Pulsed Field Gradient NMR Diffusion Research Articles

Temperature Driven Annealing of Perforations in Bicellar Model Membranes

Bicellar model membranes composed of 1,2-dimyristoylphosphatidylcholine (DMPC) and 1,2-dihexanoylphosphatidylcholine (DHPC), with a DMPC/DHPC molar ratio of 5, and doped with the negatively charged lipid 1,2-dimyristoylphosphatidylglycerol (DMPG), at DMPG/DMPC molar ratios of 0.02 or 0.1, were examined using small angle neutron scattering (SANS), (31)P NMR, and (1)H pulsed field gradient (PFG) diffusion NMR with the goal of understanding temperature effects on the DHPC-dependent perforations in these self-assembled membrane mimetics. Over the temperature range studied via SANS (300-330 K), these bicellar lipid mixtures exhibited a well-ordered lamellar phase. The interlamellar spacing d increased with increasing temperature, in direct contrast to the decrease in d observed upon increasing temperature with otherwise identical lipid mixtures lacking DHPC. (31)P NMR measurements on magnetically aligned bicellar mixtures of identical composition indicated a progressive migration of DHPC from regions of high curvature into planar regions with increasing temperature, and in accord with the "mixed bicelle model" (Triba, M. N.; Warschawski, D. E.; Devaux, P. E. Biophys. J.2005, 88, 1887-1901). Parallel PFG diffusion NMR measurements of transbilayer water diffusion, where the observed diffusion is dependent on the fractional surface area of lamellar perforations, showed that transbilayer water diffusion decreased with increasing temperature. A model is proposed consistent with the SANS, (31)P NMR, and PFG diffusion NMR data, wherein increasing temperature drives the progressive migration of DHPC out of high-curvature regions, consequently decreasing the fractional volume of lamellar perforations, so that water occupying these perforations redistributes into the interlamellar volume, thereby increasing the interlamellar spacing.

From Sulfur−Amine Solutions to Metal Sulfide Nanocrystals: Peering into the Oleylamine−Sulfur Black Box

In this Article, we present our findings on the formation of metal sulfide nanocrystals from sulfur-alkylamine solutions. By pulsed field gradient diffusion NMR along with the standard toolbox of 1D and 2D NMR, we determined that sulfur-amine solutions used as a sulfur precursor exist as alkylammonium polysulfides at low temperatures. Upon heating to temperatures used in nanocrystal synthesis, the polysulfide ions react with excess amine to generate H(2)S, which combines with the metal precursor to form metal sulfide. Four different reaction pathways were found, each of which produced H(2)S and the byproducts identified in this Article. Thioamides were identified as an intermediate and were shown to exhibit much more rapid kinetics than sulfur-alkylamine solutions at low temperatures in the synthesis of metal sulfide nanocrystals.

Microstructure of polymer hydrogels studied by pulsed field gradient NMR diffusion and TEM methods

The microstructure of various alginate gels have been studied by pulsed field gradient NMR (PFG NMR) and transmission electron microscopy (TEM). The reduced diffusivity of dendrimer diffusion within the gels has been obtained from PFG NMR diffusion experiments. The polymer strand radius, an important microstructural property, has been extracted from various diffusion models. The results agree well with the polymer strand radii obtained from image analysis of the corresponding TEM micrographs.

Tracing Pore-Space Heterogeneities in X-Type Zeolites by Diffusion Studies

Pore-space homogeneity of zeolite NaX was probed by pulsed field gradient (PFG) NMR diffusion studies with n-butane as a guest molecule. At a loading of 0.75 molecules per supercage, a wide spectrum of diffusivities was observed. Guest molecules in the (well-shaped) zeolite crystallites were thus found to experience pore spaces of quite different properties. After loading enhancement to 3 molecules per supercage, however, molecular propagation ideally followed the laws of normal diffusion in homogeneous media. At sufficiently high guest concentrations, sample heterogeneity was thus found to be of no perceptible influence on the guest mobilities anymore.

Dynamics of Water in Polyelectrolyte Multilayers: Restricted Diffusion and Cross-Relaxation

The diffusion properties of water in polyelectrolyte multilayers (PEM) are investigated by pulsed field gradient diffusion NMR. The dependence of the mean-square displacement on the observation time does not agree to Gaussian diffusion, suggesting restricted diffusion in a porous structure. However, the extraction of a pore size in a model of restricted diffusion yields a very large pore size of several micrometers. The additional influence of cross-relaxation of water and polymer spins is investigated in Goldman-Shen experiments. These demonstrate a strong influence of cross-relaxation rates on diffusion echo decays, such that pore sizes obtained from the model of restricted diffusion have to be corrected. Corrected pore sizes are about 4 microm and reflect the existence of domains of lower polymer density and thus faster water diffusion. These heterogeneities occur upon PEM preparation at high salt content for large layer numbers and are detected in the surface morphology, too.

