(13)C chemical shift and (13)C-(14)N dipolar coupling tensors determined by (13)C rotary resonance solid-state NMR

This work explores the utility of simple rotary resonance experiments for the determination of the magnitude and orientation of (13)C chemical shift tensors relative to one or more (13)C--(14)N internuclear axes from (13)C magic-angle-spinning NMR experiments. The experiment relies on simultaneous recoupling of the anisotropic (13)C chemical shift and (13)C--(14)N dipole--dipole coupling interactions using 2D rotary resonance NMR with RF irradiation on the (13)C spins only. The method is demonstrated by experiments and numerical simulations for the (13)C(alpha) spins in powder samples of L-alanine and glycine with (13)C in natural abundance. To investigate the potential of the experiment for determination of relative/absolute tensor orientations and backbone dihedral angles in peptides, the influence from long-range dipolar coupling to sequential (14)N spins in a peptide chain ((14)N(i)--(13)C(alpha)(i)--(14)N(i+1) and (14)N(i+1)--(13)C'(i)--(14)N(i) three-spin systems) as well as residual quadrupolar-dipolar coupling cross-terms is analyzed numerically.     

Three-dimensional 13C shift/1H-15N coupling/15N shift solid-state NMR correlation spectroscopy.

Triple-resonance experiments capable of correlating directly bonded and proximate carbon and nitrogen backbone sites of uniformly 13C- and 15N-labeled peptides in stationary oriented samples are described. The pulse sequences integrate cross-polarization from 1H to 13C and from 13C to 15N with flip-flop (phase and frequency switched) Lee-Goldburg irradiation for both 13C homonuclear decoupling and 1H-15N spin exchange at the magic angle. Because heteronuclear decoupling is applied throughout, the three-dimensional pulse sequence yields 13C shift/1H-15N coupling/15N shift correlation spectra with single-line resonances in all three frequency dimensions. Not only do the three-dimensional spectra correlate 13C and 15N resonances, they are well resolved due to the three independent frequency dimensions, and they can provide up to four orientationally dependent frequencies as input for structure determination. These experiments have the potential to make sequential backbone resonance assignments in uniformly 13C- and 15N-labeled proteins.     

13C-(27)Al TRAPDOR and REDOR experiments for the detection of (13)C-(27)Al dipolar interactions in solids.

We report (13)C-(27)Al double resonance experiments (REDOR and TRAPDOR) on several aluminum organic compounds with the aim of detecting (13)C-(27)Al dipolar couplings and distances in solids. The (13)C and (27)Al pulses are applied to the same probe channel because their resonance frequencies are in close proximity. The different possibilities of controlling the efficiency of the TRAPDOR approach (by varying the (27)Al RF amplitude and the MAS frequency) are investigated. The results indicate that TRAPDOR is superior to REDOR in resolving differences in (13)C-(27)Al distances when choosing the proper experimental conditions. Where known, the crystal structure data are in qualitative agreement with the distance information extracted from our experiments. The experiment should be very valuable in different fields of solid state chemistry, where the interaction of organic and inorganic sample fractions is of fundamental importance.     

(13)C-(13)C distance measurements in U-(13)C, (15)N-labeled peptides using rotational resonance width experiment with a homogeneously broadened matching condition

In this publication, we introduce a version of the rotational resonance width experiment with a homogeneously broadened matching condition. The increase in the bandwidth is achieved by the reduction of the proton decoupling power during mixing, which results in the reduction of zero-quantum relaxation, and broadens the rotational resonance condition. We show that one can achieve recoupling of the carbonyl-aliphatic side chain dipolar interactions band selectively, while avoiding the recoupling of strongly interacting C'-Calpha and C'-Cbeta spin pairs. The attenuation of the multi-spin effects in the presence of short zero-quantum relaxation enables a two-spin approximation to be employed for the analysis of the experimental data. The systematic error introduced by this approximation is estimated by comparing the results with a three-spin simulation. The experiment is demonstrated in [U-(13)C,(15)N]N-acetyl-L-Val-L-Leu dipeptide, where 11 distances, ranging from 2.5 to 6 A, were measured.     

