Deuterium NMR Spectroscopy Research Articles

Synthesis and characterization of grafted graphene oxide materials with lyotropic liquid crystalline properties detected by NMR spectroscopy

Grafted derivatives of graphene oxide (GO), namely N-vinyl-2-pyrrolidone grafted-GO (GO-g-NVP) and oleic acid grafted-GO (GO-g-OLA) were prepared by heating GO in the presence of the corresponding vinyl monomers and the AIBN radical initiator. The lyotropic mesogenic behavior of GO-g-NVP and GO-g-OLA in DMSO‑d6 was proved by deuterium NMR spectroscopy, showing these materials could be used as alignment media for obtaining NMR anisotropic parameters in organic solvent.

259-LB: Loss of AMPK Induces Elongation of Preexisting Hepatic Fatty Acids, without Affecting DNL, during Fasting–Refeeding

AMP-activated protein kinase (AMPK) is a cellular energy sensor that conserves energy by inhibiting anabolic processes, such as de novo lipogenesis (DNL). Paradoxically, fructose metabolism activates AMPK by reducing the energy state (elevated AMP/ATP ratio), yet also promotes DNL. However, the comprehensive effects of fructose metabolism and AMPK activity on hepatic DNL has not been thoroughly investigated. Here, we examine how loss of AMPK function impacts DNL in response to fasting followed by refeeding a fructose or chow diet. After a 16 hour fast, 12 liver-specific AMPKα1α2 knock-out mice (AMPK-KO) and 12 AMPK α1α2 lox/lox mice (control) were fed either high fructose or chow diets (6 per a group) and administered D2O drinking water for 7 hours prior to blood collection and tissue harvest. After Folch extraction, fatty acids in liver triglyceride (TG-FA) were analyzed by proton and deuterium NMR spectroscopy to determine 2H incorporation and estimate DNL, elongation and re-esterification fluxes. Body water enrichment (~3.5%) was similar between groups. After correcting for food intake and body weight, preliminary data indicates that the AMP/ATP ratio was greater in AMPK-KO liver compared to controls, but neither chow nor fructose diets affected the energy state. Overall, neither hepatic steatosis nor DNL differed between groups. Furthermore, loss of AMPK function did not affect the amount of various FA species (i.e. saturated, unsaturated, DHA and ω3, linoleic, and non-essential). Instead, AMPK-KO liver had increased TG-FAs that were elongated compared to controls. This effect also tended to be greater in fructose fed mice (AMPK-KO and controls) compared to chow fed mice, even though fructose fed mice consumed fewer calories. In conclusion, loss of AMPK function resulted in increased elongation of hepatic TG-FAs, but not DNL, or the amount of FAs in the liver in response to fasting and refeeding with either fructose or chow diets. Disclosure J. A. Fletcher: None. S. Deja: None. S. C. Burgess: None. X. Fu: None. M. R. Inigo: None. J. Browning: None. B. Kucejova: None. Funding National Institutes of Health (K01AA030327)

Effects of antimicrobial peptides on membrane dynamics: A comparison of fluorescence and NMR experiments

Antimicrobial peptides (AMPs) represent a promising class of compounds to fight resistant infections. They are commonly thought to kill bacteria by perturbing the permeability of their cell membranes. However, bacterial killing requires a high coverage of the cell surface by bound peptides, at least in the case of cationic and amphipathic AMPs. Therefore, it is conceivable that peptide accumulation on the bacterial membranes might interfere with vital cellular functions also by perturbing bilayer dynamics, a hypothesis that has been termed “sand in the gearbox”. Here we performed a systematic study of such possible effects, for two representative peptides (the cationic cathelicidin PMAP-23 and the peptaibol alamethicin), employing fluorescence and NMR spectroscopies. These approaches are commonly applied to characterize lipid order and dynamics, but sample different time-scales and could thus report on different membrane properties. In our case, fluorescence anisotropy measurements on liposomes labelled with probes localized at different depths in the bilayer showed that both peptides perturb membrane fluidity and order. Pyrene excimer-formation experiments showed a peptide-induced reduction in lipid lateral mobility. Finally, laurdan fluorescence indicated that peptide binding reduces water penetration below the headgroups region. Comparable effects were observed also in fluorescence experiments performed directly on live bacterial cells. By contrast, the fatty acyl chain order parameters detected by deuterium NMR spectroscopy remained virtually unaffected by addition of the peptides. The apparent discrepancy between the two techniques confirms previous sporadic observations and is discussed in terms of the different characteristic times of the two approaches. The perturbation of membrane dynamics in the ns timescale, indicated by the multiple fluorescence approaches reported here, could contribute to the antimicrobial activity of AMPs, by affecting the function of membrane proteins, which is strongly dependent on the physicochemical properties of the bilayer.

