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Solid-state NMR (ss-NMR) is a powerful tool to investigate noncrystallizable, poorly soluble molecular systems, such as membrane proteins, amyloids, and cell walls, in environments that closely resemble their physical sites of action. Rotational-echo double resonance (REDOR) is an ss-NMR methodology, which by reintroducing heteronuclear dipolar coupling under magic angle spinning conditions provides intramolecular and intermolecular distance restraints at the atomic level. In addition, REDOR can be exploited as a selection tool to filter spectra based on dipolar couplings. Used extensively as a spectroscopic ruler between isolated spins in site-specifically labeled systems and more recently as a building block in multidimensional ss-NMR pulse sequences allowing the simultaneous measurement of multiple distances, REDOR yields atomic-scale information on the structure and interaction of proteins. By extending REDOR to the determination of 1H–X dipolar couplings in recent years, the limit of measurable distances has reached ~15–20 Å, making it an attractive method of choice for the study of complex biomolecular assemblies. Following a methodological introduction including the most recent implementations, examples are discussed to illustrate the versatility of REDOR in the study of biological systems. [ABSTRACT FROM AUTHOR]

Solid-state NMR (ss-NMR) is a powerful tool to investigate noncrystallizable, poorly soluble molecular systems, such as membrane proteins, amyloids, and cell walls, in environments that closely resemble their physical sites of action. Rotational-echo double resonance (REDOR) is an ss-NMR methodology, which by reintroducing heteronuclear dipolar coupling under magic angle spinning conditions provides intramolecular and intermolecular distance restraints at the atomic level. In addition, REDOR can be exploited as a selection tool to filter spectra based on dipolar couplings. Used extensively as a spectroscopic ruler between isolated spins in site-specifically labeled systems and more recently as a building block in multidimensional ss-NMR pulse sequences allowing the simultaneous measurement of multiple distances, REDOR yields atomic-scale information on the structure and interaction of proteins. By extending REDOR to the determination of 1H–X dipolar couplings in recent years, the limit of measurable distances has reached ~15–20 Å, making it an attractive method of choice for the study of complex biomolecular assemblies. Following a methodological introduction including the most recent implementations, examples are discussed to illustrate the versatility of REDOR in the study of biological systems.

Dynamic nuclear polarization surface enhanced NMR spectroscopy has enabled the determination of high-resolution structures from surface-supported molecules, including single-site heterogeneous catalysts. Structure determinations have largely mimicked the approaches used in biomolecular NMR spectroscopy, namely, using distance measurements to constrain a conformational search. These early demonstrations made use of purpose-built software, which has limited the adoption of the technique. Herein, we describe the open-source program INTERFACES (Interpret NMR to Elucidate or Reconstruct the Full Atomistic Configurations of External Surfaces) which automates the analysis of RE(SP)DOR data as well as the structure determination for surface sites. Distances, angles, dihedral angles, complex orientation, and distance from the support can all be sampled to find all structures that agree with the experimental data. A χ2 metric is used to define the error ranges of the REDOR fits and produce structures with an arbitrary level of confidence. Structural solutions are then provided as both overlays and ORTEP-like probability ellipsoids.

Dynamic nuclear polarization NMR spectroscopy was used to investigate the effect of the antimicrobial peptide (AMP) maculatin 1.1 on E. coli cells. The enhanced 15N NMR signals from nucleic acids, proteins and lipids identified a number of unanticipated physiological responses to peptide stress, revealing that membrane-active AMPs can have a multi-target impact on E. coli cells. DNP-enhanced 15N-observed 31P-dephased REDOR NMR allowed monitoring how Mac1 induced DNA condensation and prevented intermolecular salt bridges between the main E. coli lipid phosphatidylethanolamine (PE) molecules. The latter was supported by similar results obtained using E. coli PE lipid systems. Overall, the ability to monitor the action of antimicrobial peptides in situ will provide greater insight into their mode of action.

