from peats, some cross-peaks intensities in 2D spectra and an extent of aminoacids racemization in the presence of optical lanthanide shift reagents, were corellated with data on 14C labelling and spore-pollen diagrams.
At very high magnetic fields strengths ( 14 Tesla) the xenon-131 gas phase NMR spectrum shows a well resolved quadrupolar splitting. Quadrupolar coupling of a non-spherical (S > 1/2) nuclei with an electric field gradient will occur when the electrical isotropy of the surrounding electron cloud is disturbed. The experimental results suggest that the origin of the observed splitting in the gas phase is twofold. A largely field independent portion arises from the interaction of the xenon with the gas container wall (i.e. Pyrex tube). In addition, a field dependent splitting is present, leading to a splitting of 3.9 Hz at 16.92 Tesla field strength. Comparison with multiple quantum filtered NMR of liquid xenon demonstrates that the field dependent portion does not originate from surface interactions. The discovery of a surface independent quadrupolar splitting indicates that an electrical polarization of the xenon electron cloud is induced by the magnetic field.
Deuterium quadrupolar couplings are invaluable in the study of the structure and dynamics of partially oriented materials, such as nematic liquid crystals, since local order parameters can be derived directly from these anisotropically averaged couplings. Unfortunately, the negligible deuterium chemical shifts make the assignment of the deuterium spectra in uniformly labelled materials very difficult, even though the couplings themselves are often well-resolved. To overcome this problem we have developed a two-dimensional deuterium-carbon correlation experiment. Correlation of the deuterium spectrum with the easily-assigned 13C spectrum, makes the deuterium assignment straight-forward. Additionally, overlap in the 1D spectrum is usually resolved. The experiment requires a transfer of polarization from 2H to 13C that is effective over the complete width of the deuterium spectrum, and which is dominated by one-bond transfer i.e. cross-polarization (CP) from the 2H to the 13C to which it is directly bonded. Conventional Hartmann-Hahn matching is unsuitable for this task, since the RF matching condition is strongly dependent on the value of the quadrupolar coupling. By adapting techniques based on adiabatic sweeps of the RF developed for CP in spin-1/2 systems, and by choosing the RF parameters such that the dependence of their match condition on quadrupolar coupling is minimised, we have been able to obtain broad-band CP from deuterium to 13C. The resulting Deuterium Correlation (DECOR) spectrum of the model nematic liquid crystal 5CB allows the resolution, assignment and measurement of each of the quadrupolar couplings. With short cross-polarisation times, the CP is almost exclusively one-bond, while longer cross-polarization times (i.e. slower sweeps of the linearly ramped RF) reveal longer range transfers which help to resolve remaining ambiguities, and can potentially provide additional information about the molecular structure and dynamics.
NMR techniques based on the Stejskal-Tanner Pulsed-Gradient Spin-Echo (PGSE) method allow measurement of time-dependent self-diffusion of liquids, and have many important applications, such as determining the porosity of liquid-saturated porous media [1,2]. To date, such pulsed-field gradient NMR techniques have not been fully exploited to measure gas diffusion, primarily because of the small NMR signals provided by thermally polarized gases. Recently, however, there has been a surge of interest in gas phase NMR, following the application of optical techniques using lasers to produce large nuclear spin polarizations (~10%) in the spin 1/2 noble gases (129Xe and 3He) [3]. Much of the interest in laser-polarized noble gas NMR has focused on imaging, especially of the lung gas space in animals [4] and humans, as well as dissolved phase spectroscopy and imaging of the fluid-soluble 129Xe species as it is transported to tissues in the body by the bloodstream. However, noble gas NMR diffusion measurements may also have wide applicability in both biomedical and materials systems, for example in determining the microstructure of porous and granular media (tortuosity of reservoir rocks, lung airways, etc.). We present the first systematic application of Stejskal-Tanner pulsed field gradient NMR techniques to measure gas diffusion, using both thermally polarized and laser-polarized noble gas. Diffusion coefficients over a range of diffusion times have been measured with spin, stimulated and gradient echo techniques in thermal and laser-polarized gas samples. The powerful time-dependent nature of this technique is demonstrated for the first time with gas-phase NMR, by measuring the restricted diffusion of noble gases in the pore space of randomly-packed beads. 1. P. Callaghan, et. al., Nature, 351, 467 (1991). 2. M. Hurlimann, et. al., J. Magn. Reson., A111, 169 (1994). 3. W. Happer, et. al., Phys. Rev. A, 29, 3092 (1984). 4. M.S. Albert, et. al., Nature, 370, 199 (1994).
The hydrate clathrates are a class of solids in which guest molecules occupy cages in a host structure formed from H-bonded water molecules. The normally unstable empty clathrate is stabilized by inclusion of the guest. While the general crystal structure of the type-I hydrate clathrates has already been established, the proposed mechanism for their nucleation and growth awaits experimental confirmation. This being our objective, we have studied both the melting and kinetics of formation of type I xenon hyhdrate clathrate using hyperpolarized xenon-129 NMR. The >2000 fold signal enhancement afforded by this method permits single scan observation of cage formation at less than parts-per-thousand levels. The signals arising from xenon-129 enclathrated in the tetrakaidecahedral (large) and dodecahedral (small) cages are well resolved, permitting the time dependence of each type to be measured independently. Our kinetic model incorporates the time variations of the densities of the gaseous xenon, the xenon enclathrated into the small and large cages, and the xenon-129 magnetization of all three species. The pressure and NMR data at 223 K support a mechanism whereby the hydrate nucleates by first forming a small cage. This nucleation site then serves as a template for the formation of large and additional small cages. It turns out that this mechanism is consistent with recent molecular dynamics simulations of the formation process.