NMR characterization of 1,1′‐binaphthyl‐2,2′‐diyl hydrogen phosphate binding to chiral molecular micelles

NMR spectroscopy was used to characterize the binding of the chiral compound 1,1'-binaphthyl-2,2'-diyl hydrogen phosphate (BNP) to five molecular micelles with chiral dipeptide headgroups. Molecular micelles have covalent linkages between the surfactant monomers and are used as chiral mobile phase modifiers in electrokinetic chromatography. Nuclear overhauser enhancement spectroscopy (NOESY) analyses of (S)-BNP:molecular micelle mixtures showed that in each solution the (S)-BNP interacted predominately with the N-terminal amino acid of the molecular micelle's dipeptide headgroup. NOESY spectra were also used to generate group binding maps for (S)-BNP:molecular micelle mixtures. In these maps, percentages are assigned to the (S)-BNP protons to represent the relative strengths of their interactions with a specified molecular micelle proton. All maps showed that (S)-BNP inserted into a previously reported chiral groove formed between the molecular micelle's dipeptide headgroup and hydrocarbon chain. In the resulting intermolecular complexes, the (S)-BNP protons nearest to the analyte phosphate group were found to point toward the N-terminal Halpha proton of the molecular micelle headgroup. Finally, pulsed field gradient NMR diffusion experiments were used to measure association constants for (R) and (S)-BNP binding to each molecular micelle. These K values were then used to calculate the differences in the enantiomers' free energies of binding, Delta(DeltaG). The NMR-derived Delta(DeltaG) values were found to scale linearly with electrokinetic chromatography (EKC) chiral selectivities from the literature.

Proton transport in choline dihydrogen phosphate/H3PO4 mixtures

Mixtures of the plastic crystal material choline dihydrogen phosphate [Choline][DHP] and phosphoric acid, from 4.5 mol% to 18 mol% H(3)PO(4), were investigated and shown to have significantly higher proton conductivity compared to the pure [Choline][DHP]. This was particularly evident from the electrochemical hydrogen reduction reaction and the proton NMR diffusion measurements, in addition to ionic conductivity measured from the impedance spectroscopy. The ionic conductivity was observed to increase by more than an order of magnitude in phase I (i.e. the highest temperature solid phase in [Choline][DHP]) reaching up to 10(-2) S cm(-1). The multinuclear NMR spectroscopy data suggest that, at least on the timescale of the NMR measurement, the H(+) cations and [DHP] anions are equivalent in both phases. The pulsed field gradient NMR diffusion measurements of the 18 mol% acid sample indicate that all three ions are mobile, however the H(+) diffusion coefficient is an order of magnitude higher than for the [Choline] cation or the [DHP] anion, and therefore conduction in these materials is dominated by proton conductivity. The thermal stability, as measured by TGA, is unaffected with increasing acid additions and remains high; i.e. no significant mass loss below 200 °C.

Thermodynamics of partitioning of substance P in isotropic bicelles

The temperature dependence of the partition of a neuropeptide, substance P (SP), in isotropic (q = 0.5) bicelles was investigated by using pulsed field gradient NMR diffusion technique. The partition coefficient decreases as the temperature is increased from 295 to 325 K, indicating a favorable (negative) enthalpy change upon partitioning of the peptide. Thermodynamic analysis of the data shows that the partitioning of SP at 300 K is driven by the enthalpic term (DeltaH) with the value of - 4.03 kcal mol(-1), while it is opposed by the entropic term (-TDeltaS) by approximately 1.28 kcal mol(-1) with a small negative change in heat capacity (DeltaC(p)). The enthalpy-driven process for the partition of SP in bicelles is the same as in dodecylphosphocholine (DPC) micelles, however, the negative entropy change in bicelles of flat bilayer surface is in sharp contrast with the positive entropy change in DPC micelles of highly curved surface, indicating that the curvature of the membrane surface might play a significant role in the partitioning of peptides.

