Low-resolution NMR Research Articles

Evidence for S331-G-S-L within the amyloid core of myocilin olfactomedin domain fibrils based on low-resolution 3D solid-state NMR spectra.

Myocilin-associated glaucoma is a protein-conformational disorder associated with formation of a toxic amyloid-like aggregate. Numerous destabilizing single point variants, distributed across the myocilin olfactomedin β-propeller (OLF, myocilin residues 245-504, 30 kDa) are associated with accelerated disease progression. In vitro, wild type (WT) OLF can be promoted to form thioflavin T (ThT)-positive fibrils under mildly destabilizing (37°C, pH 7.2) conditions. Consistent with the notion that only a small number of residues within a protein are responsible for amyloid formation, 3D 13C-13C solid-state NMR spectra show that OLF fibrils are likely to be composed of only about one third of the overall sequence. Here, we probe the residue composition of fibrils formed de novo from purified full-length OLF. We were able to make sequential assignments consistent with the sequence S331-G-S-L334. This sequence appears once within a previously identified amyloid-prone region (P1, G326AVVYSGSLYFQ) internal to OLF. Since nearly half of the pairs of adjacent residues (di-peptides) in OLF occur only once in the primary structure and almost all the 3-residue sequences (tri-peptides) are unique, remarkably few sequential assignments are necessary to uniquely identify specific regions of the amyloid core. This assignment approach could be applied to other systems to expand our molecular comprehension of how folded proteins undergo fibrillization.

Steady-State Free Precession sequences for high and low field NMR spectroscopy in solution: Challenges and opportunities

The receptivity of NMR spectroscopy is low when compared to other techniques. Historically, increasing the strength of the static magnetic field has been the major approach to increase NMR sensitivity. In recent years several polarization transfer protocols have been used to enhance the signal-to-noise ratio (SNR), although they require special accessories and/or sample preparation. In this paper, we consider both the challenges and opportunities of steady-state free precession (SSFP) pulse sequences as a simple and efficient alternative to enhance SNR, in standard high-resolution and benchtop low-resolution NMR spectrometers. The maximum gain in these sequences is obtained with the shortest time between the pulses (Tp). However, when Tp<T2, the SSFP signal contains FID and echo components which lead to phase, intensity, and truncation artifacts on spectra obtained by Fast Fourier transform (FT). Several phase alternation SSFP sequences were used to cancel the echo component and minimize these problems in the FT spectra. Krylov base diagonalization method (KBDM) was used to eliminate the phase and truncation problems in spectra acquired by SSFP pulse sequences and can be a viable alternative to FT. The experiments were performed in high and low resolution (bench top) NMR spectrometers and significant enhancements in SNR of low receptivity nuclei such as 13C and 15N could be achieved. The SSFP sequences were also shown to enhance SNR in nuclei with high receptivity such as 19F and 31P, in very dilute samples, as is common in environmental and biological samples.

Rheology, Sticky Chain, and Sticker Dynamics of Supramolecular Elastomers Based on Cluster-Forming Telechelic Linear and Star Polymers

We elucidate the properties of unentangled telechelic poly(isobutylene) (PIB) chains in bulk forming dynamic micellar networks mediated by endgroups capable of hydrogen-bonding and π–π interactions. The effects of the molecular architecture and type of endgroup on the properties of networks are studied by a combination of small-angle X-ray scattering (SAXS), rheology, low-resolution NMR, and dielectric spectroscopy (DS). It is found that star-shaped molecules form more time-stable networks with larger and somewhat more distantly arranged aggregates compared to their linear counterparts. Using stickers providing less hydrogen bonds speeds up terminal flow significantly, yet surprisingly, the nanoscale morphology, apparent activation energies, and the timescale of endgroup fluctuations probed by DS are very similar across the two sample series. The correlation of results from the three dynamic methods (rheology, NMR, and DS) thus fortifies previous findings for linear chains: (i) even for star molecules, terminal stress relaxation is governed by single-chain relaxation rather than the reorganization of whole micelles, and (ii) the cluster-related relaxation time accessed by DS has no trivial relation to the actual sticky bond lifetime, the determination of which is concluded to be an open problem.

