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.     

A New Two-Dimensional Pulse Sequence for T2* Measurements of Protons in C Isotopomers

A new two-dimensional pulse sequence for T₂* measurement of protons directly coupled to ¹³C spins is proposed. The sequence measures the tranverse relaxation time of heteronuclear proton single-quantum coherence under conditions of free precession and is therefore well suited to evaluate relaxation losses of proton magnetization during preparation delays of heteronuclear pulse experiments in analytical NMR. The relevant part of the pulse sequence can be inserted as a “building block” into any direct or inverse detecting H,C correlation pulse sequence if proton spin–spin relaxation is to be investigated. In this contribution, the building block is inserted into a HETCOR as well as into a HMQC pulse sequence. Experimental results for the HETCOR-based sequence are given.     

Paramagnetic Inversion of the Sign of the Interference Contribution to the Transverse Relaxation of the Imido Protons of the Coordinated Imidazoles in the UniformlyN-Labeled Cytochromec3

In the spectrum of uniformly¹⁵N-labeled cytochromec₃, the relative linewidths of the doublet peaks of the¹⁵N-coupled imido proton of the coordinated imidazole group were reversed on oxidation. This inversion was explained by the interference relaxation process between the electron–proton dipolar and¹⁵N–¹H dipolar interactions. The inversion can be used to assign the imido protons of the coordinated imidazole groups in heme proteins.     

Signal Enhancement Using 45° Water Flipback for 3D N-Edited ROESY and NOESY HMQC and HSQC

A novel implementation of the water flipback technique employing a 45° flip-angle water-selective pulse is presented. The use of this water flipback technique is shown to significantly enhance signal in 3D ¹⁵N-edited ROESY in a 20 kDa complex of the vnd/NK-2 homeodomain bound to DNA. The enhancement is seen relative to the same experiment using weak water presaturation during the recovery delay. This enhancement is observed for the signals from both labile and nonlabile protons. ROESY and NOESY pulse sequences with 45° water flipback are presented using both HMQC and HSQC for the ¹⁵N dimension. The 45° flipback pulse is followed by a gradient, a water selective 180° pulse, and another gradient to remove quadrature images and crosspeak phase distortion near the water frequency. Radiation damping of the water magnetization during the t₁ and t₂ evolution periods is suppressed using gradients. Water resonance planes from NOESY–HMQC and NOESY–HSQC spectra show that the HMQC version of the pulse sequences can provide stronger signal for very fast exchanging protons. The HSQC versions of the ROESY and NOESY pulse sequences are designed for the quantitative determination of protein–water crossrelaxation rates, with no water-selective pulses during the mixing time and with phase cycling and other measures for reducing axial artifacts in the water signal.     

Heteronuclear Multiple-Quantum Coherence NMR Spectroscopy of Im-cyt C

Some ¹³C chemical shifts of the CH_n groups in the aliphatic side chains of Im-cyt c have been determined for the first time based on the H chemical shifts of their attached protons with the aid of heteronuclear multiple-quantum coherence (HMQC) spectroscopy. Comparison of chemical shifts of these specifically assigned ¹³C and H resonances from Im-cyt c with those from cyt c indicates that ¹³C-NMR spectra may provide an opportunity to probe the electronic structure and conformational changes induced by axial ligand substitution.     

More line narrowing in TROSY by decoupling of long-range couplings: shift correlation and JNC′ coupling constant measurements

Since the introduction of RDCs in high-resolution NMR studies of macromolecules, there is a growing interest in the development of accurate, and sensitive methods for determining coupling constants. Most methods for extracting these couplings are based on the measurement of the splitting between multiplet components in J-coupled spectra. However, these methods are often unreliable since undesired multiple-bond couplings can considerably broaden the multiplet components and consequently make accurate determination of their position difficult. To demonstrate one approach to this problem, G-BIRD^(r) decoupled TROSY sequences are proposed for the measurement of ¹J_NH and ¹J_NC′ coupling constants. Resolved or unresolved splittings due to remote protons are removed by a G-BIRD^(r) module employed during t₁ and as a result, spectra with narrow, well-resolved peaks are obtained from which heteronuclear one-bond couplings can be accurately measured. Moreover, introduction of a spin-state-selective α/β-filter in the TROSY sequence allows the separation of the ¹J_NC′ doublet components into two subspectra which contain the same number of peaks as the regular TROSY spectrum. The ¹J_NC′ couplings are obtained from the displacement between the corresponding peaks in the subspectra.     

