3D NMR Experiments for MeasuringN Relaxation Data of Large Proteins: Application to the 44 kDa Ectodomain of SIV gp41

A suite of 3D NMR experiments for measuring¹⁵N–{¹H} NOE,¹⁵NT₁, and¹⁵NT_1ρvalues in large proteins, uniformly labeled with¹⁵N and¹³C, is presented. These experiments are designed for proteins that exhibit extensive spectral overlap in the 2D¹H–¹⁵N HSQC spectrum. The pulse sequences are readily applicable to perdeuterated samples, which increases the spectral resolution and signal-to-noise ratio, thereby permitting the characterization of protein dynamics to be extended to larger protein systems. Application of the pulse sequences is demonstrated on a perdeuterated¹³C/¹⁵N-labeled sample of the 44 kDa ectodomain of SIV gp41.     

EPIC- and CHANCE-HSQC: Two N-photo-CIDNP-enhanced pulse sequences for the sensitive detection of solvent-exposed tryptophan

Photochemically induced dynamic nuclear polarization (photo-CIDNP) of nuclei other than ¹H offers a tremendous potential for sensitivity enhancement in liquid state NMR under mild, physiologically relevant conditions. Photo-CIDNP enhancements of ¹⁵N magnetization are much larger than those typically observed for ¹H. However, the low gyromagnetic ratio of ¹⁵N prevents a full fruition of the potential signal-to-noise gains attainable via ¹⁵N photo-CIDNP. Here, we propose two novel pulse sequences, EPIC- and CHANCE-HSQC, tailored to overcome the above limitation. EPIC-HSQC exploits the strong ¹H polarization and its subsequent transfer to non-equilibrium N_z magnetization prior to ¹⁵N photo-CIDNP laser irradiation. CHANCE-HSQC synergistically combines ¹H and ¹⁵N photo-CIDNP. The above pulse sequences, tested on tryptophan (Trp) and the Trp-containing protein apoHmpH, were found to display up to 2-fold higher sensitivity than the reference NPE-SE-HSQC pulse train (based on simple ¹⁵N photo-CIDNP followed by N–H polarization transfer), and up to a ca. 3-fold increase in sensitivity over the corresponding dark pulse schemes (lacking laser irradiation). The observed effects are consistent with the predictions from a theoretical model of photo-CIDNP and prove the potential of ¹⁵N and ¹H photo-CIDNP in liquid state heteronuclear correlation NMR.     

Determination of Multiple φ-Torsion Angles in Proteins by Selective and Extensive C Labeling and Two-Dimensional Solid-State NMR

We describe an approach to efficiently determine the backbone conformation of solid proteins that utilizes selective and extensive ¹³C labeling in conjunction with two-dimensional magic-angle-spinning NMR. The selective ¹³C labeling approach aims to reduce line broadening and other multispin complications encountered in solid-state NMR of uniformly labeled proteins while still enhancing the sensitivity of NMR spectra. It is achieved by using specifically labeled glucose or glycerol as the sole carbon source in the protein expression medium. For amino acids synthesized in the linear part of the biosynthetic pathways, [1-¹³C]glucose preferentially labels the ends of the side chains, while [2-¹³C]glycerol labels the C^α of these residues. Amino acids produced from the citric-acid cycle are labeled in a more complex manner. Information on the secondary structure of such a labeled protein was obtained by measuring multiple backbone torsion angles φ simultaneously, using an isotropic–anisotropic 2D correlation technique, the HNCH experiment. Initial experiments for resonance assignment of a selectively ¹³C labeled protein were performed using ¹⁵N–¹³C 2D correlation spectroscopy. From the time dependence of the ¹⁵N–¹³C dipolar coherence transfer, both intraresidue and interresidue connectivities can be observed, thus yielding partial sequential assignment. We demonstrate the selective ¹³C labeling and these 2D NMR experiments on a 8.5-kDa model protein, ubiquitin. This isotope-edited NMR approach is expected to facilitate the structure determination of proteins in the solid state.     

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.     

Slice selection in solids by localized double-quantum coherence transfer

Double-quantum heteronuclear coherence transfer in solids shows a strong spatial dependence when performed in the presence of a magnetic field gradient. This is a direct consequence of the off-resonance sensitivity of the coherence transfer process and represents a new principle for localized NMR spectroscopy of quadrupole nuclei in solids. Since the slice-selective excitation is achieved simultaneously to the cross-polarization, the suggested pulse sequences avoid the use of shaped pulses, the application of which is problematic in solids. In the present work, the localization efficiency of this new slice-selection principle was analyzed in dependence on the experimental parameters for a spin system consisting of abundant spin-1/2 and rare spin-1 nuclei. The resulting slice profiles and the calculated dependences of the slice thickness for the basic coherence transfer procedures are discussed on the example of¹H?²H in monodeuterated benzene. The proposed method opens the possibility of volume-selective investigations of the structure and dynamics of materials using the well-established methodology of deuteron-NMR spectroscopy.     

