Rotational Echo Double Resonance Measurements Research Articles

Spectroscopic Characterization of Adsorbed 13CO2 on 3-Aminopropylsilyl-Modified SBA15 Mesoporous Silica.

Multiple chemisorption products are found from the interaction of CO2 with the solid-amine sorbent, 3-aminopropyl silane (APS), bound to mesoporous silica (SBA15) using solid-state NMR and FTIR spectroscopy. We employed a combination of both 15N{13C} rotational-echo double-resonance (REDOR) NMR and 13C{15N} REDOR to determine the chemical identity of these products. 15N{13C} REDOR measurements are consistent with a single 13C-15N pair and distance of 1.45 Å. In contrast, both 13C{15N} REDOR and 13C CPMAS are consistent with multiple 13C products. 13C CPMAS shows two neighboring resonances, whose chemical shifts are consistent with carbamate (at 165 ppm) and carbamic acid. The 13C{15N} REDOR experiments resonant at 165 ppm show an incomplete buildup of the REDOR data to ∼90% of the expected maximum. We conclude this 10% missing intensity corresponds to a 13C NMR species that resonates at the identical chemical shift but that is not in dipolar contact with 15N. These data are consistent with the presence of bicarbonate, HCO3-, since it is commonly observed at ∼165 ppm and lacks 15N for dipolar coupling.

The Carboxyl Terminus of Eremomycin Facilitates Binding to the Non-d-Ala-d-Ala Segment of the Peptidoglycan Pentapeptide Stem.

Glycopeptide antibiotics inhibit cell wall biosynthesis in Gram-positive bacteria by targeting the peptidoglycan (PG) pentapeptide stem structure (l-Ala-d-iso-Gln-l-Lys-d-Ala-d-Ala). Structures of the glycopeptide complexed with a PG stem mimic have shown that the d-Ala-d-Ala segment is the primary drug binding site; however, biochemical evidence suggests that the glycopeptide-PG interaction involves more than d-Ala-d-Ala binding. Interactions of the glycopeptide with the non-d-Ala-d-Ala segment of the PG stem were investigated using solid-state nuclear magnetic resonance (NMR). LCTA-1421, a double (15)N-enriched eremomycin derivative with a C-terminal [(15)N]amide and [(15)N]Asn amide, was complexed with whole cells of Staphylococcus aureus grown in a defined medium containing l-[3-(13)C]Ala and d-[1-(13)C]Ala in the presence of alanine racemase inhibitor alaphosphin. (13)C{(15)N} and (15)N{(13)C} rotational-echo double-resonance (REDOR) NMR measurements determined the (13)C-(15)N internuclear distances between the [(15)N]Asn amide of LCTA-1421 and the (13)C atoms of the bound d-[1-(13)C]Ala-d-[1-(13)C]Ala to be 5.1 and 4.8 Å, respectively. These measurements also determined the distance from the C-terminal [(15)N]amide of LCTA-1421 to the l-[3-(13)C]Ala of PG to be 3.5 Å. The measured REDOR distance constraints position the C-terminus of the glycopeptide in the proximity of the l-Ala of the PG, suggesting that the C-terminus of the glycopeptide interacts near the l-Ala segment of the PG stem. In vivo REDOR measurements provided structural insight into how C-terminally modified glycopeptide antibiotics operate.

Head-to-Tail Interaction between Amphotericin B and Ergosterol Occurs in Hydrated Phospholipid Membrane