Accurate distance measurements between proton and nitrogen can provide detailed information on the structures and dynamics of various molecules. The combination of broadband phase-modulated (PM) pulse and rotational-echo saturation-pulse double-resonance (RESPDOR) sequence at fast magic-angle spinning (MAS) has enabled the measurement of multiple 1H-14N distances with high accuracy. However, complications may arise when applying this sequence to systems with multiple inequivalent 14N nuclei, especially a single 1H sitting close to multiple 14N atoms. Due to its broadband characteristics, the PM pulse saturates all 14N atoms; hence, the single 1H simultaneously experiences the RESPDOR effect from multiple 1H-14N couplings. Consequently, no reliable H-N distances are obtained. To overcome the problem, selective 14N saturation is desired, but it is difficult because 14N is an integer quadrupolar nucleus. Alternatively, 14N overtone (OT) NMR spectroscopy can be employed owing to its narrow bandwidth for selectivity. Moreover, owing to the sole presence of two energy levels (m = ± 1), the 14N OT spin dynamics behaves similarly to that of spin-1/2. This allows the interchangeability between RESPDOR and rotational-echo double-resonance (REDOR) since their principles are the same except the degree of 14N OT population transfer; saturation for the former whereas inversion for the latter. As the ideal saturation/inversion is impractical due to the slow and orientation-dependent effective nutation of 14N OT, the working condition is usually an intermediate between REDOR and RESPDOR. The degree of 14N OT population transfer can be determined from the results of protons with short distances to 14N and then can be used to obtain long-distance determination of other protons to the same 14N site. Herein, we combine the 14N OT and REDOR/RESPDOR to explore the feasibility of selective 1H-14N distance measurements. Experimental demonstrations on simple biological compounds of L-tyrosine.HCl, N-acetyl-L-alanine, and L-alanyl-L-alanine were performed at 14.1 T and MAS frequency of 62.5 kHz. The former two consist of a single 14N site, whereas the latter consists of two 14N sites. The experimental optimizations and reliable fittings by the universal curves are described. The extracted 1H-14N distances by OT-REDOR are in good agreement with those determined by PM-RESPDOR and diffraction techniques.

Staphylococcus aureus biofilms pose a serious clinical threat as reservoirs for persistent infections. Despite this clinical significance, the composition and mechanism of formation of S. aureus biofilms are unknown. To address these problems, we used solid-state NMR to examine S. aureus (SA113), a strong biofilm-forming strain. We labeled whole cells and cell walls of planktonic cells, young biofilms formed for 12–24 h after stationary phase, and more mature biofilms formed for up to 60 h after stationary phase. All samples were labeled either by (i) [ 15 N]glycine and l -[1- 13 C]threonine, or in separate experiments, by (ii) l -[2- 13 C, 15 N]leucine. We then measured 13 C- 15 N direct bonds by C{N} rotational-echo double resonance (REDOR). The increase in peptidoglycan stems that have bridges connected to a surface protein was determined directly by a cell-wall double difference (biofilm REDOR difference minus planktonic REDOR difference). This procedure eliminates errors arising from differences in 15 N isotopic enrichments and from the routing of 13 C label from threonine degradation to glycine. For both planktonic cells and the mature biofilm, 20% of pentaglycyl bridges are not cross-linked and are potential surface-protein attachment sites. None of these sites has a surface protein attached in the planktonic cells, but one-fourth have a surface protein attached in the mature biofilm. Moreover, the leucine-label shows that the concentration of β-strands in leucine-rich regions doubles in the mature biofilm. Thus, a primary event in establishing a S. aureus biofilm is extensive decoration of the cell surface with surface proteins that are linked covalently to the cell wall and promote cell-cell adhesion. [ABSTRACT FROM AUTHOR]

Solid-state NMR spectra of whole cells and isolated cell walls of Enterococcus faecalis grown in media containing combinations of 13 C and 15 N specific labels in d - and l -alanine and l -lysine (in the presence of an alanine racemase inhibitor alaphosphin) have been used to determine the composition and architecture of the cell-wall peptidoglycan. The compositional variables include the concentrations of (i) peptidoglycan stems without bridges, (ii) d -alanylated wall teichoic acid, (iii) cross-links, and (iv) uncross-linked tripeptide and tetra/pentapeptide stems. Connectivities of l -alanyl carbonyl‑carbon bridge labels to d -[3- 13 C]alanyl and l -[ε- 15 N]lysyl stem labels prove that the peptidoglycan of E. faecalis has the same hybrid short-bridge architecture (with a mix of parallel and perpendicular stems) as the FemA mutant of Staphylococcus aureus , in which the cross-linked stems are perpendicular to one another and the cross-linking is close to the ideal 50% value. This is the first determination of the cell-wall chemical and geometrical architecture of whole cells of E. faecalis , a major source of nosocomial infections worldwide. [ABSTRACT FROM AUTHOR]