Electronic structure calculations and deuterium NMR spectroscopy reveal retinal mobility in rhodopsin activation.

G-protein coupled receptors (GPCRs) regulate a number of physiological processes, where the visual receptor rhodopsin is an excellent prototype to investigate the structural fluctuations underlying GPCR function. Several X-ray crystal structures are available for the dark and active Meta-II states of rhodopsin. However, because of dehydrating conditions used to crystalize the protein, the conformation and orientation of the retinal chromophore in its binding pocket remain uncertain. Our hypothesis was that greater retinal mobility is needed in the active Meta-II state for the interaction with effector proteins. Accordingly, we investigated the structure and orientation of retinal in the dark and the Meta-II states using density functional theory (DFT) calculations and deuterium NMR (2H NMR) spectroscopy. For the computational simulations, we modeled the protein environment by including a number of amino acid residues in the retinal binding pocket and the water molecules around it. These models were tested with different chromophore orientations and protonation states of Glu113 and Glu181. Next, we performed DFT and complete active space (CAS) calculations to characterize the electronic structure of the dark and Meta-II states of rhodopsin. 2H NMR spectroscopic studies were also conducted on the active Meta-II state that contained deuterium-labelled retinal to experimentally validate the possible retinal orientations. The experiment revealed two possible retinal orientations (“flipped” and “unflipped”) without significant steric clashes in the binding pocket of active Meta-II state. By contrast, the DFT calculations show the Meta-II state predominantly contains the “flipped” orientation while the dark-state predominantly accommodates the “unflipped” retinal orientation. Thus the chromophore of the visual receptor undergoes significant mobility changes upon light activation of rhodopsin. [1] A.V. Struts et al. (2011) Nat.Struct.Mol.Biol. 18, 392–394, [2] M.N. Ryazantsev et al. (2019) J.Membr.Biol. 252, 425–449.

Cholesterol Stiffening of Lipid Membranes.

Biomembrane order, dynamics, and other essential physicochemical parameters are controlled by cholesterol, a major component of mammalian cell membranes. Although cholesterol is well known to exhibit a condensing effect on fluid lipid membranes, the extent of stiffening that occurs with different degrees of lipid acyl chain unsaturation remains an enigma. In this review, we show that cholesterol locally increases the bending rigidity of both unsaturated and saturated lipid membranes, suggesting there may be a length-scale dependence of the bending modulus. We review our published data that address the origin of the mechanical effects of cholesterol on unsaturated and polyunsaturated lipid membranes and their role in biomembrane functions. Through a combination of solid-state deuterium NMR spectroscopy and neutron spin-echo spectroscopy, we show that changes in molecular packing cause the universal effects of cholesterol on the membrane bending rigidity. Our findings have broad implications for the role of cholesterol in lipid–protein interactions as well as raft-like mixtures, drug delivery applications, and the effects of antimicrobial peptides on lipid membranes.Graphical

Antiviral compounds investigated by solid-state deuterium NMR spectroscopy

Antiviral compounds like rimantadine, amantadine, and their derivatives are effective combatants against diseases such as influenza and dyskinesia. Characterization of structure and dynamics of these molecules is essential for understanding their antiviral activity. Here we conducted solid-state 2H NMR relaxometry to elucidate the internal dynamics and structural restraints of these compounds in the solid state. Quadrupolar-echo based relaxometry experiments were performed on powdered samples of deuterated rimantadine and amantadine.

Illuminating Disorder Induced by Glu in a Stable Arg-Anchored Transmembrane Helix.