Effects of L-spin longitudinal quadrupolar relaxation in heteronuclear recoupling and S-spin magic-angle spinning NMR

In experiments on S–L heteronuclear spin systems with evolution of the S-spin magnetization under the influence of a quadrupolar nucleus (L-spin), effects of longitudinal quadrupolar (T_1Q) relaxation of the L-spin coherence on the sub-millisecond time scale have been documented and explored, and methods for minimizing their effect have been demonstrated. The longitudinal relaxation results in heteronuclear dephasing even in the reference signal S₀ of S{L} REDOR, REAPDOR, RIDER, or SPIDER experiments, due to T_1Q-relaxation of the transiently generated S_yL_z coherence, reducing or even eliminating the observable dephasing ΔS. Pulse sequences for measuring an improved reference signal S₀₀ with minimal heteronuclear recoupling but the same number of pulses as for S₀ and S have been demonstrated. From the observed intensity ΔS₀ = S₀₀ − S₀ and the SPIDER signal ΔS/S₀, T_1Q can be estimated. Accelerated decays analogous to the dipolar S₀ curves will occur in T₂ measurements for J-coupled S–L spin pairs. Even in the absence of recoupling pulses, fast T_1Q relaxation of the unobserved nucleus shortens the transverse relaxation time T_2S,MAS of the observed nucleus, in particular at low spinning frequencies, due to unavoidable heteronuclear dipolar evolution during a rotation period. The observed spinning-frequency dependence of T_2S,MAS matches the theoretical prediction and may be used to estimate T_1Q. The effects are demonstrated on several ¹³C{¹⁴N} spin systems, including an arginine derivative, the natural N-acetylated polysaccharide chitin, and a model peptide, (POG)₁₀.     

Proton Dynamics in Letovicite, (NH4)3H(SO4)2: A H and N NMR Spectroscopic Study

The improper ferroelastic phase letovicite (NH₄)₃H(SO₄)₂ has been studied by ¹H MAS NMR as well as by static ¹⁴N NMR experiments in the temperature range of 296–425 K. The ¹H MAS NMR resonance from ammonium protons can be well distinguished from that of acidic protons. A third resonance appears just below the phase transition temperature which is due to the acidic protons in the paraelastic phase. The lowering of the second moment M₂ for the ammonium protons takes place in the same temperature range as the formation of domain boundaries, while the signals of the acidic protons suffer a line narrowing in the area of T_c. The static ¹⁴N NMR spectra confirm the temperature of the motional changes of the ammonium tetrahedra. Two-dimensional ¹H NOESY spectra indicate a chemical exchange between ammonium protons and the acidic protons of the paraphase.     

Solid-state NMR investigations of Si-29 and N-15 enriched silicon nitride

We compare ²⁹Si magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectra from the two modifications of silicon nitride, α-Si₃N₄ and β-Si₃N₄, with that of a fully (²⁹Si, ¹⁵N)-enriched sample ²⁹Si₃¹⁵N₄, as well as ¹⁵N NMR spectra of Si₃¹⁵N₄ (having ²⁹Si at natural abundance) and ²⁹Si₃¹⁵N₄. We show that the ¹⁵N NMR peak-widths from the latter are dominated by J(²⁹Si–¹⁵N) through-bond interactions, leading to significantly broader NMR signals compared to those of Si₃¹⁵N₄. By fitting calculated ²⁹Si NMR spectra to experimental ones, we obtained an estimated coupling constant J(²⁹Si–¹⁵N) of 20 Hz. We provide ²⁹Si spin-lattice (T₁) relaxation data for the ²⁹Si₃¹⁵N₄ sample and chemical shift anisotropy results for the ²⁹Si site of β-Si₃N₄. Various factors potentially contributing to the ²⁹Si and ¹⁵N NMR peak-widths of the various silicon nitride specimens are discussed. We also provide powder X-ray diffraction (XRD) and mass spectrometry data of the samples.     

Optimization of Three-Dimensional TROSY-Type HCCH NMR Correlation of Aromatic H–C Groups in Proteins