The structural information of small therapeutic compounds complexed in biological matrices is important for drug developments. However, structural studies on ligands bound to such a large and dynamic system as microtubules are still challenging. This article reports an application of the solid-state NMR technique to investigating the bioactive conformation of epothilone B, a microtubule stabilizing agent, whose analog ixabepilone was approved by the U.S. Food and Drug Administration (FDA) as an anticancer drug. First, an analog of epothilone B was designed and successfully synthesized with deuterium and fluorine labels while keeping the high potency of the drug; Second, a lyophilization protocol was developed to enhance the low sensitivity of solid-state NMR; Third, molecular dynamics information of microtubule-bound epothilone B was revealed by high-resolution NMR spectra in comparison to the non-bound epothilone B; Last, information for the macrolide conformation of microtubule-bound epothilone B was obtained from rotational-echo double-resonance (REDOR) NMR data, suggesting the X-ray crystal structure of the ligand in the P450epoK complex as a possible candidate for the conformation. Our results are important as the first demonstration of using REDOR for studying epothilones. [ABSTRACT FROM AUTHOR]

The carboxylic acid proton and the lithium coordination environments for precise and random Li-neutralized polyethylene acrylic acid P(E-AA) ionomers were explored using high speed solid-state 1H and 7Li MAS NMR. While the 7Li NMR revealed only a single Li coordination environment, the chemical shift temperature variation was dependent on the precise or random nature of the P(E-AA) ionomer. The 1H MAS NMR revealed two different carboxylic acid proton environments in these materials. By utilizing 1H-7Li rotational echo double resonance (REDOR) MAS NMR experiments, it was demonstrated that the proton environments correspond to different average 1H-7Li distances, with the majority of the protonated carboxylic acids having a close through space contact with the Li. Molecular dynamics simulations suggest that the shortest 1H-7Li distance corresponds to un-neutralized carboxylic acids directly involved in the coordination environment of Li clusters. These solid-state NMR results show that heterogeneous structural motifs need to be included when developing descriptions of these ionomer materials.

Solid state NMR (ssNMR) can characterize mineral ( 31 P) and organic ( 13 C) components of human salivary stones ( n = 8). All show apatitic 31 P spectra. 13 C ssNMR indicates more protein, of more consistent composition, than apatitic uroliths, with prominent signals from Tyr, Phe, and His. Citrate and lipids, identified by dipolar dephasing (DD), and polysaccharides are also observable in varying amounts. 13 C{ 31 P} rotational echo double resonance ( 13 C{ 31 P} REDOR) identifies carbon atoms in close (

This research, based on bibliographical and documentary approaches, aimed to recover the trajectory and memory of gender studies centers and groups belonging to REDOR, a feminist network of gender and women studies centers in the North and Northeast of Brazil. Data collection included: background and institutional location; profile and academic field of members; research, extension and training projects developed; impact on institucional policies; partnerships and social impact. Initially, we highlight the significance of the creation of these gender research centers and network, which emerged in order to legitimate a new field of knowledge. Next, we present the data that was possible to collect about 35 centers and groups. We conclude that REDOR is very important for the regions, insofar as it promotes the exchance of knowledge, in the context of the struggle for gender equity. [ABSTRACT FROM AUTHOR]

Peptidoglycan is an essential component of cell wall in Gram-positive bacteria with unknown architecture. In this review, we summarize solid-state NMR approaches to address some of the unknowns in the Gram-positive bacteria peptidoglycan architecture: 1) peptidoglycan backbone conformation, 2) PG-lattice structure, 3) variations in the peptidoglycan architecture and composition, 4) the effects of peptidoglycan bridge-length on the peptidoglycan architecture in Fem mutants, 5) the orientation of glycan strands with respect to the membrane, and 6) the relationship between the peptidoglycan structure and the glycopeptide antibiotic mode of action. Solid-state NMR analyses of Staphylococcus aureus cell wall show that peptidoglycan chains are surprisingly ordered and densely packed. The peptidoglycan disaccharide backbone adopts 4-fold screw helical symmetry with the disaccharide unit periodicity of 40 Å. Peptidoglycan lattice in the S . aureus cell wall is formed by cross-linked PG stems that have parallel orientations. The structural characterization of Fem-mutants of S . aureus with varying lengths of bridge structures suggests that the PG-bridge length is an important determining factor for the PG architecture. This article is part of a Special Issue entitled: NMR Spectroscopy for Atomistic Views of Biomembranes and Cell Surfaces. Guest Editors: Lynette Cegelski and David P. Weliky. [ABSTRACT FROM AUTHOR]

