Nuclear Magnetic Resonance Diffusion Experiments Research Articles

Hyperpolarized Laplace NMR.

Laplace nuclear magnetic resonance (NMR), dealing with NMR relaxation and diffusion experiments, reveals details of molecular motion and provides chemical resolution complementary to NMR spectra. Laplace NMR has witnessed a great progress in past decades due to the development of methodology and signal processing, and it has lots of extremely interesting applications in various fields, including chemistry, biochemistry, geology, archaeology, and medicine. The aim of this minireview is to give a pedagogically oriented overview of Laplace NMR. It does not provide a full literature review of the field, but, instead, it elucidate the benefits and features of Laplace NMR methods through few selected examples. The minireview describes also recent progress in multidimensional Laplace NMR and Laplace inversion methods. Furthermore, the potential of modern hyperpolarization methods as well as ultrafast approach to increase the sensitivity and time-efficiency of the Laplace NMR experiments is highlighted.

Hydrate Shell Growth Measured Using NMR.

Benchtop nuclear magnetic resonance (NMR) pulsed field gradient (PFG) and relaxation measurements were used to monitor the clathrate hydrate shell growth occurring in water droplets dispersed in a continuous cyclopentane phase. These techniques allowed the growth of hydrate inside the opaque exterior shell to be monitored and, hence, information about the evolution of the shell's morphology to be deduced. NMR relaxation measurements were primarily used to monitor the hydrate shell growth kinetics, while PFG NMR diffusion experiments were used to determine the nominal droplet size distribution (DSD) of the unconverted water inside the shell core. A comparison of mean droplet sizes obtained directly via PFG NMR and independently deduced from relaxation measurements showed that the assumption of the shell model-a perfect spherical core of unconverted water-for these hydrate droplet systems is correct, but only after approximately 24 h of shell growth. Initially, hydrate growth is faster and heat-transfer-limited, leading to porous shells with surface areas larger than that of spheres with equivalent volumes. Subsequently, the hydrate growth rate becomes mass-transfer-limited, and the shells become thicker, spherical, and less porous.

Finite difference diagonalization to simulate nuclear magnetic resonance diffusion experiments in porous media

A finite difference approach for computing Laplacian eigenvalues and eigenvectors in discrete porous media is derived and used to approximately solve the Bloch–Torrey equations. Neumann, Dirichlet, and Robin boundary conditions are considered and applications to simulate nuclear magnetic resonance diffusion experiments are shown. The method is illustrated with MATLAB examples and computational tests in one and two dimensions and the extension to three dimensions is outlined. © 2015 Wiley Periodicals, Inc. Concepts Magn Reson Part A 44A: 160–180, 2015.

Reduced activity of alkaline phosphatase due to host–guest interactions with humic superstructures

Nuclear Magnetic Resonance (NMR) spectroscopy was applied to directly study the interactions between the alkaline phosphatase enzyme (AP) and two different humic acids from a volcanic soil (HA-V) and a Lignite deposit (HA-L). Addition of humic matter to enzyme solutions caused signals broadening in (1)H-NMR spectra, and progressive decrease and increase of enzyme relaxation (T1 and T2) and correlation (τC) times, respectively. Spectroscopic changes were explained with formation of ever larger weakly-bound humic-enzyme complexes, whose translational and rotational motion was increasingly restricted. NMR diffusion experiments also showed that the AP diffusive properties were progressively reduced with formation of large humic-enzyme complexes. The more hydrophobic HA-L affected spectral changes more than the more hydrophilic HA-V. (1)H-NMR spectra also showed the effect of progressively greater humic-enzyme complexes on the hydrolysis of an enzyme substrate, the 4-nitrophenyl phosphate disodium salt hexahydrate (p-NPP). While AP catalysis concomitantly decreased NMR signals of p-NPP and increased those of nitrophenol, addition of humic matter progressively and significantly slowed down the rate of change for these signals. In agreement with the observed spectral changes, the AP catalytic activity was more largely inhibited by HA-L than by HA-V. Contrary to previous studies, in which humic-enzyme interactions were only indirectly assumed from changes in spectrophotometric behavior of enzyme substrates, the direct measurements of AP behavior by NMR spectroscopy indicated that humic materials formed weakly-bound host-guest complexes with alkaline phosphatase, and the enzyme catalytic activity was thereby significantly inhibited. These results suggest that the role of extracellular enzymes in soils may be considerably reduced when they come in contact with organic matter dissolved in the soil solution.