Improved methods for three-dimensional TROSY-Type HCCH correlation involving protons of negligible CSA are presented. The TROSY approach differs from the conventional approach of heteronuclear decoupling in evolution and detection periods by not mixing fast and slowly relaxing coherences and usually suppressing the former. Pervushin et al. (J. Am. Chem. Soc. 120, 6394–6400 (1998)) have proposed a 3D TROSY-type HCCH experiment where the TROSY approach is applied only in one of the ¹³C dimensions. A new pulse sequence applying the TROSY approach in both indirect dimensions is advantageous when the TROSY effect of the carbons is large or when a relatively high resolution is required. For lower resolutions or moderate TROSY effects we show that it is possible to combine the best of both worlds, namely to suppress heteronuclear couplings without mixing fast and slowly relaxing coherences while at the same time superimpose the two components and thus have both contribute to the detected signal. That is possible using the novel technique of Spin-State-Selective Time-Proportional Phase Incrementation (S³ TPPI). The new 3D S³ TPPI TROSY HCCH method is demonstrated on a ¹³C,¹⁵N-labeled protein sample, RAP 18–112 (N-terminal domain of α₂-macroglobulin receptor associated protein), at 750 MHz and average sensitivity enhancements of 10% are obtained for the cross peaks in comparison to methods based on conventional decoupling on one of the carbons or on TROSY on both carbons.     

Determination of Internucleotide JHN Couplings by the Modified 2D JNN-Correlated [N, H] TROSY

Recent developments in the direct observation of J couplings across hydrogen bonds in proteins and nucleic acids provide additional information for structure and function studies of these molecules by NMR spectroscopy. A J_NN-correlated [¹⁵N, ¹H] TROSY experiment proposed by Pervushin et al. (Proc. Natl. Acad. Sci. USA 95, 14147–14151, 1998) can be applied to measure ^hJ_HN in smaller nucleic acids in an E.COSY manner. However, it cannot be effectively applied to large nucleic acids, such as tRNA^Trp, since one of the peaks corresponding to a fast relaxing component will be too weak to be observed in the spectra of large molecules. In this Communication, we proposed a modified J_NN-correlated [¹⁵N, ¹H] TROSY experiment which enables direct measurement of ^hJ_HN in large nucleic acids.     

C, N CPMAS NMR and GIAO DFT calculations of stereoisomeric oxindole alkaloids from Cat's Claw (Uncaria tomentosa)

Oxindole alkaloids, isolated from the bark of Uncaria tomentosa [Willd. ex Schult.] Rubiaceae, are considered to be responsible for the biological activity of this herb. Five pentacyclic and two tetracyclic alkaloids were studied by solid-state NMR and theoretical GIAO DFT methods. The ¹³C and ¹⁵N CPMAS NMR spectra were recorded for mitraphylline, isomitraphylline, pteropodine (uncarine C), isopteropodine (uncarine E), speciophylline (uncarine D), rhynchophylline and isorhynchophylline. Theoretical GIAO DFT calculations of shielding constants provide arguments for identification of asymmetric centers and proper assignment of NMR spectra. These alkaloids are 7R/7S and 20R/20S stereoisomeric pairs. Based on the ¹³C CP MAS chemical shifts the 7S alkaloids (δ C3 70–71 ppm) can be easily and conveniently distinguished from 7R (δC3 74.5–74.9 ppm), also 20R (δC20 41.3–41.7 ppm) from the 20S (δC20 36.3–38.3 ppm). The epiallo-type isomer (3R, 20S) of speciophylline is characterized by a larger ¹⁵N MAS chemical shift of N4 (64.6 ppm) than the allo-type (3S, 20S) of isopteropodine (δN4 53.3 ppm). ¹⁵N MAS chemical shifts of N1–H in pentacyclic alkaloids are within 131.9–140.4 ppm.     

Recoupled Polarization-Transfer Methods for Solid-State H–C Heteronuclear Correlation in the Limit of Fast MAS

An in-depth account of the effects of homonuclear couplings and multiple heteronuclear couplings is given for a recently published technique for ¹H–¹³C dipolar correlation in solids under very fast MAS, where the heteronuclear dipolar coupling is recoupled by means of REDOR π-pulse trains. The method bears similarities to well-known solution-state NMR techniques, which form the framework of a heteronuclear multiple-quantum experiment. The so-called recoupled polarization-transfer (REPT) technique is versatile in that rotor-synchronized ¹H–¹³C shift correlation spectra can be recorded. In addition, weak heteronuclear dipolar coupling constants can be extracted by means of spinning sideband analysis in the indirect dimension of the experiment. These sidebands are generated by rotor encoding of the reconversion Hamiltonian. We present generalized variants of the initially described heteronuclear multiple-quantum correlation (HMQC) experiment, which are better suited for certain applications. Using these techniques, measurements on model compounds with ¹³C in natural abundance, as well as simulations, confirm the very weak effect of ¹H–¹H homonuclear couplings on the spectra recorded with spinning frequencies of 25–30 kHz. The effect of remote heteronuclear couplings on the spinning-sideband patterns of CH_n groups is discussed, and ¹³C spectral editing of rigid organic solids is shown to be practicable with these techniques.     