Amphotericin B (AmB) is thought to exert its antifungal activity by forming an ion-channel assembly in the presence of ergosterol. In the present study we aimed to elucidate the mode of molecular interactions between AmB and ergosterol in hydrated phospholipid bilayers using the rotational echo double resonance (REDOR) spectra. We first performed (13)C{(19)F}REDOR experiments with C14-(19)F-labeled AmB and biosynthetically (13)C-labeled ergosterol and implied that both "head-to-head" and "head-to-tail" orientations occur for AmB-ergosterol interaction in the bilayers. To further confirm the "head-to-tail" pairing, (13)C-labeled ergosterol at the dimethyl terminus (C26/C27) was synthesized and subjected to the REDOR measurements. The spectra unambiguously demonstrated the presence of a "head-to-tail" orientation for AmB-ergosterol pairing. In order to obtain information on the position of the dimethyl terminus of ergosterol in membrane, (13)C{(31)P}REDOR were carried out using the labeled ergosterol and the phosphorus atom of a POPC headgroup. Significant REDOR dephasing was observed at the C26/C27 signal of ergosterol in the presence of AmB, but not in the absence of AmB, clearly indicating that the side-chain terminus of ergosterol in the AmB complex comes close to the bilayer surface.

Local environment and distribution of alkali ions in polyelectrolyte complexes studied by solid-state NMR

Polyelectrolyte complexes (PECs) formed by the addition of substoichiometric amounts of (poly(diallyldimethyl ammonium chloride)) (PDADMAC) solutions to sodium or lithium poly(styrene sulfonate) (Na- or Li-PSS) solution contain adjustable amounts of charge balancing Li(+) or Na(+) cations, which possess ionic mobility of interest for solid electrolyte applications. Very little is known regarding the local environments and the spatial distributions of these cations and their interactions with the polyelectrolyte chains in these amorphous materials. To address such issues, the present work develops a comprehensive solid state NMR strategy based on complementary high-resolution magic-angle spinning (MAS) NMR and various dipolar spectroscopic techniques. (6,7)Li and (23)Na chemical shifts measured on a series of PECs with general composition described by B((2x-1))PSS(x)PDADMA((1-x)) (B = Li or Na and 0.53 ≤x≤ 1) reveal composition-independent local cation environments. In contrast, (7)Li{(6)Li} spin echo double resonance (SEDOR) experiments measured on (6)Li enriched materials and (7)Li{(1)H} rotational echo double resonance (REDOR) experiments are consistent with an approximately random ion distribution. The same conclusion is suggested by (23)Na{(1)H} REDOR measurements on the analogous sodium containing system indicating a non-segregated PEC structure. In apparent contrast to this conclusion, (23)Na spin echo decay spectroscopy yields nearly constant dipolar second moments over a wide composition range. This can be explained by considering that the (23)Na spin echo decays are affected by both (23)Na-(23)Na homonuclear dipolar couplings and (23)Na-(1)H heteronuclear dipolar interactions in the presence of strong homonuclear (1)H-(1)H spin exchange. In protonated Na-PSS both contributions are of comparable magnitude. In the PECs the contribution from (23)Na-(23)Na interactions decreases, while that from (23)Na-(1)H dipolar couplings with the protons from the PDADMA chains increases with decreasing Na content, resulting in superimposed opposite dependences on the ion concentration. All results for Li and Na containing PECs point at a non-phase separated polymer network with uniform ionic sites of very similar environment. The cations can be viewed as randomly distributed and located close to the polyion sulfate groups.

Rotational echo double resonance without proton decoupling under fast spinning condition

We show that rotational echo double resonance (REDOR) experiments can be carried out without proton decoupling under the conditions of fast spinning and strong rf field. Numerical simulations on a five-spin systems show that no significant attenuation of the reference signal ( S 0) is observed at a spin rate of 25 kHz, provided that the rf power is larger than 100 kHz. This approach has been validated by 31P{ 13C} REDOR measurements on isotopically labeled glyphosate. The obtained van Vleck’s second moment is in favorable agreement with the value calculated based on the crystal structure.

Design and characterization of a calixarene inclusion compound for calibration of long-range carbon–fluorine distance measurements by solid-state NMR

An inexpensive, easily synthesized calixarene:fluorotoluene host:guest inclusion complex has been designed for optimization and calibration of solid-state NMR measurements of carbon–fluorine distances using Rotational Echo DOuble Resonance (REDOR). Complexation of the fluorotoluene with the calixarene host separates the molecules such that simple two-spin behavior is observed for one site with a 4.08Å carbon–fluorine distance. Fluorotoluene dynamics within the calixarene matrix cause motional averaging of the dipolar couplings, which makes it possible to easily optimize REDOR experiments and test their accuracy for relatively long distance measurements (>6.6Å). This provides a new tool for accurate REDOR measurements of long carbon–fluorine distances, which have important applications in the characterization of fluorine-containing drugs, proteins, and polymers.