Influence of fat crystallization in W/O emulsions on the water droplet size determination by NMR diffusometry

HypothesisIt is expected that low resolution (LR) NMR diffusometry enables (more) accurate water droplet size determination for solid-fat based water-in-oil (W/O) emulsions with (sub)-micron size water droplets in comparison to liquid-oil based W/O emulsions due to hindered extra-droplet water diffusion. ExperimentsW/O emulsions with a volume-weighed mean diameter of about 1 µm and a solid fat content (SFC) ranging from 0% to 74% were produced. The aqueous phase contained the ionic marker tetraphenylphosphonium chloride (TPPCl). The water droplet size was estimated using LR and high resolution (HR) NMR diffusometry. FindingsHR-NMR diffusometry showed that the diffusion behavior of water and TPPCl was different, indicating water diffusion beyond the droplet’s interfacial boundaries. From a certain SFC onwards, a slower echo decay was observed for the water molecules, thus decreasing the overestimation of the water droplet size in (sub)micron W/O emulsions. For those emulsions, the solid fat matrix is believed to hinder extra-droplet water diffusion, which is most likely to be related to the increased tortuosity of the diffusive path in the porous fat crystal network. Using LR-NMR, it can be verified whether the water echo attenuation is mono-exponential or bi-exponential by increasing the gradient pulse duration for the maximum gradient strength, which is more convenient for routine analysis compared to HR-NMR.

Applicability of Confocal Raman Microscopy to Observe Microstructural Modifications of Cream Cheeses as Influenced by Freezing.

Confocal Raman microscopy is a promising technique to derive information about microstructure, with minimal sample disruption. Raman emission bands are highly specific to molecular structure and with Raman spectroscopy it is thus possible to observe different classes of molecules in situ, in complex food matrices, without employing fluorescent dyes. In this work confocal Raman microscopy was employed to observe microstructural changes occurring after freezing and thawing in high-moisture cheeses, and the observations were compared to those obtained with confocal laser scanning microscopy. Two commercially available cream cheese products were imaged with both microscopy techniques. The lower resolution (1 µm/pixel) of confocal Raman microscopy prevented the observation of particles smaller than 1 µm that may be part of the structure (e.g., sugars). With confocal Raman microscopy it was possible to identify and map the large water domains formed during freezing and thawing in high-moisture cream cheese. The results were supported also by low resolution NMR analysis. NMR and Raman microscopy are complementary techniques that can be employed to distinguish between the two different commercial formulations, and different destabilization levels.

Stability of the Meat Protein Type I Collagen: Influence of pH, Ionic Strength, and Phenolic Antioxidant.

The water-holding capacity (WHC) is among the key factors in determining the quality of meat and its value, which is strongly influenced by the content and quality of the connective tissue proteins like collagen. Therefore, the factors that influence the proteins’ stability, e.g., pH, ionic strength, and the antioxidants which are used to increase the meat shelf-life, also affect the WHC. The interaction of collagen, whose structure is strongly influenced by the interaction with water molecules, can be studied following the behavior of water diffusion by low-resolution 1H NMR experiments. The present study is addressed to study the collagen stability as a function of pH, ionic strength, and phenolic antioxidants like catechin. The experimental study demonstrated how the 1H NMR time domain (TD) experiments are able to evaluate the hydration properties of collagen, not only as a function of ionic strength and pH, but also in determining the ability of catechin to interact both on the surface of the collagen fibrils and inside the fibrillar domain.

Does the presence of fibres affect the microstructure and in vitro starch digestibility of commercial Italian pasta?