TROSY of side-chain amides in large proteins

By using the mixed solvent of 50% H₂O/50% D₂O and employing deuterium decoupling, TROSY experiments exclusively detect NMR signals from semideuterated isotopomers of carboxamide groups with high sensitivities for proteins with molecular weights up to 80 kDa. This isotopomer-selective strategy extends TROSY experiments from exclusively detecting backbone to both backbone and side-chain amides, particularly in large proteins. Because of differences in both TROSY effect and dynamics between ¹⁵N–H^E{D^Z} and ¹⁵N–H^Z{D^E} isotopomers of the same carboxamide, the ¹⁵N transverse magnetization of the latter relaxes significantly faster than that of the former, which provides a direct and reliable stereospecific distinction between the two configurations. The TROSY effects on the ¹⁵N–H^E{D^Z} isotopomers of side-chain amides are as significant as on backbone amides.     

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.     

ZnO nanorod arrays fabrication via chemical bath deposition: Ligand concentration effect study

A new ligand, N,N,N′,N′-tetramethylethylenediamine, has been used to grow ZnO nanorods on silicon substrates via a two steps approach. A preliminary seeding on silicon substrates has been combined with chemical bath deposition using a Zinc acetate–N,N,N′,N′-tetramethylethylenediamine aqueous solution. The used diamino ligand has been selected as Zn²⁺ complexing agent and the related hydrolysis generates the reacting ions (Zn²⁺ and OH⁻) responsible for the ZnO growth. The seed layer has been annealed at low temperature (<200 °C) and the ZnO nanorods have been grown on this ZnO amorphous layer. There is experimental evidence that the ligand concentration (ranging from 5 to 50 mM) strongly affects the alignment of ZnO nanorods on the substrate, their lateral dimension and the related surface density. Length and diameter of ZnO nanorods increase upon increasing the ligand concentration, while the nanorod density decreases. Even more important, it has been demonstrated, as proof of concept, that chemical bath deposition can be usefully combined with colloidal lithography for selective ZnO nanorod deposition. Thus, by patterning the ZnO seeded substrate with polystyrene microsphere colloidal lithography, regular Si hole arrays, spatially defined by hexagonal ZnO nanorods, have been successfully obtained.     

Triple-Resonance Experiments for Correlation of H5 and Exchangeable Pyrimidine Base Hydrogens in C,N-Labeled RNA

Triple-resonance two-dimensional H5(C5C4N)H experiments are described that provide through-bond H5 to imino/amino connectivities in uridines and cytidines in ¹³C, ¹⁵N-labeled RNAs. The experiments employ selective INEPT steps for transferring magnetization from the H5 hydrogens through the intervening C5, C4, and N3/N4 nuclei to the imino/amino hydrogens. The improved sensitivity of these experiments for assignments in a large 43-nucleotide RNA is demonstrated.     

Cation Binding Induced Changes inN CSA in a Membrane-Bound Polypeptide

Cation binding to the monovalent cation selective channel, gramicidin A, is shown to induce changes in the dipolar and chemical shift observables from uniformly aligned samples. While these changes could be the result of structural or dynamic changes, they are shown to be primarily induced by through-bond polarizability effects when cations are solvated by the carbonyl oxygens of the peptide backbone. Upon cation binding partial charges are changed throughout the peptide plane, inducing large changes in the¹³C₁chemical shifts, smaller changes in the¹⁵N chemical shifts, and even smaller effects for the¹⁵N–¹³C₁and¹⁵N–²H dipolar interactions. These conclusions are substantiated by characterizing the¹⁵N chemical shift tensors in the presence and absence of cations in fast-frozen lipid bilayer preparations of gramicidin A.     

Coherence selection in double CP MAS NMR spectroscopy

Applications of double cross-polarization (CP) magic-angle spinning (MAS) NMR spectroscopy, via (1)H/(15)N and then (15)N/(13)C coherence transfers, for (13)C coherence selection are demonstrated on a (15)N/(13)C-labeled N-acetyl-glucosamine (GlcNAc) compound. The (15)N/(13)C coherence transfer is very sensitive to the settings of the experimental parameters. To resolve explicitly these parameter dependences, we have systematically monitored the (13)C{(15)N/(1)H} signal as a function of the rf field strength and the MAS frequency. The data reveal that the zero-quantum coherence transfer, with which the (13)C effective rf field is larger than that of the (15)N by the spinning frequency, would give better signal sensitivity. We demonstrate in one- and two-dimensional double CP experiments that spectral editing can be achieved by tailoring the experimental parameters, such as the rf field strengths and/or the MAS frequency.     