Combined 17O NMR and 11B– 31P double resonance NMR studies of sodium borophosphate glasses

17O enriched sodium borophosphate glasses were prepared from isotopically enriched NaPO 3 and H 3BO 3. These glasses have been studied by 17O, 11B and 31P NMR including 17O and 11B multiple quantum magic angle sample spinning (MQMAS), 11B– 31P heteronuclear correlation (HETCOR) NMR and 11B{ 31P} rotational echo double resonance (REDOR). For comparison, the crystalline borophosphates BPO 4 and Na 5B 2P 3O 13 were included in the investigations. The latter compound shows three sharp 31P resonances at −0.2, −2 and −8 ppm and two BO 4 sites that can only be resolved by MQMAS. The 17O NMR spectra were recorded using both the static echo method at medium magnetic field (9.4 T) as well as MAS and MQMAS methods at high field (17.6 T). In total, five oxygen sites were identified in these borophosphate glasses: P–O–P, Na⋯O–P, P–O–B, B–O–B, Na⋯O–B. However, these five sites are not present simultaneously in any of the glasses. The 17O MQMAS spectra prove that P–O–B links play a major role in borophosphate glasses. These results are confirmed by the complementary 11B MAS spectra that show the presence of asymmetric and symmetric trigonal groups BO 3 a and BO 3 s and two tetrahedral BO 4 units. 11B{ 31P} REDOR NMR is used to give independent information to assign the 11B lines to structural units present in the glasses. These REDOR measurements reveal that B–O–P bonds are present for each borate unit, including the BO 3 groups. Particularly, a structural proposal for the two different BO 4 resonances is given in terms of a different number of bonded phosphate tetrahedra. The 31P MAS spectra are usually broad and not well resolved. It is shown by 11B– 31P HETCOR NMR that a possible structural assignment of a 31P signal at about −20 ppm to Q 2 units as in binary sodium phosphate glasses is wrong and that the phosphate tetrahedron belonging to this resonance must be connected to borate groups.

A solid-state NMR investigation of orexin-B

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.Key words: orexin-B, solid-state NMR, REDOR, chemical shift tensors.

19F/ 29Si distance determination and heteronuclear spin counting under fast magic-angle spinning in fluoride-containing octadecasil

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).

Determination of Molecular Geometry in Solid-State NMR: Rotational-Echo Double Resonance of Three-Spin Systems

The precise measurement of internuclear distances in solids by NMR has been widely explored using experiments that measure the dipolar coupling between labeled spin pairs. The rotational-echo double-resonance (REDOR) experiment is one of the most successful techniques for quantifying distances between heteronuclei. In the present work, REDOR is applied to the precise determination of the angle between internuclear vectors in triply labeled spin systems. The time domain REDOR signal for two heteronuclear coupled spin pairs sharing a common partner (an I2S spin system) is derived and analyzed with the aid of dipolar transforms. A two-step experimental approach for the structural analysis of I2S spin systems is then developed. Independent θ-REDOR and traditional REDOR measurements are used to obtain the heteronuclear dipolar coupling constants and the angle between them. Demonstrations are carried out on labeled polycrystalline samples of glycine and uracil. The REDOR experiment is also examined using altern...

Transmembrane interactions in the activation of the Neu receptor tyrosine kinase.