Bacterial biofilms are communities of bacterial cells surrounded by a self-secreted extracellular matrix. Biofilm formation by Vibrio cholerae , the human pathogen responsible for cholera, contributes to its environmental survival and infectivity. Important genetic and molecular requirements have been identified for V. cholerae biofilm formation, yet a compositional accounting of these parts in the intact biofilm or extracellular matrix has not been described. As insoluble and non-crystalline assemblies, determinations of biofilm composition pose a challenge to conventional biochemical and biophysical analyses. The V. cholerae extracellular matrix composition is particularly complex with several proteins, complex polysaccharides, and other biomolecules having been identified as matrix parts. We developed a new top-down solid-state NMR approach to spectroscopically assign and quantify the carbon pools of the intact V. cholerae extracellular matrix using 13 C CPMAS and 13 C{ 15 N}, 15 N{ 31 P}, and 13 C{ 31 P}REDOR. General sugar, lipid, and amino acid pools were first profiled and then further annotated and quantified as specific carbon types, including carbonyls, amides, glycyl carbons, and anomerics. In addition, 15 N profiling revealed a large amine pool relative to amide contributions, reflecting the prevalence of molecular modifications with free amine groups. Our top-down approach could be implemented immediately to examine the extracellular matrix from mutant strains that might alter polysaccharide production or lipid release beyond the cell surface; or to monitor changes that may accompany environmental variations and stressors such as altered nutrient composition, oxidative stress or antibiotics. More generally, our analysis has demonstrated that solid-state NMR is a valuable tool to characterize complex biofilm systems. This article is part of a Special Issue entitled: NMR Spectroscopy for Atomistic Views of Biomembranes and Cell Surfaces. Guest Editors: Lynette Cegelski and David P. Weliky. [ABSTRACT FROM AUTHOR]

Dynamic nuclear polarization NMR spectroscopy was used to investigate the effect of the antimicrobial peptide (AMP) maculatin 1.1 on E. coli cells. The enhanced 15 N NMR signals from nucleic acids, proteins and lipids identified a number of unanticipated physiological responses to peptide stress, revealing that membrane-active AMPs can have a multi-target impact on E. coli cells. DNP-enhanced 15 N-observed 31 P-dephased REDOR NMR allowed monitoring how Mac1 induced DNA condensation and prevented intermolecular salt bridges between the main E. coli lipid phosphatidylethanolamine (PE) molecules. The latter was supported by similar results obtained using E. coli PE lipid systems. Overall, the ability to monitor the action of antimicrobial peptides in situ will provide greater insight into their mode of action., Competing Interests: 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., (© 2022 The Author(s).)

Solid-state NMR provides insight into protein motion over time scales ranging from picoseconds to seconds. While in solution state the methodology to measure protein dynamics is well established, there is currently no such consensus protocol for measuring dynamics in solids. In this article, we perform a detailed investigation of measurement protocols for fast motions, i.e. motions ranging from picoseconds to a few microseconds, which is the range covered by dipolar coupling and relaxation experiments. We perform a detailed theoretical investigation how dipolar couplings and relaxation data can provide information about amplitudes and time scales of local motion. We show that the measurement of dipolar couplings is crucial for obtaining accurate motional parameters, while systematic errors are found when only relaxation data are used. Based on this realization, we investigate how the REDOR experiment can provide such data in a very accurate manner. We identify that with accurate rf calibration, and explicit consideration of rf field inhomogeneities, one can obtain highly accurate absolute order parameters. We then perform joint model-free analyses of 6 relaxation data sets and dipolar couplings, based on previously existing, as well as new data sets on microcrystalline ubiquitin. We show that nanosecond motion can be detected primarily in loop regions, and compare solid-state data to solution-state relaxation and RDC analyses. The protocols investigated here will serve as a useful basis towards the establishment of a routine protocol for the characterization of ps–μs motions in proteins by solid-state NMR. [ABSTRACT FROM AUTHOR]

The influenza virus fusion peptide is the N-terminal ~20 residues of the HA2 subunit of the hemagglutinin protein and this peptide plays a key role in the fusion of the viral and endosomal membranes during initial infection of a cell. The fusion peptide adopts N-helix/turn/C-helix structure in both detergent and membranes with reports of both open and closed interhelical topologies. In the present study, backbone CO-N REDOR solid-state NMR was applied to the membrane-associated fusion peptide to detect the distribution of interhelical distances. The data clearly showed a large fraction of closed and semi-closed topologies and were best-fitted to a mixture of two structures that do not exchange. One of the earlier open structural models may have incorrect G13 dihedral angles derived from TALOS analysis of experimentally correct C shifts. [ABSTRACT FROM AUTHOR]