Scaling Law of Poly(ethylene oxide) Chain Permeation through a Nanoporous Wall

This paper presents a study of the permeation of poly(ethylene oxide) (PEO) chains through the nanoporous wall of hollow polymeric capsules prepared by self-assembly of polyelectrolytes. We employ the method of pulsed field gradient (PFG) NMR diffusion to distinguish chains in different sites, i.e., in the capsule interior and free chains in the dispersion, by their respective diffusion coefficient. From a variation of the observation time, the time scale of the molecular exchange between both sites and thus the permeation rate constant is extracted from a two-site exchange model. Permeation rate constants show two different regimes with a different dependence on chain length. This suggests a transition between two different mechanisms of permeation as the molecular weight is increased. In either regime, the permeation time can be described by a scaling law tau approximately N (b) , with b = (4)/ 3 for short chains and b = (1)/ 3 for long chains. We discuss these exponents, which clearly differ from the theoretical predictions for chain translocation.

Insights in the Antibacterial Action of Poly(methyloxazoline)s with a Biocidal End Group and Varying Satellite Groups

The antimicrobial activity of poly(2-methyl-1,3-oxazoline)s (PMOX) with the antimicrobial N,N-dimethyldodecylammonium (DDA) end group is greatly dependent on the nature of the group at the distal end of the polymer, the satellite group. Three comparable PMOX with a DDA end group and different satellite groups (methyl, decyl, hexadecyl) were investigated with respect to the reasons for the huge differences in their biocidal behavior. Static light scattering (SLS) and pulsed field gradient diffusion NMR measurements revealed that the samples show comparable aggregation conduct, thus, not being responsible for the varying biological activity. Experiments using different liposomal systems as models for bacterial cell membranes have been performed. It was found that differential interactions between the respective polymers and the phospholipid membranes constitute the reason for the varying effectiveness observed in antimicrobial susceptibility determinations.

Solution Conformation, Backbone Dynamics and Lipid Interactions of the Intrinsically Unstructured Malaria Surface Protein MSP2

Merozoite surface protein 2 (MSP2), one of the most abundant proteins on the surface of the merozoite stage of Plasmodium falciparum, is a potential component of a malaria vaccine, having shown some efficacy in a clinical trial in Papua New Guinea. MSP2 is a GPI-anchored protein consisting of conserved N- and C-terminal domains and a variable central region. Previous studies have shown that it is an intrinsically unstructured protein with a high propensity for fibril formation, in which the conserved N-terminal domain has a key role. Secondary structure predictions suggest that MSP2 contains long stretches of random coil with very little α-helix or β-strand. Circular dichroism spectroscopy confirms this prediction under physiological conditions (pH 7.4) and in more acidic solutions (pH 6.2 and 3.4). Pulsed field gradient NMR diffusion measurements showed that MSP2 under physiological conditions has a large effective hydrodynamic radius consistent with an intrinsic pre-molten globule state, as defined by Uversky. This was supported by sedimentation velocity studies in the analytical ultracentrifuge. NMR resonance assignments have been obtained for FC27 MSP2, allowing the residual secondary structure and backbone dynamics to be defined. There is some motional restriction in the conserved C-terminal region in the vicinity of an intramolecular disulfide bond. Two other regions show motional restrictions, both of which display helical structure propensities. One of these helical regions is within the conserved N-terminal domain, which adopts essentially the same conformation in full-length MSP2 as in corresponding peptide fragments. We see no evidence of long-range interactions in the full-length protein. MSP2 associates with lipid micelles, but predominantly through the N-terminal region rather than the C terminus, which is GPI-anchored to the membrane in the parasite.

Diffusive diffraction phenomenon in a porous polymer material observed by NMR using radio-frequency field gradients

Pulsed field gradient NMR diffusion experiments can, in principle, lead to the "diffusive diffraction" phenomenon. In practice, its observation by gradients of the static magnetic field is difficult in real systems because they involve internal gradients due to the static magnetic field (necessary for polarizing nuclear spins). This latter drawback can be circumvented by using gradients of the radio-frequency (rf) field (the other magnetic field used in any NMR experiment). For the first time, by means of rf gradients, a so-called diffusive diffraction peak has been observed in a real porous system and its position provides a value of the mean distance between pores; this can be further complemented by the mean pore size determined from the dependence of the apparent diffusion coefficient with respect to the diffusion interval.

Cyclic modular beta-sheets.