Membrane proteins are vital for biological function and are complex to study. Even in model peptide-lipid systems, the combined influence or interaction of pairs of chemical groups still is not well understood. Disordered proteins, whether in solution or near lipid membranes, are an emerging paradigm for the initiation and control of biological function. The disorder can involve molecular orientation as well as molecular folding. This paper reports an astonishing induction of disorder when one Glu residue is introduced into a highly stable 23-residue transmembrane helix. The parent helix is anchored by a single Arg residue, tilted at a well-defined angle with respect to the DOPC bilayer normal and undergoes rapid cone precession. When Glu is introduced two residues away from Arg, with 200° (or 160°) radial separation, the helix properties change radically to exhibit a multiplicity of three or more disordered states. The helix characteristics have been monitored by deuterium (2H) NMR spectroscopy as functions of the pH and lipid bilayer composition. The disordered multistate behavior of the (Glu, Arg)-containing helix varies with the lipid bilayer thickness and pH. The results highlight a fundamental induction of protein multistate properties by a single Glu residue in a lipid membrane environment.

Site-Isolated Azobenzene-Containing Metal-Organic Framework for Cyclopalladated Catalyzed Suzuki-Miyuara Coupling in Flow.

Sites isolation of active metals centers, systematically studied in homogeneous systems, has been an alternative to develop low metal consuming, highly active next generation catalysts in heterogeneous condition. Because of the high porosity and facile synthetic procedures, MOF-based catalysts are excellent candidates for heterogenization of well-defined homogeneous catalysts. Herein, we report the direct Pd coordination on the azobenzene linker within a MOF catalyst through a postsynthetic modification method for a Suzuki-Miyaura coupling reaction. The immobilized cyclopalladated complexes in MOFs were analyzed by a series of characterization techniques including XPS, PXRD, and deuterium NMR (2H NMR) spectroscopy. The heterogeneous nature of the catalyst as well as its stability were demonstrated though "hot filtration" and recycling experiments. Furthermore, we demonstrate that the MOF packed column promoted the reaction between phenyl boronic acid and bromobenzene under microflow conditions with a 85% yield continuously for 12 h. This work sheds light on the potential of site-isolated MOF catalysts in efficient, recyclable and continuous flow systems for industrial application.

Flanking aromatic residue competition influences transmembrane peptide helix dynamics.

To address biophysical principles and lipid interactions that underlie the properties of membrane proteins, modifications that vary the neighbors of tryptophan residues in the highly dynamic transmembrane helix of GW4,20 ALP23 (acetyl-GGAW4 A(LA)6 LAW20 AGA-amide) were examined using deuterium NMR spectroscopy. It was found that L5,19 GW4,20 ALP23, a sequence isomer of the low to moderately dynamic GW5,19 ALP23, remains highly dynamic. By contrast, a removal of W4 to produce F4,5 GW20 ALP23 restores a low level of dynamic averaging, similar to that of the F4,5 GW19 ALP23 helix. Interestingly, a high level of dynamic averaging requires the presence of both tryptophan residues W4 and W20, on opposite faces of the helix, and does not depend on whether residue 5 is Leu or Ala. Aspects of helix unwinding and potential oligomerization are discussed with respect to helix dynamic averaging and the locations of particular residues at a phosphocholine membrane interface.

How cholesterol stiffens unsaturated lipid membranes

Cholesterol is an integral component of eukaryotic cell membranes and a key molecule in controlling membrane fluidity, organization, and other physicochemical parameters. It also plays a regulatory function in antibiotic drug resistance and the immune response of cells against viruses, by stabilizing the membrane against structural damage. While it is well understood that, structurally, cholesterol exhibits a densification effect on fluid lipid membranes, its effects on membrane bending rigidity are assumed to be nonuniversal; i.e., cholesterol stiffens saturated lipid membranes, but has no stiffening effect on membranes populated by unsaturated lipids, such as 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). This observation presents a clear challenge to structure-property relationships and to our understanding of cholesterol-mediated biological functions. Here, using a comprehensive approach-combining neutron spin-echo (NSE) spectroscopy, solid-state deuterium NMR (2H NMR) spectroscopy, and molecular dynamics (MD) simulations-we report that cholesterol locally increases the bending rigidity of DOPC membranes, similar to saturated membranes, by increasing the bilayer's packing density. All three techniques, inherently sensitive to mesoscale bending fluctuations, show up to a threefold increase in effective bending rigidity with increasing cholesterol content approaching a mole fraction of 50%. Our observations are in good agreement with the known effects of cholesterol on the area-compressibility modulus and membrane structure, reaffirming membrane structure-property relationships. The current findings point to a scale-dependent manifestation of membrane properties, highlighting the need to reassess cholesterol's role in controlling membrane bending rigidity over mesoscopic length and time scales of important biological functions, such as viral budding and lipid-protein interactions.