Abstract • Dry pasta is a staple food in many countries of the world and it is traditionally made of durum wheat semolina. The aim of this study was to evaluate the impact of pasta composition on microstructure, water absorption and mobility during cooking and nutritional properties of two commercial spaghetti made of durum wheat semolina (S) and whole durum wheat semolina (WS). Scanning Electron Microscopy (SEM) revealed no significant microstructural differences between the uncooked pasta samples, whereas the presence of fibre altered the integrity of the protein network in the cooked samples. Moreover, the water absorption during cooking and the water mobility at molecular level were investigated as weight increase and by low-resolution 1H NMR experiments, respectively. Compared to durum wheat spaghetti, the presence of fibre increased the water absorption while decreased the proton relaxation times 1H T2 and 1H T1 indicating an increased degree of water-polymers interaction. Lastly, the rate of starch hydrolysis, evaluated by means of in vitro methods, increased in presence of fibre, resulting in higher RDS and estimated Glycemic Index (eGI) for whole durum wheat spaghetti compared to the traditional ones. These findings were quite unexpected.

Structure, Mechanical Properties, and Dynamics of Polyethylenoxide/Nanoclay Nacre-Mimetic Nanocomposites

Nacre-mimetic nanocomposites based on high fractions of synthetic high-aspect-ratio nanoclays in combination with polymers are continuously pushing boundaries for advanced material properties, such as high barrier against oxygen, extraordinary mechanical behavior, fire shielding, and glass-like transparency. Additionally, they provide interesting model systems to study polymers under nanoconfinement due to the well-defined layered nanocomposite arrangement. Although the general behavior in terms of forming such layered nanocomposite materials using evaporative self-assembly and controlling the nanoclay gallery spacing by the nanoclay/polymer ratio is understood, some combinations of polymer matrices and nanoclay reinforcement do not comply with the established models. Here, we demonstrate a thorough characterization and analysis of such an unusual polymer/nanoclay pair that falls outside of the general behavior. Poly(ethylene oxide) (PEO) and sodium fluorohectorite form nacre-mimetic, lamellar nanocomposites that are completely transparent and show high mechanical stiffness and high gas barrier, but there is only limited expansion of the nanoclay gallery spacing when adding increasing amounts of polymer. This behavior is maintained for molecular weights of PEO varied over four orders of magnitude and can be traced back to depletion forces. By careful investigation via X-ray diffraction and proton low-resolution solid-state NMR, we are able to quantify the amount of mobile and immobilized polymer species in between the nanoclay galleries and around proposed tactoid stacks embedded in a PEO matrix. We further elucidate the unusual confined polymer dynamics, indicating a relevant role of specific surface interactions.

Using NMR Chemical Shifts and Cryo-EM Density Restraints in Iterative Rosetta-MD Protein Structure Refinement.

Cryo-EM has become one of the prime methods for protein structure elucidation, frequently yielding density maps with near-atomic or medium resolution. If protein structures cannot be deduced unambiguously from the density maps, computational structure refinement tools are needed to generate protein structural models. We have previously developed an iterative Rosetta-MDFF protocol that used cryo-EM densities to refine protein structures. Here we show that, in addition to cryo-EM densities, incorporation of other experimental restraints into the Rosetta-MDFF protocol further improved refined structures. We used NMR chemical shift (CS) data integrated with cryo-EM densities in our hybrid protocol in both the Rosetta step and the molecular dynamics (MD) simulations step. In 15 out of 18 cases for all MD rounds, the refinement results obtained when density maps and NMR chemical shift data were used in combination outperformed those of density map-only refinement. Notably, the improvement in refinement was highest when medium and low-resolution density maps were used. With our hybrid method, the RMSDs of final models obtained were always better than the RMSDs obtained by our previous protocol with just density refinement for both medium (6.9 Å) and low (9 Å) resolution maps. For all the six test proteins with medium resolution density maps (6.9 Å), the final refined structure RMSDs were lower for the hybrid method than for the cryo-EM only refinement. The final refined RMSDs were less than 1.5 Å when our hybrid protocol was used with 4 Å density maps. For four out of the six proteins the final RMSDs were even less than 1 Å. This study demonstrates that by using a combination of cryo-EM and NMR restraints, it is possible to refine structures to atomic resolution, outperforming single restraint refinement. This hybrid protocol will be a valuable tool when only low-resolution cryo-EM density data and NMR chemical shift data are available to refine structures.