N Chemical Shift Tensor Magnitude and Orientation in the Molecular Frame of Uracil Determined via MAS NMR

The potential of heteronuclear MAS NMR spectroscopy for the characterization of ¹⁵N chemical shift (CS) tensors in multiply labeled systems has been illustrated, in one of the first studies of this type, by a measurement of the chemical shift tensor magnitude and orientation in the molecular frame for the two ¹⁵N sites of uracil. Employing polycrystalline samples of ¹⁵N₂ and 2-¹³C,¹⁵N₂-labeled uracil, we have measured, via ¹⁵N–¹³C REDOR and ¹⁵N–¹H dipolar-shift experiments, the polar and azimuthal angles (θ, ψ) of orientation of the ¹⁵N–¹³C and ¹⁵N–¹H dipolar vectors in the ¹⁵N CS tensor frame. The (θ_NC, ψ_NC) angles are determined to be (92 ± 10°, 100 ± 5°) and (132 ± 3°, 88 ± 10°) for the N1 and N3 sites, respectively. Similarly, (θ_NH, ψ_NH) are found to be (15 ± 5°, −80 ± 10°) and (15 ± 5°, 90 ± 10°) for the N1 and N3 sites, respectively. These results obtained based only on MAS NMR measurements have been compared with the data reported in the literature.     

Facile post-synthesis and photocatalytic activity of N-doped ZnO–SBA-15

N-doped ZnO–SBA-15 materials (denoted as nN–xZnO–SBA-15, where n is number of urea treatments and x is the weight ratio of ZnO/(ZnO+SBA-15)) were successfully synthesized by a two-step procedure. First, xZnO–SBA-15 was prepared by impregnating SBA-15 with Zn(NO₃)₂, followed by calcinating at 550 °C. In the second step, xZnO–SBA-15 was modified n times by doping nitrogen with the assistance of urea. The resulting nN–xZnO–SBA-15 materials prepared with various numbers of urea treatments were characterized by XRD, TEM, SEM, EDS, N₂ adsorption/desorption at 77 K, diffuse reflectance UV–vis, and XPS. The results show that the nN–xZnO–SBA-15 maintains its ordered hexagonal mesostructure and exhibits light absorbance in the visible region. The nN–xZnO–SBA-15 samples were investigated with the photodegradation of methylene blue under visible light, and exhibited significant photocatalytic activity. The kinetics of the reaction obeyed the Langmuir–Hinshelwood model.     

Gradient-Enhanced One-Dimensional Proton Chemical-Shift Correlation with Full Sensitivity

High-quality spectra were obtained by implementing pulsed field gradients (PFGs) as part of 1D selective experiments. The use of PFGs for coherence rejection rather than coherence selection ensures that there is no loss of signal and the sensitivity of these experiments is the same as that of their phase-cycled predecessors. The excitation scheme chosen ensures that these experiments are highly resistant to spin–spin relaxation. The following techniques are described: 1D ge-TOCSY, 1D ge-NOESY, 1D ge-TOCSY–TOCSY, 1D ge-NOESY–NOESY, 1D ge-TOCSY–NOESY, and 1D ge-NOESY–TOCSY. Their applications, for the separation of overlapping spin systems, tracing spin-diffusion signals, and extending the transfer of magnetization beyond an individual spin system, are illustrated using oligo- and polysaccharide samples.     

Indirectly detected heteronuclear correlation solid-state NMR spectroscopy of naturally abundant 15N nuclei

Two-dimensional indirectly detected through-space and through-bond ¹H{¹⁵N} solid-state NMR experiments utilizing fast magic angle spinning (MAS) and homonuclear multipulse ¹H decoupling are evaluated. Remarkable efficiency of polarization transfer can be achieved at a MAS rate of 40 kHz by both cross-polarization and INEPT, which makes these methods applicable for routine characterizations of natural abundance solids. The first measurement of 2D ¹H{¹⁵N} HETCOR spectrum of natural abundance surface species is also reported.     