The Neu receptor tyrosine kinase is constitutively activated by a single amino acid change in the transmembrane domain of the receptor. The mutation of Val664 to glutamate or glutamine induces receptor dimerization and autophosphorylation of the receptor's intracellular kinase domain. The ability of this single mutation to activate the receptor is sequence-dependent, suggesting that specific helix-helix interactions stabilize the transmembrane dimer. We have determined the local secondary structure and interhelical contacts in the region of position 664 in peptide models of the activated receptor using solid-state rotational resonance and rotational echo double-resonance (REDOR) NMR methods. Intrahelical (13)C rotational resonance distance measurements were made between 1-(13)C-Thr662 and 2-(13)C-Gly665 on peptides corresponding to the wild-type Neu and activated Neu transmembrane sequences containing valine and glutamate at position 664, respectively. We observed similar internuclear distances (4.5 +/- 0.2 A) in both Neu and Neu*, indicating that the region near residue 664 is helical and is not influenced by mutation. Interhelical (15)N...(13)C REDOR measurements between Gln664 side chains on opposing helices were not consistent with hydrogen bonding between the side chain functional groups. However, interhelical rotational resonance measurements between 1-(13)C-Glu664 and 2-(13)C-Gly665 and between 1-(13)C-Gly665 and 2-(13)C-Gly665 demonstrated close contacts (4.3-4.5 A) consistent with the packing of Gly665 in the Neu* dimer interface. These measurements provide structural constraints for modeling the transmembrane dimer and define the rotational orientation of the transmembrane helices in the activated receptor.

Location of protons in anhydrous Keggin heteropolyacids H(3)PMo(12)O(40) and H(3)PW(12)O(40) by (1)H[(31)P]/(31)P[(1)H] REDOR NMR and DFT quantum chemical calculations.

HeteroPolyAcids (HPA's) are a class of solid acids that have broad applications in many fields of science and technology, including catalysis and chemical engineering. The proton locations within the thermally stable and commonly known Keggin unit, which is the primary structure building unit/block, has remained undetermined in anhydrous HPAs, despite numerous theoretical and experimental efforts. However, Rotational Echo DOuble Resonance (REDOR) NMR and Density Functional Theory (DFT) quantum chemical calculations offer a new opportunity to determine the exact locations of protons within the Keggin unit. The crucial experimental evidence is provided for the basic and very extensively studied acidic form of H(8-n)X(n+)M(12)O(40), X = Si, P and M = Mo, W, belonging to the Keggin structure. While showing that the acidic protons are located in the bridging oxygen positions (R(P-H) = 520 +/- 20 pm) in H(3)PMo(12)O(40) and in the terminal oxygen positions (R(P-H) = 570 +/- 20 pm) in H(3)PW(12)O(40), REDOR measurements also provide for the first time the structural basis to consistently rank the acid strength for the important class of Keggin solid catalysts.

Determination of Statherin N-Terminal Peptide Conformation on Hydroxyapatite Crystals

Proteins play an important role in inorganic crystal engineering during the development and growth of hard tissues such as bone and teeth. Although many of these proteins have been studied in the liquid state, there is little direct information describing molecular recognition at the protein−crystal interface. Here we have used 13C solid-state NMR (SSNMR) techniques to investigate the conformation of an N-terminal peptide of salivary statherin both free and adsorbed on hydroxyapatite (HAP) crystals. The torsion angle φ was determined at three positions along the backbone of the phosphorylated N-terminal 15 amino acid peptide fragment (DpSpSEEKFLRRIGRFG) by measuring distances between the backbone carbonyls carbons in the indicated adjacent amino acids using dipolar recoupling with a windowless sequence (DRAWS). Global secondary structure was determined by measuring the dipolar coupling between the 13C backbone carbonyl and the backbone 15N in the i → i + 4 residues (DpSpSEEKFLRRIGRFG) using rotational echo double resonance (REDOR). Peptides singly labeled at amino acids pS3, L8, and G12 were used for relaxation and line width measurements. The peptides adsorbed to the HAP surface have an average φ of −85° at the N-terminus (pSpS), −60° in the middle (FL) and −73° near the C-terminus (IG). The average φ angle measured at the pSpS position and the observed high conformational dispersion suggest a random coil conformation at this position. However, the FL position displays an average φ that indicates significant α-helical content, and the long time points in the DRAWS experiment fit best to a relatively narrow distribution of φ that falls within the protein data bank α-helical conformational space. REDOR measurements confirm the presence of helical content, where the distance across the LG hydrogen bond of the adsorbed peptide has been found to be 5.0 Å. The φ angle measured at the IG position falls at the upper end of the protein data bank α-helical distribution, with a best fit to a relatively broad φ distribution that is consistent with a distribution of α-helix and more extended backbone conformation. These results thus support a structural model where the N-terminus is disordered, potentially to maximize interactions between the HAP surface and the negatively charged side chains found in this region, the middle portion is largely α-helical, and the C-terminus has a more extended conformation (or a mixture of helix and extended conformations).