Microscopic State of Polymer Network Chains upon Swelling and Deformation

We use low-resolution proton NMR to probe the chain deformation in swollen and nonlinearly deformed vulcanized rubber and end-linked PDMS networks on a microscopic level, extending earlier work focusing on uniaxial stretching and isotropic dilation upon swelling toward biaxial deformation and deformation of swollen samples. Previous studies have revealed that chain deformation in bulk samples is best described by tube models, and that chains in swollen samples deform affinely after an initial desinterspersion stage, upon which entanglement-related packing effects are relieved. We test whether a subsequent deformation may also be closer to affine, and find that this is not the case. Unexpectedly, nonisotropic deformation of swollen samples also follows tube-model predictions, which is explained by a dominance of structural inhomogeneities and significant reorganization of the topological constraints active in the swollen and possibly even the bulk state.

Fat crystals: A tool to inhibit molecular transport in W/O/W double emulsions.

Water-in-oil-in-water (W/O/W) double emulsions are a promising technology for encapsulation applications of water soluble compounds with respect to functional food systems. Yet molecular transport through the oil phase is a well-known problem for liquid oil-based double emulsions. The influence of network crystallization in the oil phase of W/O/W globules was evaluated by NMR and laser light scattering experiments on both a liquid oil-based double emulsion and a solid fat-based double emulsion. Water transport was assessed by low-resolution NMR diffusometry and by an osmotically induced swelling or shrinking experiment, whereas manganese ion permeation was followed by means of T2 -relaxometry. The solid fat-based W/O/W globules contained a crystal network with about 80% solid fat. This W/O/W emulsion showed a reduced molecular water exchange and a slower manganese ion influx in the considered time frame, whereas its globule size remained stable under the applied osmotic gradients. The reduced permeability of the oil phase is assumed to be caused by the increased tortuosity of the diffusive path imposed by the crystal network. This solid network also provided mechanical strength to the W/O/W globules to counteract the applied osmotic forces.

Fat crystallization and melting in W/O/W double emulsions: Comparison between bulk and emulsified state

Water-in-oil-in-water (W/O/W) technology enables the development of an emulsion-based delivery system for functional food applications. The functionality of this system depends on the oil barrier of the double emulsion, whereby the fat matrix properties (e.g. solid fat content) play an important role. First, the effect of W/O/W emulsification on fat crystallization and melting behavior was studied and compared to bulk fat. Second, the effect of fat composition was investigated. As such, W/O/W emulsions were produced with a slow and fast crystallizing fat, i.e. soft palm mid fraction (sPMF) and partially hydrogenated sunflower oil, respectively, upon cooling and subsequent isothermal crystallization. Low resolution NMR and DSC techniques were applied, and indications about the polymorphic behavior were verified by real-time synchrotron XRD measurements. Except from increased supercooling, the overall fat crystallization dynamics in the W/O/W emulsions were similar to bulk, irrespective of the used fat. Yet, broadening of the long spacing peaks in the W/O/W emulsions indicated less ordered triacylglycerol longitudinal stacking compared to bulk. This effect was more pronounced in case of α-crystals than β’-crystals. With respect to W/O/W functionality, the melting behavior of the W/O/WsPMF emulsion showed promise as a possible carrier system with a thermo-responsive release mechanism for functional foods.

Effect of molecular exchange on water droplet size analysis as determined by diffusion NMR: The W/O/W double emulsion case

HypothesisThe accuracy of the inner water droplet size determination of W/O/W emulsions upon water diffusion measurement by diffusion NMR was evaluated. The resulting droplet size data were compared to the results acquired from the diffusion measurement of a highly water soluble marker compound with low permeability in the oil layer of a W/O/W emulsion, which provide a closer representation of the actual droplet size. Differences in droplet size data obtained from water and the marker were ascribed to extra-droplet water diffusion. ExperimentsThe diffusion data of the tetramethylammonium cation marker were measured using high-resolution pulsed field gradient NMR, whereas the water diffusion was measured using both low-resolution and high-resolution NMR. Different data analysis procedures were evaluated to correct for the effect of extra-droplet water diffusion on the accuracy of water droplet size analysis. FindingsUsing the water diffusion data, the use of a low measurement temperature and diffusion delay Δ could reduce the droplet size overestimation resulting from extra-droplet water diffusion, but this undesirable effect was inevitable. Detailed analysis of the diffusion data revealed that the extra-droplet diffusion effect was due to an exchange between the inner water phase and the oil phase, rather than by exchange between the internal and external aqueous phase. A promising data analysis procedure for retrieving reliable size data consisted of the application of Einstein’s diffusion law to the experimentally determined diffusion distances. This simple procedure allowed determining the inner water droplet size of W/O/W emulsions upon measurement of water diffusion by low-resolution NMR at or even above room temperature.