Residue-specific location of peptides in the hydrophobic core of membranes was examined using C-H REDOR and samples in which the lipids were selectively deuterated. The transmembrane topology of the KALP peptide was validated with this approach with substantial dephasing observed for deuteration in the bilayer center and reduced or no dephasing for deuteration closer to the headgroups. Insertion of β sheet HIV and helical and β sheet influenza virus fusion peptides into the hydrophobic core of the membrane was validated in samples with extensively deuterated lipids. [ABSTRACT FROM AUTHOR]

Several techniques for spectral editing of 2D C-C correlation NMR of proteins are introduced. They greatly reduce the spectral overlap for five common amino acid types, thus simplifying spectral assignment and conformational analysis. The carboxyl (COO) signals of glutamate and aspartate are selected by suppressing the overlapping amide N-CO peaks through C-N dipolar dephasing. The sidechain methine (CH) signals of valine, lecuine, and isoleucine are separated from the overlapping methylene (CH) signals of long-chain amino acids using a multiple-quantum dipolar transfer technique. Both the COO and CH selection methods take advantage of improved dipolar dephasing by asymmetric rotational-echo double resonance (REDOR), where every other π-pulse is shifted from the center of a rotor period t by about 0.15 t. This asymmetry produces a deeper minimum in the REDOR dephasing curve and enables complete suppression of the undesired signals of immobile segments. Residual signals of mobile sidechains are positively identified by dynamics editing using recoupled C-H dipolar dephasing. In all three experiments, the signals of carbons within a three-bond distance from the selected carbons are detected in the second spectral dimension via C spin exchange. The efficiencies of these spectral editing techniques range from 60 % for the COO and dynamic selection experiments to 25 % for the CH selection experiment, and are demonstrated on well-characterized model proteins GB1 and ubiquitin. [ABSTRACT FROM AUTHOR]

The carboxylic acid proton and the lithium coordination environments for precise and random Li-neutralized polyethylene acrylic acid P(E-AA) ionomers were explored using high speed solid-state 1H and 7Li MAS NMR. While the 7Li NMR revealed only a single Li coordination environment, the chemical shift temperature variation was dependent on the precise or random nature of the P(E-AA) ionomer. The 1H MAS NMR revealed two different carboxylic acid proton environments in these materials. By utilizing 1H-7Li rotational echo double resonance (REDOR) MAS NMR experiments, it was demonstrated that the proton environments correspond to different average 1H-7Li distances, with the majority of the protonated carboxylic acids having a close through space contact with the Li. Molecular dynamics simulations suggest that the shortest 1H-7Li distance corresponds to un-neutralized carboxylic acids directly involved in the coordination environment of Li clusters. These solid-state NMR results show that heterogeneous structural motifs need to be included when developing descriptions of these ionomer materials. [ABSTRACT FROM AUTHOR]

Formation of the complex between human immunodeficiency virus type-1 Tat protein and the transactivation response region (TAR) RNA is vital for transcriptional elongation, yet the structure of the Tat-TAR complex remains to be established. The NMR structures of free TAR, and TAR bound to Tat-derived peptides have been obtained by solution NMR, but only a small number of intermolecular NOEs could be identified unambiguously, preventing the determination of a complete structure. Here we show that a combination of multiple solid state NMR REDOR experiments can be used to obtain multiple distance constraints from N to C spins within the backbone and side chain guanidinium groups of arginine in a Tat-derived peptide, using F spins incorporated into the base of U23 in TAR and P spins in the P22 and P23 phosphate groups. Distances between the side chain of Arg52 and the base and phosphodiester backbone near U23 measured by REDOR NMR are comparable to distances observed in solution NMR-derived structural models, indicating that interactions of TAR RNA with key amino acid side chains in Tat are the same in the amorphous solid state as in solution. This method is generally applicable to other protein-RNA complexes where crystallization or solution NMR has failed to provide high resolution structural information. [ABSTRACT FROM AUTHOR]