The development of peptide beta-hairpins is problematic, because folding depends on the amino acid sequence and changes to the sequence can significantly decrease folding. Robust beta-hairpins that can tolerate such changes are attractive tools for studying interactions involving protein beta-sheets and developing inhibitors of these interactions. This paper introduces a new class of peptide models of protein beta-sheets that addresses the problem of separating folding from the sequence. These model beta-sheets are macrocyclic peptides that fold in water to present a pentapeptide beta-strand along one edge; the other edge contains the tripeptide beta-strand mimic Hao [JACS 2000, 122, 7654] and two additional amino acids. The pentapeptide and Hao-containing peptide strands are connected by two delta-linked ornithine (deltaOrn) turns [JACS 2003, 125, 876]. Each deltaOrn turn contains a free alpha-amino group that permits the linking of individual modules to form divalent beta-sheets. These "cyclic modular beta-sheets" are synthesized by standard solid-phase peptide synthesis of a linear precursor followed by solution-phase cyclization. Eight cyclic modular beta-sheets 1a-1h containing sequences based on beta-amyloid and macrophage inflammatory protein 2 were synthesized and characterized by 1H NMR. Linked cyclic modular beta-sheet 2, which contains two modules of 1b, was also synthesized and characterized. 1H NMR studies show downfield alpha-proton chemical shifts, deltaOrn delta-proton magnetic anisotropy, and NOE cross-peaks that establish all compounds but 1c and 1g to be moderately or well folded into a conformation that resembles a beta-sheet. Pulsed-field gradient NMR diffusion experiments show little or no self-association at low (</=2 mM) concentrations. Changes to the residues in the Hao-containing strands of 1c and 1g improve folding and show that folding of the structures can be enhanced without altering the sequence of the pentapeptide strand. Well-folded cyclic modular beta-sheets 1a, 1b, and 1f each have a phenylalanine directly across from Hao, suggesting that cyclic modular beta-sheets containing aromatic residues across from Hao are better folded.

The Cold Denatured State Is Compact but Expands at Low Temperatures: Hydrodynamic Properties of the Cold Denatured State of the C-terminal Domain of L9

A point mutation of a small globular protein, the C-terminal domain of L9 destabilizes the protein and leads to observable cold-denaturation at temperatures above zero. The cold denatured state is in slow exchange with the native state on the NMR time scale, and this allows the hydrodynamic properties of the cold unfolded state and the native state to be measured under identical conditions using pulsed-field gradient NMR diffusion measurements. This provides the first experimental measurement of the hydrodynamic properties of a cold unfolded protein and its folded form under identical conditions. Hydrodynamic radii of the cold-induced unfolded states were measured for a set of temperatures ranging from 2 °C to 25 °C at pD 6.6 in the absence of denaturant. The cold unfolded state is compact compared to the urea or acid unfolded state and a trend of increasing radii of hydration is observed as the temperature is lowered. These observations are confirmed by experiments on the same protein at pD 8.0, where it is more stable, in the presence of a modest concentration of urea. The expansion of the cold-denatured state at lower temperatures is consistent with the temperature dependence of hydrophobic interactions.

Solution NMR techniques for investigating colloidal nanocrystal ligands: A case study on trioctylphosphine oxide at InP quantum dots

Colloidal semiconductor nanocrystals or quantum dots are an important building block in bottom-up nanotechnology. They consist of an inorganic, crystalline core surrounded by a monolayer of organic ligands. As these ligands can be modified or exchanged for others, they provide a convenient way to give the quantum dots functionality. Here, we show that solution NMR is a useful tool to investigate the ligands of colloidal nanocrystals using InP quantum dots with trioctylphospine oxide ligands. Combining H 1 – C 13 HSQC spectroscopy with pulsed field gradient diffusion NMR, an unequivocal identification of the resonances of the bound ligands is possible. This leads to the determination of the diffusion coefficient of the nanocrystals in solutions and allows to verify capping exchange procedures. By calibrating the surface area of the NMR resonances using a solute of known concentration, the density of ligands at the nanocrystal surface can be quantified. This demonstrates that a dynamic equilibrium exists between bound and free ligands. Analysis of the corresponding adsorption isotherm – determined using H 1 NMR – leads to an estimation of the free energy of adsorption and the free energy of ligand–ligand interaction at the nanocrystals surface.