Impact of Acyl Chain Mismatch on the Formation and Properties of Sphingomyelin-Cholesterol Domains

Lateral segregation and the formation of lateral domains are well-known phenomena in ternary lipid bilayers composed of an unsaturated (low gel-to-liquid phase transition temperature (Tm)) phospholipid, a saturated (high-Tm) phospholipid, and cholesterol. The formation of lateral domains has been shown to be influenced by differences in phospholipid acyl chain unsaturation and length. Recently, we also showed that differential interactions of cholesterol with low- and high-Tm phospholipids in the bilayer can facilitate phospholipid segregation. Now, we have investigated phospholipid-cholesterol interactions and their role in lateral segregation in ternary bilayers composed of different unsaturated phosphatidylcholines (PCs) with varying acyl chain lengths, N-palmitoyl-D-erythro-sphingomyelin (PSM), and cholesterol. Using deuterium NMR spectroscopy, we determined how PSM was influenced by the acyl chain composition in surrounding PC environments and correlated this with the affinity of cholestatrienol (a fluorescent cholesterol analog) for PSM in the different PC environments. Results from a combination of time-resolved fluorescence measurements of trans-parinaric acid and Förster resonance energy transfer experiments showed that the relative affinity of cholesterol for phospholipids determined the degree to which the sterol promoted domain formation. From Förster resonance energy transfer, deuterium NMR, and differential scanning calorimetry results, it was clear that cholesterol also influenced both the thermostability of the domains and the degree of order in and outside the PSM-rich domains. The results of this study have shown that the affinity of cholesterol for both low-Tm and high-Tm phospholipids and the effects of low- and high-Tm phospholipids on each other influence both lateral structure and domain properties in complex bilayers. We envision that similar effects also contribute to lateral heterogeneity in even more complex biological membranes.

Peculiarities in the Director Reorientation and the NMR Spectra Evolution in a Nematic Liquid Crystals under the Effect of Crossed Electric and Magnetic Fields

The illustrative description of the field-induced peculiarities of the director reorientation in the microsized nematic volumes under the effect of crossed magnetic B and electric E fields have been proposed. The most interesting feature of such configuration is that the nematic phase becomes unstable after applying the strong E . The theoretical analysis of the reorientational dynamics of the director field provides an evidence for the appearance of the spatially periodic patterns in response to applied large E directed at an angle α to B . The feature of this approach is that the periodic distortions arise spontaneously from a homogeneously aligned nematic sample that ultimately induces a faster response than in the uniform mode. The nonuniform rotational modes involve additional internal elastic distortions of the conservative nematic system and, as a result, these deformations decrease of the viscous contribution U vis to the total energy U of the nematic phase. In turn, that decreasing of U vis leads to decrease of the effective rotational viscosity coefficient γ eff ( α ) . That is, a lower value of γ eff ( α ) , which is less than one in the bulk nematic phase, gives the less relaxation time τ on ( α ) ∼ γ eff ( α ) , when α is bigger than the threshold value α th . The results obtained by Deuterium NMR spectroscopy confirm theoretically obtained dependencies of τ on ( α ) on α .

Novel F13,F15 Gramicidin Subunits Predicted to Cross Bilayer Membranes and form Ion Channels

