Pulsed Field Gradient NMR Experiments Research Articles

Induction of protein-like molecular architecture by self-assembly processes

One of the most intriguing self-assembly processes is the folding of peptide chains into native protein structures. We have developed a method for building protein-like structural motifs that incorporate sequences of biological interest. A lipophilic moiety is attached onto an Nα-amino group of a peptide chain, resulting in a ‘peptide-amphiphile’. The alignment of amphiphilic compounds at the lipid–solvent interface is used to facilitate peptide alignment and structure initiation and propagation. Peptide-amphiphiles containing potentially triple-helical structural motifs have been synthesized. The resultant head group structures have been characterized by circular dichroism and NMR spectroscopies. Evidence for a self-assembly process of peptide-amphiphiles has been obtained from: (a) circular dichroism spectra and melting curves characteristic of triple-helices, (b) one- and two-dimensional NMR spectra indicative of stable triple-helical structure at low temperatures and melted triple-helices at high temperatures, and (c) pulsed-field gradient NMR experiments demonstrating different self-diffusion coefficients between proposed triple-helical and non-triple-helical species. The peptide-amphiphiles described here provide a simple approach for building stable protein structural motifs using peptide head groups.

Diffusion of ammonia in silicalite studied by QENS and PFG NMR

Ammonia diffusivities in silicalite have been studied by quasi-elastic neutron scattering (QENS) and pulsed-field gradient (PFG) NMR for temperatures between 200 and 500 K and loadings from 1.5 up to 4.3 molecules per unit cell. The diffusion coefficients obtained by both techniques increase with increasing ammonia concentration. The QENS diffusivities refer to only a certain fraction of molecules, because during the time scale of the measurement the other part of molecules is essentially immobile, in interaction with silanol groups. During the much larger time scale of the PFG NMR experiment, ammonia molecules assume both states of mobility, leading to an average diffusivity which is smaller than the diffusivity of the mobile molecules recorded by QENS. The difference between the diffusivities derived from both techniques decreases when the proportion of immobile molecules is taken into account. The residence time of ammonia in interaction with silanol groups is about two orders of magnitude longer than with oxygen atoms.

Polymer additives mixture analysis using pulsed-field gradient NMR spectroscopy

The analysis of mixtures is a problem commonly faced in analytical chemistry. In this work, pulsed-field gradient NMR (PFG-NMR) experiments were used to analyze mixtures of polymer additives and simple polymer solutions. PFG-NMR experiments were also utilized to determine diffusion coefficients of the individual components of a mixture and in this way facilitate resonance assignments. This strategy is particularly useful for molecules containing isolated protons that are not amenable to the standard two-dimensional NMR experiments based on J-coupling. PFG-NMR was also used to edit the NMR spectra of polymer solutions by eliminating the resonances of fast-diffusing components such as low molecular weight additives or residual solvent. Diffusion ordered spectroscopy (DOSY) was used for the analysis of the PFG-NMR data. The DOSY method produces a two-dimensional spectrum that correlates chemical shift with the calculated diffusion coefficient, and simplifies the analysis of many complex mixtures. For well resolved resonances, the correct diffusion coefficient is unambiguously determined. The DOSY analysis was able to resolve the diffusion coefficients of most of the components in the mixtures examined. However, in some cases of overlapped resonances and components with similar diffusion coefficients, a single diffusion coefficient was determined which was the weighted average of the two components. ©1998 John Wiley & Sons, Ltd.

Minimal Lipidation Stabilizes Protein-Like Molecular Architecture

Peptide-amphiphiles with collagen-model head groups and dialkyl chain tails have been shown previously to self-assemble into highly ordered polyPro II-like triple-helical structures when dissolved in aqueous subphases. In the present study, we have examined peptide-amphiphiles containing monoalkyl chain tails for similar self-assembly behaviors. The structure of a collagen-model peptide has been characterized with and without an N-terminal hexanoic acid (C6) modification. Evidence for a self-assembly process of both the peptide and peptide-amphiphile has been obtained from (a) circular dichroism spectra and melting curves characteristic of triple-helices, (b) one-dimensional NMR spectra indicative of stable triple-helical structure at low temperatures and melted triple helices at high temperatures, and (c) pulsed-field gradient NMR experiments demonstrating different self-diffusion coefficients between proposed triple-helical and non-triple-helical species. The peptide-amphiphile appeared to form monomeric triple helices. The thermal stability of the collagen-like structure in the peptide-amphiphile was found to increase as the monoalkyl tail chain length is increased over a range of C6 to C16. The assembly process driven by the hydrophobic tail, albeit monoalkyl or dialkyl, may provide a general method for creating well-defined protein molecular architecture. Peptide-amphiphile structures possessing these alkyl moieties have the potential to be used for biomaterial surface modification to improve biocompatibility or, by mimicing fusion of viral envelopes with cellular membranes, as drug delivery vehicles.