Probing hydrogen bonds in the antibody-bound HIV-1 gp120 V3 loop by solid state NMR REDOR measurements.

We describe solid state NMR measurements on frozen solutions of the complex of the 24-residue HIV-1 gp120 V3 loop peptide RP135 with the Fab fragment of the anti-gp120 antibody 0.5beta, using rotational echo double resonance (REDOR). In order to probe possible hydrogen bonding between arginine side chains and glycine backbone carbonyls in the region of the conserved Gly-Pro-Gly-Arg (GPGR) motif of the V3 loop, RP135 samples were prepared with 15N labels at the eta nitrogen positions of arginine side chains and 13C labels at glycine carbonyl positions and 13C-detected 13C-15N REDOR measurements were performed on peptide/antibody complexes of these labeled samples. Such hydrogen bonding was previously observed in a crystal structure of the V3 loop peptide/antibody complex RP142/59.1 [Ghiara et al. (1994) Science, 264, 82-85], but is shown by the REDOR measurements to be absent in the RP135/0.5beta complex. These results confirm the antibody-dependent conformational differences in the GPGR motif suggested by previously reported solid state NMR measurements of phi and psi backbone dihedral angles in the RP135/0.53 complex. In addition, we describe REDOR measurements on the helical synthetic peptide MB(i+4)EK in frozen solution that establish our ability to detect 13C-15N dipole-dipole couplings in the distance range appropriate to these hydrogen bonding studies. We also report the results of molecular modeling calculations on the central portion RP135, using a combination of the solid state NMR restraints of Weliky et al. [Nat. Struct. Biol., 6, 141-145, 1999] and the liquid state NMR restraints of Tugarinov et al. (Nat. Struct. Biol., 6, 331-335, 1999]. The dynamics calculations demonstrate the mutual compatibility of the two sets of experimental structural restraints and reduce ambiguities in the solid state NMR restraints that result from symmetry and signal-to-noise considerations.

Long-distance rotational echo double resonance measurements for the determination of secondary structure and conformational heterogeneity in peptides

The utility of rotational echo double resonance (REDOR) NMR spectroscopy for determining the conformations of linear peptides has been examined critically using a series of crystalline and amorphous samples. The focus of the present work was the evaluation of long-distance (>5 Å) interactions using 13 C– 15 N dephasing. Detailed studies of specifically labeled melanostatin and synthetic analogs of the α-factor yeast mating hormone show that nitrogen-dephased, carbon-observe REDOR measurements are reliable for distances up to 6.0 Å, and that dipolar interactions can be detected for distances up to 7 Å. By contrast, nitrogen-observe REDOR gives reliable results only for distances shorter than 5.0 Å. To measure distances accurately, REDOR data must be corrected for the effects of natural-abundance spins. These corrections are particularly important for measuring long distances, which are of the greatest value for determining peptide secondary structure. We have developed a spherical shell model for calculating the effect of these background spins. The REDOR studies also indicate that in a lyophilized powder, the tridecapeptide α-factor mating pheromone from Saccharomyces cerevisiae (WHWLQLKPGQPMY) probably exists as a distribution of different turn structures around the KPGQ region. This finding revises previous solid-state NMR studies on this peptide, which concluded α-factor assumes a distorted type-I β-turn in the Pro-Gly central region of the molecule [J.R. Garbow, M. Breslav, O. Antohi, F. Naider, Biochemistry, 33 (1994) 10094].