Combination of (13)C-(13)C COSY and DARR (COCODARR) in solid-state NMR

In this work, we describe a new 2D (13)C-(13)C correlation experiment in solids, in which (13)C-(13)C J-correlation (COSY) and dipolar correlation (DARR) are recorded at the same time. The sequence is similar to COCONOSY in the liquid-state NMR, in which (1)H-(1)H COSY and NOESY spectra are obtained in a single experiment. The combined COSY and DARR experiment facilitates assignment of (13)C signals in solids.     

Sensitivity-enhanced E.COSY-type HSQC experiments for accurate measurements of one-bond 15N-1H(N) and 15N-13C' and two-bond 13C'-1H(N) residual dipolar couplings in proteins

Novel E.COSY-type HSQC experiments are presented for the accurate measurement of one-bond 15N-1H(N) and 15N-13C(') and two-bond 13C(')-1H(N) residual dipolar couplings in proteins. Compared with existing experiments, the (delta,J)-E.COSY experiments described here are composed of fewer pulses and the resulting spectra exhibit 1.4 times the sensitivity of coupled HSQC spectra. Since residual dipolar couplings play increasingly important roles in structural NMR, the proposed methods should find wide spread application for structure determination of proteins and other biological macromolecules.     

The role of (15)N CSA and CSA/dipole cross-correlation in (15)N relaxation in solid proteins

The influence of the (15)N CSA on (15)N longitudinal relaxation is investigated for an amide group in solid proteins in powder form under MAS. This contribution is determined to be typically 20-33% of the overall longitudinal relaxation rate, at 11.74 and 16.45 T, respectively. The improved treatment is used to analyze the internal dynamics in the protein Crh, in the frame of a motional model of diffusion in a cone, using the explicit average sum approach. Significant variations with respect to the determined dynamics parameters are observed when properly accounting for the contribution of (15)N CSA fluctuations. In general, the fit of experimental data including CSA led to the determination of diffusion times (tau(w)) which are longer than when considering only an (15)N-(1)H dipolar relaxation mechanism. CSA-Dipole cross-correlation is shown to play little or no role in protonated solids, in direct contrast to the liquid state case.     

Dual 13C, 15N labelling of terrestrial slugs (Deroceras reticulatum)

A simple, rapid and cost-effective laboratory method is described for labelling terrestrial slugs simultaneously with 13C and 15N. Slugs (Deroceras reticulatum) were provided with a mixture of [U-13C6]glucose, 15N-enriched lettuce powder, and wheat bran. Assimilation efficiencies for 13C (24.2%) and 15N (27.4%) were not affected by feeding regimes offering ad libitum or restricted access to unlabelled food during the labelling period. Body tissue was significantly more highly enriched in 13C but significantly less in 15N than cutaneous mucus after 15 days.     

Efficient measurement of (3)J(N,Cgamma) and (3)J(C',Cgamma) coupling constants of aromatic residues in (13)C, (15)N-labeled proteins

An NMR pulse sequence is proposed for the simultaneous determination of side chain chi1 torsion-angle related (3)J(N,Cgamma) and (3)J(C', Cgamma) couplings in aromatic amino acid spin systems. The method is of the quantitative J correlation type and takes advantage of attenuated (15)N and (1)H transverse relaxation by means of the TROSY principle. Unlike previously developed schemes for the measurement of either of the two coupling types, spectra contain internal reference peaks that are usually recorded in separate experiments. Therefore, the desired information is extracted from a single rather than four data sets. The new method is demonstrated with uniformly (13)C/(15)N labeled Desulfovibrio vulgaris flavodoxin, which contains 14 aromatic out of 147 total amino acid residues.     

13C-14N REAPDOR and 13C-2D theta-REDOR NMR on a blend of tri-p-tolylamine and bisphenol-A-polycarbonate

Solid-state NMR experiments are performed on amorphous blends of tri-p-tolylamine (TTA) and polycarbonate. Amines that are similar to TTA are blended with polycarbonates and used as photoconducting components in xerography. Because of its simple molecular structure, TTA is a charge-transporting molecule that is used as a model to understand charge transport in photoconducting materials. Charge transfer between amine molecules must depend on the separation and relative orientation between amine molecules. However, little information is known about the structural relationship between the amine molecules and the polymer host, presumably because of the difficulties associated with characterizing amorphous materials. We report the results of 13C-14N rotational-echo, adiabatic-passage, double-resonance (REAPDOR) and 13C-2D theta-rotational-echo, double-resonance (theta-REDOR) magic-angle spinning NMR experiments on a TTA/polycarbonate blend. The experiments provide information about the organization of TTA relative to polycarbonate.     