Abstract: Epicatechin gallate (ECg), a green tea polyphenol, has various physiological effects. Our previous nuclear Overhauser effect spectroscopy (NOESY) study using solution NMR spectroscopy demonstrated that ECg strongly interacts with the surface of phospholipid bilayers. However, the dynamic behavior of ECg in the phospholipid bilayers has not been clarified, especially the dynamics and molecular arrangement of the galloyl moiety, which supposedly has an important interactive role. In this study, we synthesized [13C]-ECg, in which the carbonyl carbon of the galloyl moiety was labeled by 13C isotope, and analyzed it by solid-state NMR spectroscopy. Solid-state 31P NMR analysis indicated that ECg changes the gel-to-liquid-crystalline phase transition temperature of DMPC bilayers as well as the dynamics and mobility of the phospholipids. In the solid-state 13C NMR analysis under static conditions, the carbonyl carbon signal of the [13C]-ECg exhibited an axially symmetric powder pattern. This indicates that the ECg molecules rotate about an axis tilting at a constant angle to the bilayer normal. The accurate intermolecular–interatomic distance between the labeled carbonyl carbon of [13C]-ECg and the phosphorus of the phospholipid was determined to be 5.3±0.1Å by 13C–31P rotational echo double resonance (REDOR) measurements. These results suggest that the galloyl moiety contributes to increasing the hydrophobicity of catechin molecules, and consequently to high affinity of galloyl-type catechins for phospholipid membranes, as well as to stabilization of catechin molecules in the phospholipid membranes by cation–π interaction between the galloyl ring and quaternary amine of the phospholipid head-group. [Copyright &y& Elsevier]

Rhodopsin is the visual pigment of the vertebrate rod photoreceptor cell and is the only member of the G protein coupled receptor family for which a crystal structure is available. Towards the study of dynamics in rhodopsin, we report NMR-spectroscopic investigations of α,ɛ-15N-tryptophan labeled rhodopsin in detergent micelles and reconstituted in phospholipids. Using a combination of solid state 13C,15N-REDOR and HETCOR experiments of all possible 13C′ i-1 carbonyl/15N i -tryptophan isotope labeled amide pairs, and H/D exchange 1H,15N-HSQC experiments conducted in solution, we assigned chemical shifts to all five rhodopsin tryptophan backbone 15N nuclei and partially to their bound protons. 1H,15N chemical shift assignment was achieved for indole side chains of Trp351.30 and Trp1754.65. 15N chemical shifts were found to be similar when comparing those obtained in the native like reconstituted lipid environment and those obtained in detergent micelles for all tryptophans except Trp1754.65 at the membrane interface. The results suggest that the integrated solution and solid state NMR approach presented provides highly complementary information in the study of structure and dynamics of large membrane proteins like rhodopsin. [ABSTRACT FROM AUTHOR]

Abstract: Solid-state NMR spectroscopic techniques were used to investigate the secondary structure of the transmembrane peptide phospholamban (TM-PLB), a sarcoplasmic Ca2+ regulator. 13C cross-polarization magic angle spinning spectra of 13C carbonyl-labeled Leu39 of TM-PLB exhibited two peaks in a pure 1-palmitoyl-2-oleoyl-phosphocholine (POPC) bilayer, each due to a different structural conformation of phospholamban as characterized by the corresponding 13C chemical shift. The addition of a negatively charged phospholipid (1-palmitoyl-2-oleoylphosphatidylglycerol (POPG)) to the POPC bilayer stabilized TM-PLB to an α-helical conformation as monitored by an enhancement of the α-helical carbonyl 13C resonance in the corresponding NMR spectrum. 13C–15N REDOR solid-state NMR spectroscopic experiments revealed the distance between the 13C carbonyl carbon of Leu39 and the 15N amide nitrogen of Leu42 to be 4.2±0.2Å indicating an α-helical conformation of TM-PLB with a slight deviation from an ideal 3.6 amino acid per turn helix. Finally, the quadrupolar splittings of three 2H labeled leucines (Leu28, Leu39, and Leu51) incorporated in mechanically aligned DOPE/DOPC bilayers yielded an 11°±5° tilt of TM-PLB with respect to the bilayer normal. In addition to elucidating valuable TM-PLB secondary structure information, the solid-state NMR spectroscopic data indicates that the type of phospholipids and the water content play a crucial role in the secondary structure and folding of TM-PLB in a phospholipid bilayer. [Copyright &y& Elsevier]

Some key aspects of the secondary structure of solid orexin-B, a 28 amino-acid peptide, have been investigated by solid-state NMR spectroscopy. The 13C–15N dipolar coupling between the carbonyl carbon of Leu11 and the nitrogen of Leu15, as determined by rotational echo double resonance (REDOR) experiments, is 35 Hz, indicating that these nuclei are separated by approximately 4.5 Å. This distance is consistent with the α-helical structure determined for this segment of orexin-B by solution NMR measurements. REDOR measurements of the dipolar coupling between the carbonyl carbon of Ala17 and the nitrogen of Ala22 support the contention in an earlier solution NMR study that a bend exists between the two α helices of orexin-B. However, in the solid state the internuclear distance (6.4 Å) is significantly greater than that observed for orexin-B in aqueous solution. In addition to the distance measurements, the principal components of the amide carbonyl carbon chemical shift (CS) tensors for Leu11 and Ala17 and of the amide nitrogen CS tensors for Leu15 and Ala22 are reported. There are only minor differences between the amide carbonyl carbon CS tensors for Leu11 and Ala17 and between the nitrogen CS tensors for Leu15 and Ala22. [ABSTRACT FROM AUTHOR]