Membrane Interaction of Disease-Related Dynorphin A Variants

The membrane interaction properties of two single-residue variants, R6W and L5S, of the 17-amino acid neuropeptide dynorphin A (DynA) were studied by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. Corresponding gene mutations have recently been discovered in humans and causatively linked to a neurodegenerative disorder. The peptides were investigated in buffer and in isotropic solutions of q = 0.3 bicelles with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or DMPC (0.8) and 1,2-dimyristoyl-sn-glycero-3-phospho(1'-rac-glycerol) (DMPG) (0.2). The CD results and the NMR secondary chemical shifts show that R6W-DynA has a small α-helical fraction in buffer, which increases in the presence of bicelles, while L5S-DynA is mainly unstructured under all conditions studied here. R6W-DynA has an almost complete association with zwitterionic bicelles (∼90%, as probed by NMR diffusion experiments), similar to the behavior of wtDynA, while L5S-DynA has a weaker association (∼50%). For all peptides, the level of bicelle association is increased in negatively charged bicelles. The L5A-DynA peptide adopts a very shallow position in the headgroup region of the bicelle bilayer, as studied by paramagnetic spin relaxation enhancement experiments using paramagnetic probes. Similarly, the results show that R6W-DynA is more deeply buried in the bilayer, with only the C-terminal residues exposed to solvent, again more similar to the case of wild-type DynA. We suggest that the results presented here may explain the differences in cell toxicity of these disease-related neuropeptide variants.

Stabilization and Structure Determination of the Recombinant C-Terminal Domain of Nephila Clavipes Dragline Silk

Spider silk, as simple as it may look, holds a secret which lies in the processing of the silk proteins from a highly concentrated soluble state to the insoluble fiber. The conserved C-terminal domain of MaSp1, the dragline silk main protein, appears to play a key role in the control of solubility and fiber formation initiated by changes in ionic composition, mechanical stimuli or pH variations along the secretory gland ducts. Therefore, the study of its structure is necessary to understand this remarkable molecular behaviour. We have established a purification protocol to prevent the aggregation of the recombinant Nephila clavipes MaSp1 C-terminal domain. We have also investigated the structure of the protein using solution nuclear magnetic resonance (NMR), dynamic light scattering (DLS) and fluorescence spectroscopy. NMR diffusion and DLS experiments provide information about the oligomerization and aggregation of the protein. Further NMR experiments with the doubly 13C/15N labelled protein will lead to the complete determination of the protein structure.

