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Extensive deuteration is frequently used in solid-state NMR studies of biomolecules because it dramatically reduces both homonuclear (1H-1H) and heteronuclear (1H-13C and 1H-15N) dipolar interactions. This approach greatly improves resolution, enables low-power rf decoupling, and facilitates 1H-detected experiments even in rigid solids at moderate MAS rates. However, the resolution enhancement is obtained at some cost due the reduced abundance of protons available for polarization transfer. Although deuterium is a useful spin-1 NMR nucleus, in typical experiments the deuterons are not directly utilized because the available probes are usually triple-tuned to 1H,13C and 15N. Here we describe a 1H/13C/2H/15N MAS ssNMR probe designed for solid-state NMR of extensively deuterated biomolecules. The probe utilizes coaxial coils, with a modified Alderman-Grant resonator for the 1H channel, and a multiply resonant solenoid for 13C/2H/15N. A coaxial tuning-tube design is used for all four channels in order to efficiently utilize the constrained physical space available inside the magnet bore. Isolation among the channels is likewise achieved using short, adjustable transmission line elements. We present benchmarks illustrating the tuning of each channel and isolation among them and the magnetic field profiles at each frequency of interest. Finally, representative NMR data are shown demonstrating the performance of both the detection and decoupling circuits.

Solid‐state NMR (SSNMR) spectroscopy of integer‐spin quadrupolar nuclei is important for the molecular‐level characterization of a variety of materials and biological solids; of the integer spins, 2H (S = 1) is by far the most widely studied, due to its usefulness in probing dynamical motions. SSNMR spectra of integer‐spin nuclei often feature very broad powder patterns that arise largely from the effects of the first‐order quadrupolar interaction; as such, the acquisition of high‐quality spectra continues to remain a challenge. The broadband adiabatic inversion cross‐polarization (BRAIN‐CP) pulse sequence, which is capable of cross‐polarization (CP) enhancement over large bandwidths, has found success for the acquisition of SSNMR spectra of integer‐spin nuclei, including 14N (S = 1), especially when coupled with Carr–Purcell/Meiboom–Gill pulse sequences featuring frequency‐swept WURST pulses (WURST‐CPMG) for T2‐based signal enhancement. However, to date, there has not been a systematic investigation of the spin dynamics underlying BRAIN‐CP, nor any concrete theoretical models to aid in its parameterization for applications to integer‐spin nuclei. In addition, the BRAIN‐CP/WURST‐CPMG scheme has not been demonstrated for generalized application to wideline or ultra‐wideline (UW) 2H SSNMR. Herein, we provide a theoretical description of the BRAIN‐CP pulse sequence for spin‐1/2 → spin‐1 CP under static conditions, featuring a set of analytical equations describing Hartmann–Hahn matching conditions and numerical simulations that elucidate a CP mechanism involving polarization transfer, coherence exchange, and adiabatic inversion. Several experimental examples are presented for comparison with theoretical models and previously developed integer‐spin CP methods, demonstrating rapid acquisition of 2H NMR spectra from efficient broadband CP. [ABSTRACT FROM AUTHOR]

We introduce a 13C–2H Rotational Echo DOuble Resonance (REDOR) technique that uses the difference between on-resonance and off-resonance 2H irradiation to detect dynamic segments in deuterated molecules. By selectively inverting specific regions of the 2H magic-angle spinning (MAS) sideband manifold to recouple some of the deuterons to nearby carbons, we distinguish dynamic and rigid residues in 1D and 2D 13C spectra. We demonstrate this approach on deuterated GB1, H/D exchanged GB1, and perdeuterated bacterial cellulose. Numerical simulations reproduce the measured mixing-time and 2H carrier-frequency dependence of the REDOR dephasing of bacterial cellulose. Combining numerical simulations with experiments thus allow the extraction of motionally averaged quadrupolar couplings from REDOR dephasing values. [ABSTRACT FROM AUTHOR]