NMR study of materials for immobilisation of nuclear waste. We present an NMR investigation on thorium phosphate hydrogen phosphate (TPHP), which is a precursor for thorium phosphate diphosphate (TPD), that is one of the four retained matrix for the study of the actinide immobilisation. As XRD cannot work, it is particularly difficult to define the TPHP chemical formula: Th2(PO4)2HPO4 H2O or Th4(PO4)4P2O7 3 H2O. In this contribution, we show that a local technique as NMR can bring an answer to this problem. To cite this article : J. Emery et al., C. R. Chimie 7 (2004). [Copyright &y& Elsevier]

19F/29Si rotational-echo double-resonance (REDOR), θ-REDOR and Hartmann–Hahn cross-polarization (CP) NMR techniques have been applied under fast magic-angle spinning (MAS) to a powder sample of fluoride-containing octadecasil. The internuclear distance of the 19F–29Si spin pairs formed by the silicons in the D4R units (T-1 site) and the fluoride anions deduced directly from the CP and REDOR measurements is in very good agreement with the X-ray crystal structure. Moreover, heteronuclear spin-counting θ-REDOR experiments unambiguously show that the 19F and the T-1 29Si nuclei form fairly isolated spin pairs, whereas multiple-spin behaviour is clearly evidenced at the T-2 site. To cite this article: P. Bertani et al., C. R. Chimie 7 (2004). [Copyright &y& Elsevier]

Spiders synthesize several kinds of silk fibers. In the primary structure of spider silk, one of the major ampullate (dragline, frame) silks, spidroin 1, and flagelliform silk (core fibers of adhesive spiral), there are common repeated X-Gly-Gly (X = Ala, Leu, Pro, Tyr, Glu, and Arg) sequences, which are considered to be related to the elastic character of these fibers. In this paper, two dimensional spin diffusion solid-state NMR under off magic angle spinning (OMAS), 13C chemical shift contour plots, and Rotational Echo DOuble Resonance (REDOR) were applied to determine the torsion angles of one Ala and two kinds of Gly residues in the Ala-Gly-Gly sequence of 13C=O isotope-labeled (Ala-Gly-Gly)10. The torsion angles were determined to be (φ, ψ) = (-90°, 150° ) within an experimental error of ±10° for each residue. This conformation is characterized as 31 helix which is in agreement with the structure proposed from the X-ray powder diffraction pattern of poly(Ala-Gly-Gly). The 31 helix of (Ala-Gly-Gly)10 does not change by formic acid treatment although (Ala-Gly)15 easily changes from the silk I conformation (the structure of Bombyx mori silk fibroin before spinning in the solid state) to silk II conformation (the structure of the silk fiber after spinning) by such treatment. Thus, the 31 helix conformation of (Ala-Gly-Gly)10 is considered very stable. Furthermore, the torsion angles of the 16th Leu residue of (Leu-Gly-Gly)10 were also determined as (φ, ψ) = (-90°, 150° ) and this peptide is also considered to take 31 helix conformation. [ABSTRACT FROM AUTHOR]

An approach to the determination of the 2-13C′ chemical shift (CS) tensor orientation in pyrimidine bases via heteronuclear MAS NMR spectroscopy is presented. Considering a dipolar coupled spin 1/2 network of the type S1-I-S2 consisting of directly bonded heteronuclear spins, we have carried out numerical simulations to assess the sensitivity of I-S REDOR spinning sidebands to the Euler angles defining the orientation of the I-S1 and I-S2 dipolar vectors in the I spin CS tensor principal axes system. Our investigations clearly demonstrate the potential of I-S REDOR studies in IS1S2 systems for obtaining with high reliability and accuracy the I spin chemical shift tensor orientation in the molecular frame spanned by the two internuclear vectors I-S1 and I-S2. The significant contribution to the observed REDOR sideband intensities from anti-phase operator terms which are present at the start of the data acquisition is illustrated. The procedure for the recording and analysis of the I-S REDOR spectra in IS1S2 systems is presented and the measurement of the 2-13C′ CS tensor orientation in a polycrystalline sample of [1,3-15N2, 2-13C] uracil, which is one of the four bases in RNA, is experimentally demonstrated. [ABSTRACT FROM AUTHOR]