Xenon Self-Diffusion in Organic Polymers by Pulsed Field Gradient NMR Spectroscopy

Xenon self-diffusion coefficients of commercially available organic polymers have been determined by application of pulsed field gradient (PFG) NMR spectroscopy. Xenon diffusion in the elastomeric terpolymer ethylene-propylene-diene (EPDM) was found to be about 20 times faster than in the more rigid semi-crystalline poly(propylene) (PP). In a polymer blend of both materials consisting of 80% PP and 20% EPDM both diffusion coefficients are in the same range and between the values of the pure materials. As SEM pictures show, this incompatible blend consists of small EPDM domains (1-3 Im) within the PP matrix. The root mean square displacement of xenon within the time interval of the PFG NMR experiment is greater than the domain sizes of EPDM. These results also evidence that xenon in this blend is mainly absorbed in EPDM domains and in the PP matrix near the PP/EPDM interface.

Phase Behavior and Characterization of Micellar and Cubic Phases in the Nonionic Surfactant C〈17〉E〈84〉/Water System. A PFG NMR, SAXS, Cryo-TEM, and Fluorescence Study

An extensive study of the diffusion behavior of the C〈17〉E〈84〉/water system is presented. The surfactant, when mixed with water, forms a micellar phase below ≈13 wt % and a cubic phase between ≈20−60 wt % (at 25 °C). In addition to the pulsed field gradient (PFG) NMR technique (used to determine the self-diffusion coefficients), the system has been investigated by small-angle X-ray scattering (SAXS), cryo-transmission electron microscopy (cryo-TEM), and time-resolved fluorescence quenching (TRFQ). The self-diffusion coefficient (D) and the transverse relaxation time (T2) of the surfactant molecules decrease significantly when going from the micellar to the cubic phase. The results of the PFG NMR, SAXS, cryo-TEM, and TRFQ experiments show that the cubic phase is composed of discrete aggregates. On the basis of the rapid transverse 1H NMR relaxation in the cubic phase, it is argued that the micellar building blocks of the cubic phase are nonspherical. Furthermore, the SAXS data for four different concentrations of the surfactant (25, 35, 45, and 55 wt %) in the cubic phase can be indexed to the space group Im3m. If the data from the NMR and the SAXS measurements are combined, a lifetime of the surfactant monomers in the micelles of 8 and 7 ms was obtained at 25 and 35 wt % C〈17〉E〈84〉, respectively.

A Model for Diffusive Transport through a Spherical Interface Probed by Pulsed-Field Gradient NMR

In biological systems, because of higher intracellular viscosity and/or the restriction of the diffusion space inside cells, the (apparent) diffusion coefficient of an intracellular species (e.g., water) is generally smaller than when it is in the extracellular medium. This difference affects the spin-echo signal attenuation in the pulsed field gradient NMR experiment and thus affords a means of separating the intracellular from the extracellular species, thereby providing a basis for studying transmembrane transport. Such experiments have commonly been analyzed using the macroscopic model of Kärger (see Adv. Magn. Reson. 21:1–89 (1988)). In our previous study, we considered a microscopic model of diffusive transport through a spherical interface using the short gradient pulse approximation (J. Magn. Reson. A114:39–46 (1995)). The spins in the external medium were modeled with the “partially absorbing wall” condition or as having a small but finite lifetime. In the present paper, we extend our treatment to the case in which there is no limitation upon the lifetime in either medium. We also consider a simple modification of Kärger’s model that more properly accounts for the restricted intracellular diffusion. Importantly, it was found that the exact solution within the short gradient pulse approximation developed here and the modified Kärger model are in close agreement in the (experimentally relevant) long-time limit. The results of this study show that when there is no limitation upon the lifetime of the transported species in either phase, the spin-echo attenuation curve is very sensitive to transport.