Solution NMR Spectroscopy as a Useful Tool to Investigate Colloidal Nanocrystal Dispersions from the Capping Ligand's Point of View

AbstractColloidal semiconductor nanocrystals or quantum dots are an important building block in bottom-up nanotechnology. They consist of an inorganic, crystalline core surrounded by a monolayer of organic ligands. As these ligands can be modified or exchanged for others, they provide a convenient way to give the quantum dots functionality. Here, we show that solution NMR techniques, including diffusion pulsed field gradient spectroscopy, is a very useful tool to investigate the ligands of colloidal nanocrystals. This is demonstrated using InP quantum dots with trioctylphospine oxide ligands as an example. Combining 1H-13C HSQC spectroscopy with pulsed field gradient diffusion NMR, an unequivocal identification of the resonances of the bound ligands is possible. This leads to the determination of the diffusion coefficient of the nanocrystals in solution and allows to verify capping exchange procedures. By calibrating the surface area of the NMR resonances using a solute of known concentration, the density of ligands at the nanocrystal surface can be quantified. We could demonstrate that a dynamic equilibrium exists between bound and free ligands. Analysis of the corresponding adsorption isotherm - determined using 1H NMR - leads to an estimation of the free energy of adsorption and the free energy of ligand-ligand interaction at the nanocrystals surface. Similar investigations are in progress on capped PbSe and ZnO2 nanoparticles. Preliminary results strongly support the generic nature of the approach described for the case of TOPO capped InP nanocrystals.

In Situ 1H NMR Study on the Trioctylphosphine Oxide Capping of Colloidal InP Nanocrystals

We used trioctylphosphine oxide (TOPO) capped colloidal InP nanocrystals (Q-InP|TOPO) to explore the potential of solution 1H NMR spectroscopy in studying in situ the capping and capping exchange of sterically stabilized colloidal nanocrystals. The spectrum of Q-InP|TOPO shows resonances of free TOPO, superimposed on broadened spectral features. The latter were assigned to TOPO adsorbed at Q-InP by means of pulsed field gradient diffusion NMR and 1H-13C HSQC spectroscopy. The diffusion coefficient of Q-InP|TOPO nanocrystals was inferred from the decay of the adsorbed TOPO NMR signal. The corresponding hydrodynamic diameter correlates well with the diameter of Q-InP. By using the resolved methyl resonance of adsorbed TOPO, the packing density of TOPO at the InP surface can be estimated. Spectral hole burning was used to demonstrate explicitly that the adsorbed TOPO resonances are heterogeneously broadened. Exchange of the TOPO capping by pyridine was demonstrated by the disappearance of the resonances for adsorbed TOPO and the appearance of pyridine resonances in the 1H NMR spectrum. These results show that solution NMR spectroscopy should be considered a powerful technique for the in situ study of the capping of sterically stabilized colloidal nanocrystals.

Binding and Folding of Melittin in the Presence of Ganglioside GM1 Micelles

In this work we examine the binding and folding of the membrane-active peptide, melittin in the presence of ganglioside GM1 micelle. The membrane bilayer is capable of inducing folding to small proteins and peptides upon binding. Using two-dimensional NMR techniques we have shown that at low concentration, GM1 micelle is able to induce an extended helical conformation to MLT. The pulsed-field gradient diffusion NMR study indicates that the peptide partition into GM1 micelle along with about 32% binding. While looking for the binding between MLT and GM1 using saturation transfer difference NMR spectroscopy, Val5, Leu9, Thr11, Ile17, Ser18, and Trp19 have been identified as the residues that are in close proximity to GM1 micelles.

Sticking Probability on Zeolites

The sticking coefficient, i.e., the probability that, on hitting the surface of a nanoporous particle (zeolite), a molecule shall be able to enter the intracrystalline space, is a key quantity for the application of such materials in heterogeneous catalysis and molecular sieving. On the basis of pulsed field gradient NMR diffusion measurements and molecular dynamics simulations, typical values of this probability are found to be close to one. They exceed previous estimates on the basis of IR uptake measurements by many orders of magnitude.

Interaction of ferulic acid derivatives with human erythrocytes monitored by pulse field gradient NMR diffusion and NMR relaxation studies

Ferulic acid (Fer), a natural anti-oxidant and chemo-protector, is able to suppress experimental carcinogenesis in the forestomach, lungs, skin, tongue and colon. Several Fer derivatives have been suggested as promising candidates for cancer prevention, being the biological activity related also to the capacity of partitioning between aqueous and lipid phases. In the present work, pulsed field gradient (PFG) NMR diffusion measurement and NMR relaxation rates have been adopted for investigating the interaction of three Fer derivatives (Fer-C11, Fer-C12 and Fer-C13) with human erythrocytes. Binding to the erythrocyte membrane has been shown for all derivatives, which displayed a similar interaction mode such that the aromatic moiety and the terminal part of the alkyl chain were the most affected. Quantitative analysis of the diffusion coefficients was used to show that Fer-C12 and Fer-C13 display higher affinity for the cell membrane when compared with Fer-C11. These findings agree with the higher anti-oxidant activity of the two derivatives.