The Dynamic Structure of Filamentous Tau

Filaments of the protein tau are a characteristic occurrence in Alzheimer disease and many other neurodegenerative disorders and the distribution of tau filaments correlates well with the loss of neurons and cognitive functions in Alzheimer disease. Filament formation of tau filaments is based on structural transitions from random coil to b-structure, to give the paired helical filaments (PHFs) which share common characteristics of amyloid fibrils. Protease digestion and solvent-accessibility studies demonstrated that the “core” of PHFs is mainly built from repeat sequences in the C-terminal half of the tau protein. The PHF core is surrounded by a “fuzzy coat”, of more than 200 residues that come from the N-terminal half of the protein as well as the C-terminus (Figure 1a). Electron paramagnetic resonance and nuclear magnetic resonance (NMR) suggested that residues within the fuzzy coat are highly flexible. Biochemical studies have shown that the fuzzy coat is important for tau aggregation as well as neurotoxicity. Herein we characterized the dynamic structure of PHFs formed by 441-residue tau (htau40), the longest isoform of tau present in the human central nervous system (Figure 1a), at single-residue level using NMR spectroscopy. We aggregated N-labeled htau40 into insoluble filaments. NMR diffusion experiments demonstrated that the observed NMR signals arises from aggregated tau protein with a molecular mass of more than 1 MDa (Figure 1b). In a two-dimensional heteronuclear single quantum coherence (HSQC) spectrum employing high-resolution magic-angle spinning (HR-MAS) (see Figure S1 in the Supporting Information), we observed about 260 signals (Figure 1c and Figure S2 and S3 in the Supporting Information). Sequencespecific resonance assignment of 244 of these signals (BMRB accession number: 17920; see Figure S2 in the Supporting Information) identified most of the residues in the N-terminal domain up to Thr212 and at the C-terminus starting at Val399. No signals were detected for residues between Thr212 and Val399, suggesting that residues in the central domain are too immobile to be detected by liquid-state NMR spectroscopy in agreement with previous studies. Comparison with monomeric htau40 revealed that the NMR resonances of many residues were strongly reduced in filamentous htau40 (Figure 1d,e). Most strikingly, the sections His121– Lys130 and Met1–Gly37 that are separated from the fibril core by 170 residues or more, showed changes in position and intensity of NMR signals (Figure 1e and Figure S2c in the Supporting Information). The presence of chemical exchange in these regions was further supported by N spin relaxation measurements (Figure 1 f and Supporting Figure S3). In agreement with chemical exchange, additional peaks were observed in close proximity to several of these residues (Figure 1 f and Figure S4 in the Supporting Information). The additional signals could not be connected in triple-resonance experiments or using exchange spectroscopy because of low signal-to-noise and signal overlap. Therefore, we assigned the additional peaks to the residue for which the assigned crosspeak of the major peak set had the greatest similarity in chemical shifts and paramagnetic relaxation enhancement (see Figure S5 in the Supporting Information). This procedure indicates that the additional peaks arise from residues at the Nand C-terminus. No peak doubling was observed at the Nand C-terminus in monomeric tau (see Figure S4 in the Supporting Information), highlighting the specificity of the multiple conformations in PHF tau. We revealed the identity of the PHF-specific conformations through measurements of paramagnetic relaxation enhancements (PREs), in which nitroxide spin labels are attached to cysteine residues at various positions in the PHF tau. The resulting broadening of amide resonances caused by enhanced relaxations rate through the paramagnetic nitroxide label, is quantified through the intensity ratios in the paramagnetic and diamagnetic states (Figure 2). The PRE effect scales as the inverse sixth power of the distance between the unpaired electron of the nitroxide unit and the NMR spin, providing a powerful probe of distances. N spin relaxation times (Figure 1 f) indicate that the fuzzy coat of PHFs is highly dynamic on a broad scale suggesting that the correlation time of the electron—amide proton internuclear vector is comparable to that of small water soluble proteins. Initially, we measured PRE broadening for PHFs with a [*] S. Bibow, Dr. M. D. Mukrasch, Prof. Dr. C. Griesinger, Prof. Dr. M. Zweckstetter Department of NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry Am Fassberg 11, 37077 Gcttingen (Germany) E-mail: mzwecks@gwdg.de

Determination of the Defining Boundary in Nuclear Magnetic Resonance Diffusion Experiments

While nuclear magnetic resonance diffusion experiments are widely used to resolve structures confining the diffusion process, it has been elusive whether they can exactly reveal these structures. This question is closely related to x-ray scattering and to Kac's "hear the drum" problem. Although the shape of the drum is not "hearable," we show that the confining boundary of closed pores can indeed be detected using modified Stejskal-Tanner magnetic field gradients that preserve the phase information and enable imaging of the average pore in a porous medium with a largely increased signal-to-noise ratio.