Gramicidin A (formyl-L-Val-Gly-L-Ala-D-Leu-L-Ala-D-Val-L-Val-D-Val-L-Trp9-D-Leu-L-Trp11-D-Leu-L-Trp13-D-Leu-L-Trp15-ethanolamine) functions as a membrane-spanning channel for monovalent cations. Each channel is composed of two subunits joined by six hydrogen bonds. The four Trp residues anchor each subunit to the aqueous interface and prevent subunits from crossing a lipid-bilayer membrane. Nevertheless, it has been observed that replacing Trp residues 13 and 15 with Phe results in subunits (designated [Phe13,15]gA) that are capable of crossing the membrane, presumably in a double-stranded conformation, to engage in channel formation. Our goal is to modify [Phe13,15]gA to increase the channel lifetime in biological membranes, while retaining its ability to cross membranes. To this end, the length of [Phe13,15]gA was increased to allow for better matching in thicker biological membranes, and Ala was introduced near the N-terminus to increase channel stability. Comparative biophysical experiments with three novel peptides, endo-Gly0B-Gly0A-[Phe13,15]gA, endo-Gly0B-D-Ala0A-[Phe13,15]gA and endo-D-Ala0A-[Phe13,15]gA, were performed in DMPC and DOPC bilayers. Deuterated alanines were included to facilitate analysis by solid-state deuterium NMR spectroscopy. The 2H NMR spectra in mechanically aligned DMPC bilayers suggest a single major membrane orientation for each peptide, with slightly different backbone torsion angles for Ala3 and Ala5. By contrast, the spectra in DOPC are poorly resolved. Circular dichroism spectra in DMPC and DOPC vesicles suggest mixtures of single- and double-stranded conformations. The Trp fluorescence emission λ-maxima for endo-Gly0B-D-Ala0A-[Phe13,15]gA (335 nm) and endo-D-Ala0A-[Phe13,15]gA (336 nm) in DOPC vesicles are shifted to higher wavelengths compared to [Phe13,15]gA (330 nm). Experiments to assess channel lifetimes and membrane-crossing ability of each peptide are underway.

Rhodopsin Hydration Dynamics Studied by Solid-State Deuterium NMR Spectroscopy

Rhodopsin Hydration Dynamics Studied by Solid-State Deuterium NMR Spectroscopy

Proton and Deuterium NMR Study of the CBC9CB Dimer System.

Using proton and deuterium NMR spectroscopy, this work provides a description of the molecular orientational order of the mesogenic groups in both the Ntb and the classical nematic phase (N) of the liquid crystal dimer CB-C9-CB. The proton NMR data were collected at high field (7 T) with the nematic domains aligned by the field. Deuterium NMR data obtained from aligned samples at 11.7 T, published by Hoffmann , A. Soft Matter 2015 , 11 , 850 , were also considered in this study. Using the first-order perturbation theory, we carried out detailed simulations of the proton spectra from the terminal mesogenic groups along with the quadrupolar splittings from the carbon-deuterium bonds in the first chain positions, which allow for the determination of the Saupe order tensor for the mesogenic groups. We show that the main mechanism that induces the change of the 1H NMR spectrum and the quadrupolar splittings at the N-Ntb phase transition is the change of the orientation of the most ordered molecular frame (eigenframe of the Saupe tensor), along with the onset of the molecular biaxiality parameter, D. This orientation change is associated with the achiral symmetry breaking at the N-Ntb phase transition.

Dynamics of Cetyltrimethylammonium Bromide Head Groups in Bulk by Solid-State Deuterium NMR Spectroscopy.

Variable temperature, solid-state deuterium (2H) NMR spectroscopy has been used to probe the rather complex head group dynamics of the surfactant cetyltrimethylammonium bromide- d9 (CTAB- d9) in bulk. Heating and cooling runs were made as the surfactant underwent supercooling. 2H NMR line shape simulations were used to identify the hierarchy of the molecular motions of CTAB as a function of temperature. Fast continuous methyl rotations about the N-Cmethyl axes and 3-fold jumps about the main chain C-N axis were present at all of the temperatures from -40 to 120 °C. With heating, the spectra were consistent with CTAB molecules starting 180° flips about the hydrocarbon chain molecular axis around 0 °C, which continued to flip with increasing flip rates up to 80 °C. At 90 °C, the flips changed to rotation of the CTAB molecules about the hydrocarbon chain axis and that rotation continued to 120 °C. Comparison of spectra of bulk CTAB at different temperatures from heating and cooling runs revealed that the rotation about the long axis of the hydrocarbon chains started at around 90 °C on heating, however, it does not freeze out until between 70 and 80 °C because of supercooling.