Cr(VI)/silica catalyst was initiated by alkanes and aromatics, sometimes isotopically labeled, and their fate was monitored through D-NMR, GC-MS, etc. • Phillips Cr(VI)/silica catalysts can be initiated by non-olefinic hydrocarbons • Oxygenated ligands are formed as well as Cr alkyl or hydride initiating species • Polymer character, and Cr activity, are controlled by ligands left after initiation • D-labeling of the reductant indicates that it can initiate the first PE chain The Phillips Cr(VI)/silica catalyst, used worldwide to manufacture polyethylene, must first be reduced and alkylated to display polymerization activity. This is accomplished commercially by exposure to ethylene, in a reaction that is still uncertain after 60 years of investigation. Here we report that other non-olefinic hydrocarbons can also be used in this self-alkylation mechanism, and we track, through isotopic labeling and other analyses, the fate of such hydrocarbons when used to initiate (reduce and alkylate) Cr(VI). A part of the reductant hydrocarbon is converted into oxygenated ligands, which remain attached as a permanent component of the active catalyst, thus influencing its behavior. Another part is used to alkylate the Cr, and it becomes incorporated into the first polymer chain. Each hydrocarbon reductant produces a unique catalyst, with unique ligands, displaying its own activity and sometimes producing distinctive polymer. The implication of this situation, in contrast to more traditional findings from the field, is one of opportunity to create novel varieties of new Phillips catalysts. [ABSTRACT FROM AUTHOR]

The effect of inserting a neutral water-soluble adsorbing polymer on the flexibility of amphiphilic bilayers in a lamellar phase is investigated. The L α system is a stack of charged undulating bilayers composed of sodium dodecyl sulfate (SDS) and octanol separated by aqueous solutions of polyethylene glycol (PEG). The mean bending elastic modulus κ is determined from the quadrupole splittings in the solid state NMR spectra of the perdeuterated octanol chains embedded in the membranes that undergo collective fluctuations. Parameters for describing the membrane behavior (bilayer thickness, elastic compressibility modulus, order parameter) are obtained by supplementing the NMR data with complementary experiments (x-ray scattering), NMR spectral simulations, and theoretical considerations. A fairly complete picture of the membrane rigidity emerges for any location in the lamellar phase thanks to a broad sweep of the lamellar domain by systematically varying the membrane fraction along dilution lines as well as the polymer composition. The most remarkable result is the difference between dilute and semi-dilute regimes. In the dilute PEG solution, no (or slight positive shift) polymer contribution to the rigidity curvature of the layered system is noted within the experimental resolution (≤0.3 k B T) and κ remains around 2.7 k B T. In contrast, the membrane rigidity increases steadily upon polymer addition once the crossover concentration c p * is exceeded, amounting to a 60% increase in κ at polymer concentration 2.5 c p * in the aqueous interlayers. These results are discussed with regard to the theoretical expectation of membrane rigidification upon irreversible polymer adsorption. [ABSTRACT FROM AUTHOR]

Methionine side chains are flexible entities which play important roles in defining hydrophobic interfaces. We utilize deuterium static solid-state NMR to assess rotameric inter-conversions and other dynamic modes of the methionine in the context of a nine-residue random-coil peptide (RC9) with the low-complexity sequence GGKGMGFGL. The measurements in the temperature range of 313 to 161 K demonstrate that the rotameric interconversions in the hydrated solid powder state persist to temperatures below 200 K. Removal of solvation significantly reduces the rate of the rotameric motions. We employed 2 H NMR line shape analysis, longitudinal and rotation frame relaxation, and chemical exchange saturation transfer methods and found that the combination of multiple techniques creates a significantly more refined model in comparison with a single technique. Further, we compare the most essential features of the dynamics in RC9 to two different methionine-containing systems, characterized previously. Namely, the M35 of hydrated amyloid-β 1-40 in the three-fold symmetric polymorph as well as Fluorenylmethyloxycarbonyl (FMOC)-methionine amino acid with the bulky hydrophobic group. The comparison suggests that the driving force for the enhanced methionine side chain mobility in RC9 is the thermodynamic factor stemming from distributions of rotameric populations, rather than the increase in the rate constant., (© 2024 Wiley-VCH GmbH.)