NMR of heparin API: investigation of unidentified signals in the USP-specified range of 2.12–3.00 ppm

This article addresses the identification and quantification of the chemical species resulting in resonances at 2.17 and 2.25ppm in the (1)H nuclear magnetic resonance (NMR) spectrum of pharmaceutical-grade heparin sodium. The NMR signals in question were first confirmed to arise from chemical moieties covalently attached to the heparin molecule through NMR diffusion experiments as well as chemical treatment of heparin active pharmaceutical ingredient (API) containing the resonances. The material responsible for the extra NMR signals was then demonstrated by NMR spiking studies to be something other than oversulfated chondroitin sulfate and was finally identified as an O-acetylation product of heparin through (13)C labeling experiments with subsequent NMR analysis. The extent of O-acetylation was quantified using three orthogonal techniques: (1)H NMR, ion chromatography, and headspace gas chromatography/mass spectrometry. The results of this work showed good agreement between the three quantitative methods developed to analyze the signals in the United States Pharmacopeia-specified region of 2.12-3.00ppm for heparin API.

NMR methods to monitor the enzymatic depolymerization of heparin

Heparin and the related glycosaminoglycan, heparan sulfate, are polydisperse linear polysaccharides that mediate numerous biological processes due to their interaction with proteins. Because of the structural complexity and heterogeneity of heparin and heparan sulfate, digestion to produce smaller oligosaccharides is commonly performed prior to separation and analysis. Current techniques used to monitor the extent of heparin depolymerization include UV absorption to follow product formation and size exclusion or strong anion exchange chromatography to monitor the size distribution of the components in the digest solution. In this study, we used 1H nuclear magnetic resonance (NMR) survey spectra and NMR diffusion experiments in conjunction with UV absorption measurements to monitor heparin depolymerization using the enzyme heparinase I. Diffusion NMR does not require the physical separation of the components in the reaction mixture and instead can be used to monitor the reaction solution directly in the NMR tube. Using diffusion NMR, the enzymatic reaction can be stopped at the desired time point, maximizing the abundance of larger oligosaccharides for protein-binding studies or completion of the reaction if the goal of the study is exhaustive digestion for characterization of the disaccharide composition. In this study, porcine intestinal mucosa heparin was depolymerized using the enzyme heparinase I. The unsaturated bond formed by enzymatic cleavage serves as a UV chromophore that can be used to monitor the progress of the depolymerization and for the detection and quantification of oligosaccharides in subsequent separations. The double bond also introduces a unique multiplet with peaks at 5.973, 5.981, 5.990, and 5.998 ppm in the 1H-NMR spectrum downfield of the anomeric region. This multiplet is produced by the proton of the C-4 double bond of the non-reducing end uronic acid at the cleavage site. Changes in this resonance were used to monitor the progression of the enzymatic digestion and compared to the profile obtained from UV absorbance measurements. In addition, in situ NMR diffusion measurements were explored for their ability to profile the different-sized components generated over the course of the digestion. FigureDOSY spectra of intact (blue) and digested (red) heparin illustrating the differences in their diffusion coefficients.

NMR Structure of Navel Orangeworm Moth Pheromone-Binding Protein (AtraPBP1): Implications for pH-Sensitive Pheromone Detection,