One- and two-dimensional C–H/N–H dipolar correlation experiments under fast magic-angle spinning for determining the peptide dihedral angle φ

A recently proposed ¹³C–¹H recoupling sequence operative under fast magic-angle spinning (MAS) [K. Takegoshi, T. Terao, Solid State Nucl. Magn. Reson. 13 (1999) 203–212.] is applied to observe ¹³C–¹H and ¹⁵N–¹H dipolar powder patterns in the ¹H–¹⁵N–¹³C–¹H system of a peptide bond. Both patterns are correlated by ¹⁵N-to-¹³C cross polarization to observe one- or two-dimensional (1D or 2D) correlation spectra, which can be simulated by using a simple analytical expression to determine the H–N–C–H dihedral angle. The 1D and 2D experiments were applied to N-acetyl[1,2-¹³C,¹⁵N] -valine, and the peptide φ angle was determined with high precision by the 2D experiment to be ±155.0°±1.2°. The positive one is in good agreement with the X-ray value of 154°±5°. The 1D experiment provided the value of φ=±156.0°±0.8°.     

Enhancement of upconversion luminescence in Tm/Er/Yb-codoped glass ceramic containing LiYF4 nanocrystals

A transparent Er³⁺–Tm³⁺–Yb³⁺ tri-doped oxyfluoride glass ceramics containing LiYF₄ nanocrystals were prepared. Under 980 nm laser diode (LD) pumping, intensive red, green and blue upconversion (UC) was obtained. The blue, green, and red UC radiations correspond to the transitions ¹G₄→³H₆ of Tm³⁺, ²H_11/2/⁴S_3/2→⁴I_15/2, and ⁴F_9/2→⁴I_15/2 of Er³⁺ ions, respectively. This is similar to that in Tm³⁺–Yb³⁺ and/or Er³⁺–Yb³⁺ co-doped glass ceramics. However, the blue UC radiations of the Er³⁺–Yb³⁺ co-doped glass ceramics is two-photon process due to cooperative energy transfer. The UC mechanisms were proposed based on spectral, kinetic, and pump power dependence analyses.     

Molecular dynamics and information on possible sites of interaction of intramyocellular metabolites in vivo from resolved dipolar couplings in localized H NMR spectra

Proton NMR resonances of the endogenous metabolites creatine and phosphocreatine ((P)Cr), taurine (Tau), and carnosine (Cs, β-alanyl-l-histidine) were studied with regard to residual dipolar couplings and molecular mobility. We present an analysis of the direct ¹H–¹H interaction that provides information on motional reorientation of subgroups in these molecules in vivo. For this purpose, localized ¹H NMR experiments were performed on m. gastrocnemius of healthy volunteers using a 1.5-T clinical whole-body MR scanner. We evaluated the observable dipolar coupling strength SD₀ (S = order parameter) of the (P)Cr-methyl triplet and the Tau-methylene doublet by means of the apparent line splitting. These were compared to the dipolar coupling strength of the (P)Cr-methylene doublet. In contrast to the aliphatic protons of (P)Cr and Tau, the aromatic H2 (δ = 8 ppm) and H4 (δ = 7 ppm) protons of the imidazole ring of Cs exhibit second-order spectra at 1.5 T. This effect is the consequence of incomplete transition from Zeeman to Paschen-Back regime and allows a determination of SD₀ from H2 and H4 of Cs as an alternative to evaluating the multiplet splitting which can be measured directly in high-resolution ¹H NMR spectra. Experimental data showed striking differences in the mobility of the metabolites when the dipolar coupling constant D₀ (calculated with the internuclear distance known from molecular geometry in the case of complete absence of molecular dynamics and motion) is used for comparison. The aliphatic signals involve very small order parameters S ≈ (1.4 − 3) × 10⁻⁴ indicating rapid reorientation of the corresponding subgroups in these metabolites. In contrast, analysis of the Cs resonances yielded S ≈ (113 − 137) × 10⁻⁴. Thus, the immobilization of the Cs imidazole ring owing to an anisotropic cellular substructure in human m. gastrocnemius is much more effective than for (P)Cr and Tau subgroups. Furthermore, ¹H NMR experiments on aqueous model solutions of histidine and N-acetyl-l-aspartate (NAA) enabled the assignment of an additional signal component at δ = 8 ppm of Cs in vivo to the amide group at the peptide bond. The visibility of this proton could result from hydrogen bonding which would agree with the anticipated stronger motional restriction of Cs. Referring to the observation that all dipolar-coupled multiplets resolved in localized in vivo ¹H NMR spectra of human m. gastrocnemius collapse simultaneously when the fibre structure is tilted towards the magic angle (θ ≈ 55°), a common model for molecular confinement in muscle tissue is proposed on the basis of an interaction of the studied metabolites with myocellular membrane phospholipids.     