Effect of Chain Branching on Orientational Ordering in Glycolipid Self-assembly by 2H-NMR using Extrinsic Probes

The structure and dynamics of molecular solids, liquid crystals, and thin films of membrane lipids may be elucidated from the use of deuterium NMR (2H-NMR) spectroscopy. The 2H-NMR spectrum of a deuterated material (either selectively or fully) is rather simpler to interpret compared with the corresponding proton NMR (1H-NMR). Here, we apply this technique to understand the effect of chain branching on orientational order of the glycolipid self-assembly by using deuterated probes. The 2H-NMR resonance of n-octanol-d17 in n-octyl-β-D-glucopyranoside (β-GlcOC8) and the branched-chain glycolipid 2-hexyldecyl-β-D-glucopyranoside (β-GlcOC10C6) were measured at different temperatures. We also measured the 2H-NMR spectrum of 2-ethyl-n-hexanol-α,α-d2, a branched-chain alcohol which is selectively deuterated at α-position in β-GlcOC8. All systems gave quadrupolar splittings in their respective 2H-NMR spectra, which are attributed to the formation of anisotropic phase. The n-octanol-d17 dissolved in β-GlcOC8 give us spectra in the lamellar phase and when used with a branched-chain lipid, i.e. β-GlcOC10C6, gave us new spectra in the inverse hexagonal phase. The magnitude of the quadrupolar splittings of the n-octanol-d17 is indicative of the degree of orientational ordering in the given system. The results show that the quadrupolar splitting is larger in β-GlcOC8 lipid than in β-GlcOC10C6.

Field-induced peculiarities in the dynamics of the director reorientation in confined nematic liquid crystals

The peculiarities in the dynamics of the director reorientation in confined nematic liquid crystals (LCs) under the influence of a strong electric field E directed at an angle α to the magnetic field B have been investigated theoretically on the basis of the hydrodynamic theory including the director motion with appropriate boundary and initial conditions. Analysis of the numerical results for the turn-on process, when the strong electric field E is suddenly applied, provides an evidence for the appearance of the spatially periodic patterns in confined LC film. Calculations show that when the angle α tends to be 90°, the optimum wavelength, giving the fastest response, provides the lower effective viscosity γeffα, which is less than one in the bulk nematic phase. So, a lower value of γeffα gives a faster relaxation times, as observed experimentally by using the time-resolved deuterium NMR spectroscopy.

Integrated Computational Protocol for the Analysis of Quadrupolar Splittings from Natural‐Abundance Deuterium NMR Spectra in (Chiral) Oriented Media

AbstractThe front cover artwork is provided by Dr. Armando Navarro‐Vázquez from the department of fundamental chemistry of the Universidade Federal de Pernambuco (Brazil) as well as Dr. Philippe Berdagué and Dr. Philippe Lesot (DR CNRS), both from the Institut de Chimie Moléculaire et des Matériaux d'Orsay of the Université de Paris‐Sud/Paris‐Saclay (France). Graphically inspired by the famous word game «Scrabble», the image illustrates, through selected keywords, the analytical potential of a hyphenated computational protocol for investigating (achiral, prochiral or chiral) organic molecules by combining deuterium NMR spectroscopy at natural abundance level and chiral oriented systems, as described in their article. Read the full text of the article at 10.1002/cphc.201601423.

Integrated Computational Protocol for the Analysis of Quadrupolar Splittings from Natural-Abundance Deuterium NMR Spectra in (Chiral) Oriented Media.

Despite its low natural abundance, deuterium NMR spectroscopy in weakly oriented (chiral) solvents gives easy access to deuterium residual quadrupolar couplings (2 H-RQCs). These are formally equivalent to one-bond residual dipolar couplings ((13 C-1 H)-RDCs) for calculation of the Saupe tensor, and provide similar information for the study of molecular structure and orientational behavior. Because the quadrupolar interaction is one order of magnitude larger than the dipolar one, 2 H-RQC analysis is a much more sensitive tool for the detection of subtle structural differences and also tiny differences in molecular alignment, such as those observed for different enantiomers in chirally aligned media. To promote the analytical advantages of anisotropic, natural-abundance deuterium NMR (NAD NMR) spectroscopy in the organic chemistry community, we describe a 2 H-RQC/DFT-based integrated computational protocol for the evaluation of the order parameters of aligned solutes by using singular-value decomposition. Several examples of 2 H-RQC-assisted analysis of chiral and prochiral molecules dissolved in various polypeptide lyotropic chiral liquid crystals are reported. The role of the molecular shape in the ordering mechanism was investigated through the determination of intertensor angles between alignment tensors and inertia tensors by using the proposed protocol.