Numerous theories of molecular ordering in the nematic phase exist that attempt to incorporate the features of weak attractions, excluded volume (aspect ratio), and rigid and non-rigid segments, as well as assess entropic and enthalpic effects. Para-quinquephenyl represents a model rodlike molecule, but study of this liquid crystal presents challenges and surprises given the high temperature of the nematic phase. We present NMR experiments throughout the nematic range on two specifically deuterium-labelled quinquephenyl isotopomers, which we combine with high fidelity molecular dynamics simulations to explore and quantify various aspects of orientational order. The striking agreement between experiment and simulation provides new insights into the role of molecular flexibility and correlations (clustering, director fluctuations) on observed order parameters. We compare several nematic theories with our observations and discuss implications for further theoretical understanding. [ABSTRACT FROM AUTHOR]

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 iswell 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-snglycero- 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-compressibilitymodulus andmembrane 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 controllingmembrane bending rigidity over mesoscopic length and time scales of important biological functions, such as viral budding and lipid-protein interactions. [ABSTRACT FROM AUTHOR]

[Display omitted] • We demonstrate the methodology of 2H off-resonance rotating frame relaxation. • The method is applied for protein methyl groups under MAS and static powders. • The systems are amyloid fibrils, FMOC-methionine, a fragment of apolipoprotein-B. We demonstrate the feasibility of deuterium solid-state NMR off-resonance rotating frame relaxation measurements for studies of slow motions in biomolecular solids. The pulse sequence, which includes adiabatic pulses for magnetization alignment, is illustrated for static and magic-angle spinning conditions away from rotary resonances. We apply the measurements for three systems with selective deuterium labels at methyl groups: a) a model compound, Fluorenylmethyloxycarbonyl methionine-D 3 amino acid, for which the principles of the measurements and corresponding motional modeling based on rotameric interconversions are demonstrated; b) amyloid-β 1-40 fibrils labeled at a single alanine methyl group located in the disordered N-terminal domain. This system has been extensively studied in prior work and here serves as a test of the method for complex biological systems. The essential features of the dynamics consist of large-scale rearrangements of the disordered N-terminal domain and the conformational exchange between the free and bound forms of the domain, the latter one due to transient interactions with the structured core of the fibrils. and c) a 15-residue helical peptide which belongs to the predicted α-helical domain near the N-terminus of apolipoprotein B. The peptide is solvated with triolein and incorporates a selectively labeled leucine methyl groups. The method permits model refinement, indicating rotameric interconversions with a distribution of rate constants. [ABSTRACT FROM AUTHOR]

Using rheo-NMR, pulsed-field-gradient NMR diffusometry and solution rheology, we observe temperature- and composition-dependent orientational order as well as director dynamics and molecular transport in a nematic wormlike micelle (WLM) system composed of cetyltrimethylammonium bromide in D2O. We measure, for the first time over a comprehensive nematic range in WLMs, four of the five independent anisotropic viscosities by fitting nonlinear rheo-NMR director realignment data and comparing with traditional solution rheology measurements. Additionally, we extract self-diffusion coefficients in three orthogonal directions for the aligned WLM unimers as well as for the D2O solvent molecules. Continued study and enhanced understanding of complex fluid dynamics in these anisotropic shear-modulated fluid systems can lead to advances in lubricants, coatings, oil extraction, drug delivery and tissue engineering. [ABSTRACT FROM AUTHOR]

The influenza M2 protein not only forms a proton channel but also mediates membrane scission in a cholesterol-dependent manner to cause virus budding and release. The atomic interaction of cholesterol with M2, as with most eukaryotic membrane proteins, has long been elusive. We have now determined the cholesterol-binding site of the M2 protein in phospholipid bilayers using solid-state NMR spectroscopy. Chain-fluorinated cholesterol was used to measure cholesterol proximity to M2 while sterol-deuterated cholesterol was used to measure bound-cholesterol orientation in lipid bilayers. Carbon-fluorine distance measurements show that at a cholesterol concentration of 17 mol%, two cholesterol molecules bind each M2 tetramer. Cholesterol binds the C-terminal transmembrane (TM) residues, near an amphipathic helix, without requiring a cholesterol recognition sequencemotif. DeuteriumNMR spectra indicate that bound cholesterol is approximately parallel to the bilayer normal, with the rough face of the sterol rings apposed to methyl-rich TMresidues. The distance- and orientation-restrained cholesterol-binding site structure shows that cholesterol is stabilized by hydrophobic interactionswith the TM helix and polar and aromatic interactions with neighboring amphipathic helices. At the 1:2 binding stoichiometry, lipid 31P spectra show an isotropic peak indicative of high membrane curvature. This M2-cholesterol complex structure, together with previously observed M2 localization at phase boundaries, suggests that cholesterol mediates M2 clustering to the neck of the budding virus to cause the necessary curvature for membrane scission. The solid-state NMR approach developed here is generally applicable for elucidating the structural basis of cholesterol's effects on membrane protein function. [ABSTRACT FROM AUTHOR]