Influence of internal water phase gelation on the shear- and osmotic sensitivity of W/O/W-type double emulsions

It has already been hypothesized and in some cases proven that gelation of the internal water droplets of W/O/W-type double emulsions might allow the application of a higher shear in the second emulsification step while maintaining a high internal water content. In this contribution, thermal denaturation of a concentrated whey protein isolate solution, which is used as the internal water phase, was investigated. Low-resolution NMR T2-relaxometry measurements (using a CPMG-sequence) clearly showed that this thermal treatment effectively gelled the internal water phase. Using this approach, double emulsion droplets with a small average diameter (D43 of about 5 μm) and narrow particle size distribution were produced while a high internal water content was maintained. Moreover, our results indicated that gelation could not prevent osmotic shrinking of the double emulsion droplets upon dilution in hypertonic solution. However, gelation of the internal water phase clearly reduced the degree of osmotic swelling of the double emulsion droplets upon hypotonic dilution. However, this effect is probably due to droplet-globule coalescence occurring at lower internal water volume increases in case the internal water phase is gelled.

Effect of molecular exchange on water droplet size analysis in W/O emulsions as determined by diffusion NMR

Using NMR diffusometry, the diffusion of water and tetramethylammonium chloride was recorded in order to determine the water droplet size distribution in W/O emulsions. This study aimed at evaluating the effect of extradroplet diffusion of water on the estimated droplet size distribution upon comparison to the real droplet size distribution. The latter originated from the diffusion behavior of the tetramethylammonium cation (TMA+), which is known to have a much lower permeability through the oil phase as compared to water. Whereas both low-resolution and high-resolution pulsed field gradient NMR revealed that the water droplet size overestimation could be reduced selecting either a lower measurement temperature during diffusion analysis, or a smaller diffusion delay value Δ, still comparison to TMA+ diffusion indicated that artefacts were unavoidable even at low Δ and temperature. In order to correct for this extradroplet water diffusion phenomenon, different data analysis methods were evaluated. The previously described Pfeuffer exchange model could only partly compensate for the effect of extradroplet diffusion on the water droplet size determination. On the other hand, accurate water droplet size analysis results were obtained by correcting the experimentally determined diffusion distances based on Einstein’s diffusion law. As such, reliable data could be obtained by low resolution NMR based on water diffusion at or even above room temperature.

Basic principles of static proton low-resolution spin diffusion NMR in nanophase-separated materials with mobility contrast

We review basic principles of low-resolution proton NMR spin diffusion experiments, relying on mobility differences in nm-sized phases of inhomogeneous organic materials such as block-co- or semicrystalline polymers. They are of use for estimates of domain sizes and insights into nanometric dynamic inhomogeneities. Experimental procedures and limitations of mobility-based signal decomposition/filtering prior to spin diffusion are addressed on the example of as yet unpublished data on semicrystalline poly(ϵ-caprolactone), PCL. Specifically, we discuss technical aspects of the quantitative, dead-time free detection of rigid-domain signals by aid of the magic-sandwich echo (MSE), and magic-and-polarization-echo (MAPE) and double-quantum (DQ) magnetization filters to select rigid and mobile components, respectively. Such filters are of general use in reliable fitting approaches for phase composition determinations. Spin diffusion studies at low field using benchtop instruments are challenged by rather short 1H T1 relaxation times, which calls for simulation-based analyses. Applying these, in combination with domain sizes as determined by small-angle X-ray scattering, we have determined spin diffusion coefficients D for PCL (0.34, 0.19 and 0.032nm2/ms for crystalline, interphase and amorphous parts, respectively). We further address thermal-history effects related to secondary crystallization. Finally, the state of knowledge concerning the connection between D values determined locally at the atomic level, using 13C detection and CP- or REDOR-based “1H hole burning” procedures, and those obtained by calibration experiments, is summarized. Specifically, the non-trivial dependence of D on the magic-angle spinning (MAS) frequency, with a minimum under static and a local maximum under moderate-MAS conditions, is highlighted.