In vivo detection of (15)N-coupled protons in rat brain by ISIS localization and multiple-quantum editing.

Three-dimensional image-selected in vivo spectroscopy (ISIS) was combined with phase-cycled (1)H-(15)N heteronuclear multiple-quantum coherence (HMQC) transfer NMR for localized selective observation of protons J-coupled to (15)N in phantoms and in vivo. The ISIS-HMQC sequence, supplemented by jump-return water suppression, permitted localized selective observation of 2-5 micromol of [(15)N(indole)]tryptophan, a precursor of the neurotransmitter serotonin, through the (15)N-coupled proton in 20-40 min of acquisition in vitro at 4.7 T. In vivo, the amide proton of [5-(15)N]glutamine was selectively observed in the brain of spontaneously breathing (15)NH(4)(+)-infused rats, using a volume probe with homogeneous (1)H and (15)N fields. Signal recovery after three-dimensional localization was 72-82% in phantoms and 59 +/- 4% in vivo. The result demonstrates that localized selective observation of (15)N-coupled protons, with complete cancellation of all other protons except water, can be achieved in spontaneously breathing animals by the ISIS-HMQC sequence. This sequence performs both volume selection and heteronuclear editing through an addition/subtraction scheme and predicts the highest intrinsic sensitivity for detection of (15)N-coupled protons in the selected volume. The advantages and limitations of this method for in vivo application are compared to those of other localized editing techniques currently in use for non-exchanging protons.     

"BEST" homonuclear adiabatic decoupling for 13C- and 15N-double-labeled proteins.

The cyclic irradiation sidebands appearing in homonuclear adiabatic decoupling are calculated in detail, which reveals the origin of the antisymmetric sidebands. The sidebands can be inverted by inserting an initial decoupling with a different period, but the same f1rms as the main decoupling that is required for Bloch-Siegert shift compensation. The sidebands can be eliminated in a broad decoupling range by adding spectra of opposite sidebands. Based on this scheme, an offset-independent double-adiabatic decoupling, named Bloch-Siegert Shift Eliminated and Cyclic Sideband Trimmed Double-Adiabatic Decoupling, or "BEST" decoupling for short, is constructed, which not only compensates the Bloch-Siegert shift as shown earlier by Zhang and Gorenstein (1998) but also eliminates residual sidebands effectively.     

Precise value for the three-bond 13C-1H coupling constant in acetone

A method based on the utilization of continuous space-filling curves is applied to a two-dimensional elastic scattering problem involving a central Yukawa potential. The Lippmann-Schwinger equation is brought into the form of a one-dimensional inhomogeneous integral equation and solved numerically. The results agree to within about 5 per cent with those obtained by a recent direct numerical solution based on two-dimensional quadratures.     

Short-term changes in delta(13)C and delta(15)N signatures of water discharged from grazed grasslands

The composition of dissolved organic matter (DOM) in a soil is the product of a variety of soil processes. Changes in the composition of DOM in water discharged from soil should, therefore, give an important insight into modifications in these soil processes. We hypothesise that these processes in soils, under different grassland management regimes, would be affected to different extents by the short-term disturbance of a storm event and that evidence of this could be detected in delta(13)C and delta(15)N signatures in drainage and surface runoff waters. During a storm event we collected discharge waters from 1 ha grassland lysimeters, with or without artificial drainage, which received contrasting fertiliser inputs, and delta(13)C and delta(15)N signatures were determined. Changes in (13)C enrichment during the storm event were clearly identifiable, as were differences between plots for (13)C and (15)N, illustrating that this technique has potential to be a useful tool for identifying and investigating short- and long-term changes in soil organic matter dynamics. Copyright 1999 John Wiley & Sons, Ltd.     