The navel orangeworm, Amyelois transitella (Walker), is an agricultural insect pest that can be controlled by disrupting male-female communication with sex pheromones, a technique known as mating disruption. Insect pheromone-binding proteins (PBPs) provide fast transport of hydrophobic pheromones through the aqueous sensillar lymph and promote sensitive delivery of pheromones to receptors. Here we present the three-dimensional structure of a PBP from A. transitella (AtraPBP1) in solution at pH 4.5 determined by nuclear magnetic resonance (NMR) spectroscopy. Pulsed-field gradient NMR diffusion experiments, multiangle light scattering, and (15)N NMR relaxation analysis indicate that AtraPBP1 forms a stable monomer in solution at pH 4.5 in contrast to forming mostly dimers at pH 7. The NMR structure of AtraPBP1 at pH 4.5 contains seven alpha-helices (alpha1, L8-L23; alpha2, D27-F36; alpha3, R46-V62; alpha4, A73-M78; alpha5, D84-S100; alpha6, R107-L125; alpha7, M131-E141) that adopt an overall main-chain fold similar to that of PBPs found in Antheraea polyphemus and Bombyx mori. The AtraPBP1 structure is stabilized by three disulfide bonds formed by C19/C54, C50/C108, and C97/C117 and salt bridges formed by H69/E60, H70/E57, H80/E132, H95/E141, and H123/D40. All five His residues are cationic at pH 4.5, whereas H80 and H95 become neutral at pH 7.0. The C-terminal helix (alpha7) contains hydrophobic residues (M131, V133, V134, V135, V138, L139, and A140) that contact conserved residues (W37, L59, A73, F76, A77, I94, V111, and V115) suggested to interact with bound pheromone. Our NMR studies reveal that acid-induced formation of the C-terminal helix at pH 4.5 is triggered by a histidine protonation switch that promotes rapid release of bound pheromone under acidic conditions.

Simultaneous convection compensation and solvent suppression in biomolecular NMR diffusion experiments

Thermal convection and high intensity solvent resonances can significantly hamper diffusion estimates in pulsed gradient spin-echo nuclear magnetic resonance diffusion experiments on biomolecule samples. To overcome these two problems, a new double functional NMR diffusion sequence, double echo PGSTE-WATERGATE, is presented. The new sequence provides excellent convection compensation and solvent suppression (with a suppression factor in excess of at least 10(5) in a single scan) in biomolecular NMR diffusion experiments. Due to its stimulated echo nature, the new sequence is much less susceptible to spin-spin relaxation than Hahn spin-echo based sequences. Furthermore, the new sequence is not susceptible to spin diffusion due to the application of bipolar pulsed gradients. The new sequence is also much easier to set up compared to previously developed stimulated echo based convection compensation and solvent suppression sequence. The utility of the new sequence is demonstrated on an aqueous lysozyme sample.

1H NMR signal broadening in spectra of alkane molecules adsorbed on MFI-type zeolites

The anisotropic behavior of C 1–C 6 alkane molecules adsorbed in MFI zeolite was studied by 1H nuclear magnetic resonance (NMR) using single-pulse excitation, Carr–Purcell–Meiboom–Gill (CPMG) pulse sequence, Hahn echo (HE) pulse sequence, and magic-angle spinning. The molecular order parameter was obtained by both static 2H NMR spectroscopy and molecular simulations. This yields an order parameter in the range of 0.28–0.42 for linear alkanes in MFI zeolite, whereas the parameter equals zero for FAU zeolite with a cubic symmetry. Thus, in the case of a zeolite with a non-cubic symmetry like MFI, the mobility of the molecules in one crystallite cannot fully average the dipolar interaction. As a consequence, transverse nuclear magnetization as revealed in the echo attenuation notably deviates from a mono-exponential decay. This information is of particular relevance for the performance of pulsed field gradient (PFG) NMR diffusion experiments, since the occurrence of non-exponential magnetization attenuation could be taken as an indication of the existence of different molecules or of molecules in different states of mobility.

NMR Structure of DREAM: Implications for Ca2+-Dependent DNA Binding and Protein Dimerization,