Influence of molecular exchange on the enclosed water volume fraction of W/O/W double emulsions as determined by low-resolution NMR diffusometry and T2-relaxometry

Low-resolution T2-relaxometry and pfg-NMR diffusometry were applied for determination of the enclosed water volume fraction of liquid oil and solid fat based W/O/W emulsions. Whereas pfg-NMR diffusometry enables the discrimination between internal and external water based on differences in diffusion behavior, the T2-relaxometry method is based on the relaxation behavior differences of the internal and external water upon addition of an external water-soluble paramagnetic probe, such as manganese dichloride. Provided that data-acquisition and data-analysis were optimized to eliminate interfering effects, such as oil phase contribution, incomplete signal relaxation or exchange between the internal and external water phase, both investigated NMR methods provided similar results. As compared to most alternative techniques, low-resolution NMR does not require the physical separation of both water phases. In addition, both low-resolution NMR techniques do not require any tracer addition during multiple emulsion preparation, and hence can also be applied on existing formulations. Whereas T2 relaxation is faster and less affected by NMR parameters, pfg-NMR has the advantage that it does not require any sample pretreatment.

Study of Water States in Different Hydrated and Consolidated Clay Using Nuclear Magnetic Resonance

ABSTRACT The aim of the present work was to test low-resolution NMR to characterize and quantify the state of water in some clay materials such as illite, montmorillonite, and bentonite with water content and in a compacted state. Low-field 1H-NMR spectroscopy (0.54 T) was used to determine the most mobile fraction of water in different types of clay materials by analyzing free induction decay (FID) and Carr-Purcell-Meiboom-Gill (CPMG) relaxation curves at 20°C, −7°C, and −25°C. Results have shown that a fast exchange occurs between the first bound monolayer of water and the following ones for illite, montmorillonite, and bentonite; a multiexponential decay of some CPMG relaxation curves was also observed. FID measurements at −25°C showed that 83% of the water in illite (41% water content), 44% in montmorillonite (31% w.c.), and 52% in bentonite (27% w.c.) is frozen at this temperature. Treatment of signals obtained with bentonite compacted in capillaries (28% w.c.) was complicated by a signal coming from the capillaries themselves and an increase in internal magnetic field gradients, which led to a dual quantification whose most likely result is 39% of freezable water.

Evaluation of the effect of homogenization energy input on the enclosed water volume of concentrated W/O/W emulsions by low-resolution T2-relaxometry

The usefulness of low-resolution NMR for W/O/W double emulsion characterization was evaluated. Whereas pfg-NMR diffusion experiments enabled the detection of phase inversion and water droplet diameter distribution in the primary W/O emulsion, T2 relaxation measurements allowed discriminating internal and external water when a paramagnetic agent, such as manganese dichloride was added to the W/O/W emulsion.The study aimed to entrap a maximal amount of water in the W/O emulsion in order to obtain a concentrated W/O/W emulsion. In addition, the impact of homogenization duration and intensity on the enclosed water volume of the double emulsion was investigated. The results indicated that high-intensity process conditions drastically decreased the yield of the W/O/W emulsion.

Microstructure of cellulose: NMR relaxation study

The models of structural organization of cellulose are reviewed within the framework of the semi-crystalline concept. Special attention is given to two crystalline modifications of cellulose: Iα and Iβ. The results of low-resolution 1H NMR studies of the supramolecular structure of cellulose are considered. The mathematical model describing the line shape of the solid-state NMR spectrum of cellulose is presented. A scheme for the structure of cellulose microfibrils that fits most of the known model concepts is proposed. It is shown that this scheme can be used to determine the major supramolecular parameters of this polymer.