Spin echo diffraction in disordered media with single length scales

The spin echo diffraction observed in a pulsed field gradient NMR experiment is studied by simulation in computer generated two-dimensional disordered porous media characterized by a single length scale such as their mean pore diameter. In the propagator formulation of the pulsed field gradient experiments carried out in fluids confined inside porous media one expects to see a diffraction pattern characterizing the above length scale. We have explored the possibility of using this echo diffraction technique as a practical noninvasive tool to extract information about the pore geometries. It turns out that even for a disordered medium, the echodiffraction pattern picks up the dominant length scale. @S0163-1829~97!12417-1#

Detection of Insulin Aggregates with Pulsed-Field Gradient Nuclear Magnetic Resonance Spectroscopy

Self-association of metal-free human insulin in aqueous solution at pH 9.4 has been studied using pulsed-field gradient (PFG) NMR spectroscopy. The diffusion coefficients were measured for two different insulin concentrations, 0.72 mM in which the dimer is the predominant species and 3 mM which is a mixture of dimer and tetramer. In the more concentrated solution, exchange between the two aggregates is slow on the NMR chemical shift time scale. As a result, the dimer and tetramer give rise to separated resonances in the aromatic region of the 1H NMR spectrum. The diffusion coefficient determined for the tetramer by PFG NMR is 1.07 × 10 −6 cm 2 s −1 at 298 K, while that for the dimer is 1.38 × 10 −6 cm 2 s −1. The hydrodynamic diameters calculated for the tetramer and dimer are 46 and 36 Å, respectively, which are in good agreement with those measured by dynamic light scattering. The advantage of PFG NMR for the measurement of insulin aggregation in these solutions results from the chemical shift selectivity which allows diffusion coefficients to be directly calculated for the different aggregates. The T 1 and T 2 relaxation times of the dimer and tetramer aromatic protons were also measured in order to optimize the parameters of the PFG NMR experiment and correctly interpret the results of the measured spectra.

New pulsed field gradient NMR experiments for the detection of bound water in proteins

New H(2)O-selective homonuclear and heteronuclear 2D NMR experiments have been designed for the observation of protein hydration (PHOGSY, Protein Hydration Observed by Gradient Spectroscop Y). These experiments utilize selective excitation of the H(2)O resonance and pulsed field gradients for coherence selection and efficient H(2)O suppression. The method allows for a rapid and sensitive detection of H(2)O molecules in labelled and unlabelled proteins. In addition it opens a way to measure the residence time of water bound to proteins. Its application to uniformly (15)N-labelled FKBP-12 (FK-506 binding protein) is demonstrated.

Pulsed field gradient spin-echo NMR experiments in micellar solutions of the water/cetyltrimethylammonium bromide system

Pulsed Field Gradient NMR experiments, in their original time domain, have been carried out to separate both water and surfactant self-diffusion coefficients in the micellar solution of the system cetyltrimethylammonium bromide/water. These results have been supported by those obtained by NMR experiments performed with a Fourier Transform spectrometer. The water self-diffusion coefficients were used to calculate the obstruction effect of the micellar aggregates in accordance with a stoichiometric model already used for lyotropic mesophases. It was found that the micellar aggregates change shape with the concentration and are characterized by an average hydration number of approximately 9 +/- 3.

Analysis of mixtures based on molecular size and hydrophobicity by means of diffusion-ordered 2D NMR

Diffusion-ordered 2D NMR spectroscopy (DOSY) has been applied to the analysis of aqueous mixtures. Pulsed field gradient NMR (PFG-NMR) experiments were performed with special provisions for minimizing the effects of pulse-induced eddy currents and with sample spinning during the data acquisition period. The FT PFG-NMR data sets were processed with the data inversion program SPLMOD to resolve and identify the solutes on the basis of diffusion coefficients that normally are related to molecular size. Contour plots were then constructed in which conventional 1D NMR spectra were displayed in one dimension and diffusion spectra in the other

Pore geometry information via pulsed field gradient NMR

Studies of echo attenuation at long diffusion times in pulsed field gradient NMR experiments on a variety of rock core samples are interpreted in the light of recent theoretical analysis of the effect of pore geometry and surface relaxation. This study is motivated by the need to test the applicability of that theory to real rock systems.

7Li and 23Na nuclear magnetic resonance studies of transport and diffusion in liposomes. Comparison of transport rate constants estimated using pulsed field gradient and magnetization-transfer procedures

The ‘two-region approximation’ to pulsed field gradient (PFG) NMR studies of heterogeneous systems has been applied to the estimation of the rates of ionophore-mediated transmembrane exchange of Li+ in liposomes. Transport modulation of restricted diffusion, originally observed for hypophosphite in human erythrocytes, was also seen in the suspensions of liposomes with Na+ and Li+ transport. There was good agreement between the transmembrane exchange rate constants estimated from PFG and magnetization-transfer 7Li NMR experiments. Thus PFG experiments are likely to be a viable alternative to ‘dynamic’ NMR methods for measuring rapid transport rates in those biological systems which do not display a significant difference in chemical shift, or relaxation rate, of the transmembrane-distributed species.