Extensive deuteration is frequently used in solid-state NMR studies of biomolecules because it dramatically reduces both homonuclear ( 1 H- 1 H) and heteronuclear ( 1 H- 13 C and 1 H- 15 N) dipolar interactions. This approach greatly improves resolution, enables low-power rf decoupling, and facilitates 1 H-detected experiments even in rigid solids at moderate MAS rates. However, the resolution enhancement is obtained at some cost due the reduced abundance of protons available for polarization transfer. Although deuterium is a useful spin-1 NMR nucleus, in typical experiments the deuterons are not directly utilized because the available probes are usually triple-tuned to 1 H, 13 C and 15 N. Here we describe a 1 H/ 13 C/ 2 H/ 15 N MAS ssNMR probe designed for solid-state NMR of extensively deuterated biomolecules. The probe utilizes coaxial coils, with a modified Alderman-Grant resonator for the 1 H channel, and a multiply resonant solenoid for 13 C/ 2 H/ 15 N. A coaxial tuning-tube design is used for all four channels in order to efficiently utilize the constrained physical space available inside the magnet bore. Isolation among the channels is likewise achieved using short, adjustable transmission line elements. We present benchmarks illustrating the tuning of each channel and isolation among them and the magnetic field profiles at each frequency of interest. Finally, representative NMR data are shown demonstrating the performance of both the detection and decoupling circuits. [ABSTRACT FROM AUTHOR]

We compared the dynamics of key methionine methyl groups in the water-accessible hydrophobic cavity of amyloid fibrils and Fluorenylmethyloxycarbonyl-Methionine (FMOC-Met), which renders general hydrophobicity to the environment without the complexity of the protein. Met35 in the hydrated cavity was recently found to undergo a dynamical cross-over from the dominance of methyl rotations at low temperatures to the dominance of diffusive motion of methyl axis at high temperatures. Current results indicate that in FMOC-Met this cross-over is suppressed, similar to what was observed for the dry fibrils, indicating that hydration of the cavity is driving the onset of the dynamical transition. [ABSTRACT FROM AUTHOR]

2H NMR order parameters of the acyl chain of phospholipid membranes are an important indicator of the effects of molecules on membrane order, mobility, and permeability. So far, the evaluation procedures are case-by-case studies for every type of small molecule with certain types of membranes. Rapid screening of the effects of a variety of drugs would be invaluable if it were possible. Unfortunately, to date there is no practical or theoretical approach to this as there is with other experimental parameters, e.g., chemical shifts from 1H and 13C NMR. We aim to remedy this situation by introducing a model based on graph neural networks (GNN) capable of predicting 2H NMR order parameters of lipid membranes in the presence of different molecules based on learned molecular features. Rapid prediction of these parameters would allow fast assessment of potential effects of drugs on lipid membranes, which is important for further drug development and provides insight into potential side effects. We conclude that the graph network-based model presented in this work can predict order parameters with sufficient accuracy, and we are confident that the concepts presented are a suitable basis for future research. We also make our model available to the public as a web application at https://proteinformatics.uni-leipzig.de/g2r/. [Display omitted] • 2H NMR chain order are an important tool to characterize the membrane interaction. • molecular features can be extracted by graph neural networks. • machine learning on molecular features can be used to predict membrane impact. • graph neural networks can predict 2H NMR order parameters of lipid membranes. [ABSTRACT FROM AUTHOR]

This work proposes a new approach to characterize cell membranes in intact cells by 2 H solid-state nuclear magnetic resonance (NMR) in only a few hours using magic-angle spinning (MAS) and spectral moment analysis. The method was first validated on model dipalmitoylphosphatidylcholine (DPPC) membranes, allowing the detection of lipid fluctuations below the main transition temperature. Then the lipid dynamics in Escherichia coli membranes was compared in bacteria grown under different diets. More specifically, deuterated palmitic acid was used to isotopically label the phospholipid acyl chains in bacteria membranes, with or without the presence of protonated oleic acid. Our results showed improved lipid fluidity when bacteria were grown in the presence of oleic acid, which helps preserving the natural fatty acid profile in E. coli membranes. The MAS 2 H solid-state NMR study of membranes combined with spectral moment analysis showed to be a fast method compatible with in vivo bacterial studies, and should also be applicable to other micro-organisms to obtain molecular information on living cells by solid-state NMR. [ABSTRACT FROM AUTHOR]