A solid-state NMR investigation of the structure of mesoporous silica nanoparticle supported rhodium catalysts

A detailed study of the chemical structure of mesoporous silica catalysts containing rhodium ligands and nanoparticles (RhP-MSN) was carried out by multi-dimensional solid-state NMR techniques. The degree of functionalization of the rhodium–phosphinosilyl complex to the surface of the RhP-MSN channels was determined by ²⁹Si NMR experiments. The structural assignments of the rhodium–phosphinosilyl complex were unambiguously determined by employing the novel, indirectly detected heteronuclear correlation (¹³C–¹H and ³¹P–¹H idHETCOR) techniques, which indicated that oxidation of the attached phosphinosilyl groups and detachment of Rh was enhanced upon syngas conversion.     

Improved Resolution from Double Constant-Time Evolution of 3D and 4D Triple-Resonance Experiments

Triple-resonance NMR experiments are nearly essential for performing backbone assignments of proteins larger than 15 kDa. Our work extends the double constant-time (2CT) evolution scheme to triple-resonance 3D and 4D experiments. The modifications needed to accomplish 2CT evolution in triple resonance experiments are straight forward, are completely general, and consequently, will yield increased resolution for all out-and-back experiments. We expect that the increased resolution of experiments presented here will be useful in the study of larger proteins (>30 kDa) and in the study of highly helical proteins where¹HN,¹⁵N, and¹³C dimensions are poorly dispersed.     

Structural Assignment of Isomeric S‐ and S,N‐1‐Alkoxycarbonylalkylated 6‐Amino‐2‐thiouracil Derivatives by Means of 1H and 13C NMR Spectroscopy

Abstract

The structures of new isomeric 2‐alkoxycarbonylalkylthio‐ and 2‐alkoxy‐ carbonylalkylthio‐1‐alkoxycarbonylalkyl‐6‐aminouracils (1–21) have been established on the basis of the ¹H NMR and ¹³C NMR spectroscopic data. The ¹H NMR and ¹³C NMR spectra of 1–21 have been fully assigned by a combination of two‐dimensional experiments [heteronuclear multiple quantum coherence (HMQC) and heteronuclear multiple bond correlation (HMBC)]. The ¹³C NMR spectra have been shown to be able to differentiate between isomers.     

Recoupling in solid state NMR using γ prepared states and phase matching

The paper describes two-dimensional solid state NMR experiments that use powdered dephased antiphase coherence (γ preparation) to encode chemical shifts in the indirect dimension. Both components of this chemical shift encoded gamma-prepared states can be refocused into inphase coherence by a recoupling element. This helps to achieve sensitivity enhancement in 2D NMR experiments by quadrature detection. The powder dependence of the gamma-prepared states allows for manipulating them by suitable insertion of delays in the recoupling periods. This helps to design experiments that suppress diagonal peaks in 2D spectra, leading to improved resolution. We describe some new phase modulated heteronuclear and homonuclear recoupling pulse sequences that simplify the implementation of the described experiments based on γ prepared states. Recoupling in the heteronuclear spin system is achieved by matching the difference in the amplitude of the sine/cosine modulated phase on the two rf-channels to the spinning frequency while maintaining the same power on the two rf-channels.     

Site-specific ϕ- and ψ-torsion angle determination in a uniformly/extensively 13C- and 15N-labeled peptide

A solid-state rotational-echo double resonance (REDOR) NMR method was introduced to identify the ?- and ψ-torsion angle from a ¹H–¹⁵N or ¹H–¹³C′ spin system of alanine-like residues in a selectively, uniformly, or extensively ¹⁵N-/¹³C-labeled peptide. When a C_α(i) or a ¹⁵N peak is site-specifically obtainable in the NMR spectrum of a uniformly ¹⁵N/¹³C-labeled sample system, the ψ- or ?-torsion angle specified by the conformational structure of peptide geometry involving ¹⁵N(i)–¹H_αi–¹⁵N(i + 1) or ¹³C′(i − 1)–¹H^Ni–¹³C′(i) spin system can be identified based on ¹³C_α- or ¹⁵N-detected ¹H_α–¹⁵N or ¹H_N–¹³C REDOR experiment. This method will conveniently be utilized to identify major secondary motifs, such as α-helix, β-sheet, and β-turn, from a uniformly ¹⁵N-/¹³C-labled peptide sample system. When tested on a ¹³C-/¹⁵N-labeled model system of a three amino acid peptide Gly–[U–¹³C, ¹⁵N]Ala–[U–¹³C, ¹⁵N]Leu, the ψ-angle of alanine obtained experimentally, ψ = −40 ± 30°, agreed reasonably well with the X-ray determined angle, ψ = −39°.