Being a major metabolite for maintaining cellular homeostasis, as well as an important structural component in lipid membrane, cholesterol also plays critical roles in the life cycles of some viruses, including human immunodeficiency virus-1 (HIV-1). The involvement of cholesterol in HIV-1 infectivity, assembly and budding has made it an important research target. Viral protein R (Vpr) is an accessory protein of HIV-1, which is involved in many major events in the life cycle of HIV-1. In addition to its multi-functional roles in the HIV-1 life cycle, it is shown to interact with lipid membrane and form a cation-selective channel. In this work, we examined the effect of cholesterol on the interaction of Vpr and lipid membrane. Using calcein release assay, we found that the membrane permeability induced by the membrane binding of Vpr was significantly reduced in the presence of cholesterol in membrane. In addition, using solid-state NMR (ssNMR) spectroscopy, Vpr was shown to experience multiple chemical environments in lipid membrane, as indicated by the broad line shape of carbonyl 13C resonance of Cys-76 residue ranging from 165–178 ppm, which can be attributed to the existence of complex Vpr-membrane environments. We further showed that the presence of cholesterol in membrane will alter the distribution of Vpr in the complex membrane environments, which may explain the change of the Vpr induced membrane permeability in the presence of cholesterol. [ABSTRACT FROM AUTHOR]

Rotational-Echo Double-Resonance (REDOR) NMR on strategically 13C and 15N labeled samples is used to study the conformation of the LGXQ (X = S, G, or N) motif in the major ampullate gland dragline silk from the spider Nephila clavipes. A method is described for calculating REDOR dephasing curves suitable for background subtractions, using probability distributions of nitrogen atoms surrounding a given carbon site, which are developed from coordinates in the Brookhaven Protein Data Bank. The validity of the method is established by comparison to dephasings observed from natural abundance 13C peaks for G and A. Straightforward fitting of universal REDOR dephasing curves to the background corrected peaks of interest provide results which are not self-consistent, and a more sophisticated analysis is developed which better accounts for 15N labels which have scrambled from the intended positions. While there is likely some heterogeneity in the structures formed by the LGXQ sequences, the data indicate that they all form compact turn-like structures. [ABSTRACT FROM AUTHOR]

Accurate distance measurements between proton and nitrogen can provide detailed information on the structures and dynamics of various molecules. The combination of broadband phase-modulated (PM) pulse and rotational-echo saturation-pulse double-resonance (RESPDOR) sequence at fast magic-angle spinning (MAS) has enabled the measurement of multiple 1 H- 14 N distances with high accuracy. However, complications may arise when applying this sequence to systems with multiple inequivalent 14 N nuclei, especially a single 1 H sitting close to multiple 14 N atoms. Due to its broadband characteristics, the PM pulse saturates all 14 N atoms; hence, the single 1 H simultaneously experiences the RESPDOR effect from multiple 1 H- 14 N couplings. Consequently, no reliable H-N distances are obtained. To overcome the problem, selective 14 N saturation is desired, but it is difficult because 14 N is an integer quadrupolar nucleus. Alternatively, 14 N overtone (OT) NMR spectroscopy can be employed owing to its narrow bandwidth for selectivity. Moreover, owing to the sole presence of two energy levels ( m = ± 1), the 14 N OT spin dynamics behaves similarly to that of spin-1/2. This allows the interchangeability between RESPDOR and rotational-echo double-resonance (REDOR) since their principles are the same except the degree of 14 N OT population transfer; saturation for the former whereas inversion for the latter. As the ideal saturation/inversion is impractical due to the slow and orientation-dependent effective nutation of 14 N OT, the working condition is usually an intermediate between REDOR and RESPDOR. The degree of 14 N OT population transfer can be determined from the results of protons with short distances to 14 N and then can be used to obtain long-distance determination of other protons to the same 14 N site. Herein, we combine the 14 N OT and REDOR/RESPDOR to explore the feasibility of selective 1 H- 14 N distance measurements. Experimental demonstrations on simple biological compounds of L-tyrosine.HCl, N-acetyl-L-alanine, and L-alanyl-L-alanine were performed at 14.1 T and MAS frequency of 62.5 kHz. The former two consist of a single 14 N site, whereas the latter consists of two 14 N sites. The experimental optimizations and reliable fittings by the universal curves are described. The extracted 1 H- 14 N distances by OT-REDOR are in good agreement with those determined by PM-RESPDOR and diffraction techniques., Competing Interests: YN is employed by JEOL RESONANCE Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a past co-authorship with one of the authors YN., (Copyright © 2021 Duong, Gan and Nishiyama.)