[Display omitted] • 2H rotating frame NMR relaxation measurements for dynamics studies in rotating solids. • Experimental systems are DMSO-D6 and amyloid-beta fibrils. • Theoretical/computational considerations within 2-site jump models are presented. We demonstrate experimental and computational approaches for measuring 2H rotating frame NMR relaxation for solid samples under magic angle spinning (MAS) conditions. The relaxation properties of the deuterium spin-1 system are dominated by the reorientation of the anisotropic quadrupolar tensors, with the effective quadrupolar coupling constant around 55 kHz for methyl groups. The technique is demonstrated using the model compound dimethyl-sulfone at MAS rates of 10 and 60 kHz as well as for an amyloid fibril sample comprising an amyloid-β (1–40) protein with a selective methyl group labeled in the disordered domain of the fibrils, at an MAS rate of 8 kHz. For both systems, the motional parameters fall well within the ranges determined by other techniques, thus validating its feasibility. Experimental and computational factors such as i) the probe's radio frequency inhomogeneity profiles, ii) rotary resonances at conditions for which the spin-lock field strength matches the half- or full-integer of the MAS rate, iii) the choice of MAS rates and spin-lock field strengths, and iv) simulations that account for the interconversion of multiple coherences for the spin-1 system under MAS and deviations from the analytical Redfield treatment are thoroughly considered. [ABSTRACT FROM AUTHOR]

We provide an NMRPipe macro to meet an increasing need in membrane biophysics for facile de-Pake-ing of axially symmetric deuterium, and to an extent phosphorous, static lineshapes. The macro implements the development of McCabe & Wassall (1997), and is run as a simple replacement for the usual Fourier transform step in an NMRPipe processing procedure.

Abstract: Natural rubber (NR) film was crosslinked by γ-irradiation under uniaxial extension. The degree of deformation defined as λ′=l′/l 0′ (l 0′ and l′ being the lengths of relaxed and uniaxially deformed sample, respectively) was constant and equal to 1.50, and the total irradiation dose was 300kGy. Deuterium NMR spectroscopy, known as an extremely sensitive method to detect anisotropic molecular motions, was applied for the study of dynamic behaviour of polyisoprene chain segments. Perdeuterated cyclohexane was used as deuterium probe. NMR spectra indicate that segmental motions in NR films crosslinked under deformation are anisotropic. Moreover, spectra of NR films stretched in the direction parallel and perpendicular to the direction of deformation applied during the crosslinking process show an intrinsic difference in orientational behaviour of chain segments. [Copyright &y& Elsevier]

Understanding how non-lipid components of bacteria affect antimicrobial peptide (AMP)-induced membrane disruption is important for a comprehensive understanding of AMP mechanisms and informing AMP-based drug development. This study investigates how lipopolysaccharide (LPS) affects membrane disruption by the AMP MSI-78 and compares the results to the effect of TP2, a cell-penetrating peptide that crosses membrane bilayers without permeabilizing them. We destabilize the LPS layer of Escherichia coli (E. coli) cells via chelation of the stabilizing divalent cations. 2 H NMR spectra of E. coli demonstrate that EDTA concentrations of 2.5 mM and 9.0 mM alone have very minor effects on lipid acyl chain order. Interestingly, we find that E. coli pre-treated with 9.0 mM EDTA before treatment with MSI-78 are more sensitive to AMP-induced acyl chain disruption, indicating that intact LPS reduces MSI-78-induced membrane disruption in E. coli. Surprisingly, we also found that at the level of 2 H NMR, the peptide-induced acyl chain disruption is similar for MSI-78 and TP2, although MSI-78 permeabilizes the bilayer and TP2 does not. Furthermore, LPS disruption appears to protect the bacteria from TP2, although it sensitizes them to MSI-78., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)