DREAM (calsenilin/KChIP3) is an EF-hand calcium-binding protein that binds to specific DNA sequences and regulates Ca2+-induced transcription of prodynorphin and c-fos genes. Here, we present the atomic-resolution structure of Ca2+-bound DREAM in solution determined by nuclear magnetic resonance (NMR) spectroscopy. Pulsed-field gradient NMR diffusion experiments and 15N NMR relaxation analysis indicate that Ca2+-bound DREAM forms a stable dimer in solution. The structure of the first 77 residues from the N-terminus could not be determined by our NMR analysis. The C-terminal DREAM structure (residues 78-256) contains four EF-hand motifs arranged in a tandem linear array, similar to that seen in KChIP1, recoverin, and other structures of the neuronal calcium sensor (NCS) branch of the calmodulin superfamily. Mg2+ is bound at the second EF-hand, whereas Ca2+ is bound functionally at the third and fourth sites. The first and second EF-hands form an exposed hydrophobic groove on the protein surface lined by side-chain atoms of L96, F100, F114, I117, Y118, F121, F122, Y151, L155, L158, and L159 that are highly conserved in all NCS proteins. An exposed leucine near the C-terminus (L251) is suggested to form intermolecular contacts with leucine residues in the hydrophobic groove (L155, L158, and L159). Positively charged side chains of Arg and Lys (Lys87, Lys90, Lys91, Arg98, Lys101, Arg160, and Lys166) are clustered on one side of the protein surface and may mediate electrostatic contacts with DNA targets. We propose that Ca2+-induced dimerization of DREAM may partially block the putative DNA-binding site, which may suggest as to how Ca2+ abolishes DREAM binding to DNA to activate the transcription of prodynorphin and other downstream genes in pain control.

Automated sample preparation station for studying self-diffusion in porous solids with NMR spectroscopy

In studies of gas diffusion in porous solids with nuclear magnetic resonance (NMR) spectroscopy the sample preparation procedure becomes very important. An apparatus is presented here that pretreats the sample ex situ and accurately sets the desired pressure and temperature within the NMR tube prior to its introduction in the spectrometer. The gas manifold that supplies the NMR tube is also connected to a microbalance containing another portion of the same sample, which is kept at the same temperature as the sample in the NMR tube. This arrangement permits the simultaneous measurement of the adsorption loading on the sample, which is required for the interpretation of the NMR diffusion experiments. Furthermore, to ensure a good seal of the NMR tube, a hybrid valve design composed of titanium, a Teflon® seat, and Kalrez® O-rings is utilized. A computer controlled algorithm ensures the accuracy and reproducibility of all the procedures, enabling the NMR diffusion experiments to be performed at well controlled conditions of pressure, temperature, and amount of gas adsorbed on the porous sample.

Epitope Mapping and Competitive Binding of HSA Drug Site II Ligands by NMR Diffusion Measurements

It is important to characterize drug-albumin binding during drug discovery and lead optimization as strong binding may reduce bioavailability and/or increase the drug's in vivo half-life. Despite knowing about the location of human serum albumin (HSA) drug binding sites and the residues important for binding, less is understood about the binding dynamics between exogenous drugs and endogenous fatty acids. In contrast to highly specific antibody-antigen interactions, the conformational flexibility of albumin allows the protein to adopt multiple conformations of approximately equal energy in order to accommodate a variety of ligands. Nuclear magnetic resonance (NMR) diffusion measurements are a simple way to quantitatively describe ligand-protein interactions without prior knowledge of the number of binding sites or the binding stoichiometry. This method can also provide information about ligand orientation at the binding site due to buildup of exchange-transferred NOE (trNOE) on the diffusion time scale of the experiment. The results of NMR diffusion and NOE experiments reveal multiple binding interactions of HSA with dansylglycine, a drug site II probe, and caprylate, a medium-chain fatty acid that also has primary affinity for HSA's drug site II. Interligand NOE (ilNOE) detected in the diffusion analysis of a protein solution containing both ligands provides insight into the conformations adopted by these ligands while bound in common HSA binding pockets. The results demonstrate the ability of NMR diffusion experiments to identify ternary complex formation and show the potential of this method for characterizing other biologically important ternary structures, such as enzyme-cofactor-inhibitor complexes.