Simple and accurate determination of global tau(R) in proteins using (13)C or (15)N relaxation data

In the study of protein dynamics by (13)C or (15)N relaxation measurements different models from the Lipari-Szabo formalism are used in order to determine the motion parameters. The global rotational correlation time tau(R) of the molecule must be estimated prior to the analysis. In this Communication, the authors propose a new approach in determining an accurate value for tau(R) in order to realize the best fit of R(2) for the whole sequence of the protein, regardless of the different type of motions atoms may experience. The method first determines the highly structured regions of the sequence. For each corresponding site, the Lipari-Szabo parameters are calculated for R(1) and NOE, using an arbitrary value for tau(R). The chi(2) for R(2), summed over the selected sites, shows a clear minimum, as a function of tau(R). This minimum is used to better estimate a proper value for tau(R).     

Triple resonance experiments for aligned sample solid-state NMR of (13)C and (15)N labeled proteins

Initial steps in the development of a suite of triple-resonance (1)H/(13)C/(15)N solid-state NMR experiments applicable to aligned samples of (13)C and (15)N labeled proteins are described. The experiments take advantage of the opportunities for (13)C detection without the need for homonuclear (13)C/(13)C decoupling presented by samples with two different patterns of isotopic labeling. In one type of sample, the proteins are approximately 20% randomly labeled with (13)C in all backbone and side chain carbon sites and approximately 100% uniformly (15)N labeled in all nitrogen sites; in the second type of sample, the peptides and proteins are (13)C labeled at only the alpha-carbon and (15)N labeled at the amide nitrogen of a few residues. The requirement for homonuclear (13)C/(13)C decoupling while detecting (13)C signals is avoided in the first case because of the low probability of any two (13)C nuclei being bonded to each other; in the second case, the labeled (13)C(alpha) sites are separated by at least three bonds in the polypeptide chain. The experiments enable the measurement of the (13)C chemical shift and (1)H-(13)C and (15)N-(13)C heteronuclear dipolar coupling frequencies associated with the (13)C(alpha) and (13)C' backbone sites, which provide orientation constraints complementary to those derived from the (15)N labeled amide backbone sites. (13)C/(13)C spin-exchange experiments identify proximate carbon sites. The ability to measure (13)C-(15)N dipolar coupling frequencies and correlate (13)C and (15)N resonances provides a mechanism for making backbone resonance assignments. Three-dimensional combinations of these experiments ensure that the resolution, assignment, and measurement of orientationally dependent frequencies can be extended to larger proteins. Moreover, measurements of the (13)C chemical shift and (1)H-(13)C heteronuclear dipolar coupling frequencies for nearly all side chain sites enable the complete three-dimensional structures of proteins to be determined with this approach.     

Novel peak assignments of in vivo (13)C MRS in human brain at 1.5 T

(13)C MRS studies at natural abundance and after intravenous 1-(13)C glucose infusion were performed on a 1.5-T clinical scanner in four subjects. Localization to the occipital cortex was achieved by a surface coil. In natural abundance spectra glucose C(3beta,5beta), myo-inositol, glutamate C(1,2,5), glutamine C(1,2,5), N-acetyl-aspartate C(1-4,C=O), creatine CH(2), CH(3), and C(C=N), taurine C(2,3), bicarbonate HCO(-)(3) were identified. After glucose infusion (13)C enrichment of glucose C(1alpha,1beta), glutamate C(1-4), glutamine C(1-4), aspartate C(2,3), N-acetyl-aspartate C(2,3), lactate C(3), alanine C(3), and HCO(-)(3) were observed. The observation of (13)C enrichment of resonances resonating at >150 ppm is an extension of previously published studies and will provide a more precise determination of metabolic rates and substrate decarboxylation in human brain.     

Assignments and signs of 13C-31P coupling constants from off-resonance proton decoupled 13C spectra

The grand canonical ensemble is used to derive the first quantum correction (FQC) to the radial distribution function (RDF) for a fluid whose molecules interact with a potential which is an analytic function of distance. The low-order cluster integrals appearing in the density expansion of the RDF are evaluated on the Lennard-Jones (12-6) potential. The FQC to the isothermal compressibility and to the equation of state is discussed.