The 2D {P} Spin-Echo-Difference Constant-Time [C, H]-HMQC Experiment for Simultaneous Determination of JH3′P and JC4′P in C-Labeled Nucleic Acids and Their Protein Complexes

A two-dimensional {³¹P} spin-echo-difference constant-time [¹³C, ¹H]-HMQC experiment (2D {³¹P}-sedct-[¹³C, ¹H]-HMQC) is introduced for measurements of ³J_C4′P and ³J_H3′P scalar couplings in large ¹³C-labeled nucleic acids and in DNA–protein complexes. This experiment makes use of the fact that ¹H–¹³C multiple-quantum coherences in macromolecules relax more slowly than the corresponding ¹³C single-quantum coherences. ³J_C4′P and ³J_H3′P are related via Karplus-type functions with the phosphodiester torsion angles β and ε, respectively, and their experimental assessment therefore contributes to further improved quality of NMR solution structures. Data are presented for a uniformly ¹³C, ¹⁵N-labeled 14-base-pair DNA duplex, both free in solution and in a 17-kDa protein–DNA complex.     

An α/β-HSQC-α/β Experiment for Spin-State Selective Editing of IS Cross Peaks

A generalized version of the TROSY experiment allows the spin-state selective editing of the four multiplet components of¹⁵N–¹H cross peaks of amide groups in proteins into four different subspectra, with no penalty in sensitivity. An improvement by in sensitivity results, if only two of the four multiplet components are selected. Use of the experiment for the measurement of¹J_HNcoupling constants is discussed. A water flip-back version of the experiment is demonstrated with a 45 kDa fragment of¹⁵N/²H labeledStaphylococcus aureusgyrase B.     

The First in Vivo Observation of C–N Coupling in Mammalian Brain

[5-¹³C,¹⁵N]Glutamine, with ¹J(¹³C–¹⁵N) of 16 Hz, was observed in vivo in the brain of spontaneously breathing rats by ¹³C MRS at 4.7 T. The brain [5-¹³C]glutamine peak consisted of the doublet from [5-¹³C,¹⁵N]glutamine and the center [5-¹³C,¹⁴N]glutamine peak, resulting in an apparent triplet with a separation of 8 Hz. The time course of formation of brain [5-¹³C,¹⁵N]glutamine was monitored in vivo with a time resolution of 20–35 min. This [5-¹³C,¹⁵N]glutamine was formed by glial uptake of released neurotransmitter [5-¹³C]glutamate and its reaction with ¹⁵NH₃ catalyzed by the glia-specific glutamine synthetase. The neurotransmitter glutamate C5 was selectively¹³C-enriched by intravenous [2,5-¹³C]glucose infusion to ¹³C-label whole-brain glutamate C5, followed by [¹²C]glucose infusion to chase ¹³C from the small and rapidly turning-over glial glutamate pool, leaving ¹³C mainly in the neurotransmitter [5-¹³C]glutamate pool, which is sequestered in vesicles until release. Hence, the observed [5-¹³C,¹⁵N]glutamine arises from a coupling between ¹³C of neuronal origin and ¹⁵N of glial origin. Measurement of the rate of brain [5-¹³C,¹⁵N]glutamine formation provides a novel noninvasive method of studying the kinetics of neurotransmitter uptake into glia in vivo, a process that is crucial for protecting the brain from glutamate excitotoxicity.     

Separating Structure and Dynamics in CSA/DD Cross-Correlated Relaxation: A Case Study on Trehalose and Ubiquitin

We present two new sensitivity enhanced gradient NMR experiments for measuring interference effects between chemical shift anisotropy (CSA) and dipolar coupling interactions in a scalar coupled two-spin system in both the laboratory and rotating frames. We apply these methods for quantitative measurement of longitudinal and transverse cross-correlation rates involving interference of ¹³C CSA and ¹³C–¹H dipolar coupling in a disaccharide, α,α- -trehalose, at natural abundance of ¹³C as well as interference of amide ¹⁵N CSA and ¹⁵N–¹H dipolar coupling in uniformly ¹⁵N-labeled ubiquitin. We demonstrate that the standard heteronuclear T₁, T₂, and steady-state NOE autocorrelation experiments augmented by cross-correlation measurements provide sufficient experimental data to quantitatively separate the structural and dynamic contributions to these relaxation rates when the simplifying assumptions of isotropic overall tumbling and an axially symmetric chemical shift tensor are valid.