Nuclear magnetic resonance studies on hydrolysis kinetics and micellar growth in solutions of surface‐active betaine esters

AbstractA series of surface‐active betaine esters of medium‐ to long‐chain alcohols has been synthesized. The hydrophobic parts used were decyl, dodecyl, tetradecyl, and oleyl chains. Ethyl betainate was prepared as a non‐surface‐active reference compound. The surfactants were characterized by critical micellization concentration (CMC), and base‐catalyzed hydrolysis was studied by using 1H nuclear magnetic resonance (NMR). Micellar catalysis was shown to have a strong influence on the hydrolysis. This means that the hydrolysis rates are concentration dependent. For dodecyl betainate, the effect of the degradation products (dodecanol and betaine) on the micellar shape was investigated by NMR diffusion experiments. The degradation products were shown to induce micellar growth.

Simulations of NMR-detected diffusion in suspensions of red cells: the "signatures" in q-space plots of various lattice arrangements

Coherence effects from pulsed field-gradient spin-echo (PGSE) nuclear magnetic resonance diffusion experiments have been observed and characterized for diffusants in many heterogeneous systems, ranging from porous materials to cell suspensions. The resulting coherence patterns appear in plots of the normalized PGSE signal intensities as a function of the spatial wave vector Q in a so-called q-space plot. The origin of these phenomena and their mathematical and physical underpinnings are now well established. We have conducted a number of studies of diffusion-coherence phenomena in suspensions of red blood cells and have made extensive use of computer simulations of molecular diffusion in virtual lattices of cells to aid in the interpretation and analysis of experimental data. In the current work we extended the canonical model used in these studies to investigate the effect that varying the packing arrangement of cells in the suspension has on the coherence patterns, as seen in q-space plots. We show that changes in the packing arrangement of cells are reflected in the q-space plots and in the results of diffusion tensor analysis and thus we speculate upon the possible clinical importance of these findings.

Simulations of Molecular Diffusion in Lattices of Cells: Insights for NMR of Red Blood Cells

The pulsed field-gradient spin-echo (PGSE) nuclear magnetic resonance (NMR) experiment, conducted on a suspension of red blood cells (RBC) in a strong magnetic field yields a q-space plot consisting of a series of maxima and minima. This is mathematically analogous to a classical optical diffraction pattern. The method provides a noninvasive and novel means of characterizing cell suspensions that is sensitive to changes in cell shape and packing density. The positions of the features in a q-space plot characterize the rate of exchange across the membrane, cell dimensions, and packing density. A diffusion tensor, containing information regarding the diffusion anisotropy of the system, can also be derived from the PGSE NMR data. In this study, we carried out Monte Carlo simulations of diffusion in suspensions of “virtual” cells that had either biconcave disc (as in RBC) or oblate spheroid geometry. The simulations were performed in a PGSE NMR context thus enabling predictions of q-space and diffusion tensor data. The simulated data were compared with those from real PGSE NMR diffusion experiments on RBC suspensions that had a range of hematocrit values. Methods that facilitate the processing of q-space data were also developed.

NMR studies of the aggregation of glucagon-like peptide-1: formation of a symmetric helical dimer

Nuclear magnetic resonance (NMR) spectroscopy reveals that higher-order aggregates of glucagon-like peptide-1-(7–36)-amide (GLP-1) in pure water at pH 2.5 are disrupted by 35% 2,2,2-trifluoroethanol (TFE), and form a stable and highly symmetric helical self-aggregate. NMR spectra show that the helical structure is identical to that formed by monomeric GLP-1 under the same experimental conditions [Chang et al., Magn. Reson. Chem. 37 (2001) 477–483; Protein Data Bank at RCSB code: 1D0R], while amide proton exchange rates reveal a dramatic increase of the stability of the helices of the self-aggregate. Pulsed-field gradient NMR diffusion experiments show that the TFE-induced helical self-aggregate is a dimer. The experimental data and model calculations indicate that the dimer is a parallel coiled coil, with a few hydrophobic residues on the surface that may cause aggregation in pure water. The results suggest that the coiled coil dimer is an intermediate state towards the formation of higher aggregates, e.g. fibrils.