The Role of Coherence Transfer Efficiency in Design of TROSY-Type Multidimensional NMR Experiments

An improved method for TROSY-type (Pervushin et al., Proc. Natl. Acad. Sci. USA 94, 12366-12371 (1997)) heteronuclear two-dimensional correlation involving protons of negligible CSA is presented. Rather than applying a simple INEPT sequence for back-transfer to protons (Pervushin et al., J. Am. Chem. Soc. 120, 6394-6400 (1998)), we replace the pi/2 proton pulse in INEPT by a spin-state-selective coherence transfer element (Sorensen et al., J. Biomol. NMR 10, 181-186 (1997)) and maintain broadband decoupling during acquisition. Theoretically that results in a sensitivity enhancement of a factor of 2. The new method is demonstrated using a (13)C,(15)N-labeled protein sample, RAP 18-112 (N-terminal domain of alpha(2)-macroglobulin receptor associated protein), at 750 MHz.     

Spin-State-Selective TPPI: A New Method for Suppression of Heteronuclear Coupling Constants in Multidimensional NMR Experiments

A novel multidimensional NMR pulse sequence tool, spin-state-selective time-proportional phase incrementation (S(3) TPPI), is introduced. It amounts to application of different TPPIs on the two components of doublets so that their frequencies can be manipulated independently. The chief application is for suppression of large heteronuclear one-bond coupling constants in indirect dimensions of multidimensional experiments without interchanging the two transverse magnetization components of doublets as conventional decoupling does, which is advantageous when they relax at different rates such as by partial compensation of dipolar and CSA relaxation contributions. For experimental confirmation we use a sample of (15)N-labeled neural cell adhesion molecule modules 1 and 2, a protein with a molecular weight of about 20 kDa. The new tool is general and can be combined with many multidimensional NMR experiments for proteins.     

Suppression of Diagonal Peaks in TROSY-Type H NMR NOESY Spectra of N-Labeled Proteins

A novel method for suppression of diagonal peaks in the amide region of NOESY NMR spectra of ¹⁵N-labeled proteins is presented. The method is particularly useful for larger proteins at high magnetic fields where interference between dipolar and chemical shift anisotropy relaxation mechanisms results in large TROSY effects, i.e., large differences in ¹H^N linewidths depending on the spin state of attached ¹⁵N nuclei. In this limit the new TROSY NOESY method does not compromise sensitivity. It is demonstrated using a perdeuterated ¹⁵N-labeled protein sample, Neural Cell Adhesion Molecule 213–308 (NCAM) from rat, in H₂O at 800 MHz.     

Three-Dimensional C Shift/H–N Coupling/N 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.     

J Coupling between C and H across Hydrogen Bonds in Proteins

Two new two- or three-dimensional NMR methods for measuring ^3hJ_C′N and ^2hJ_C′H coupling constants across hydrogen bonds in proteins are presented. They are tailored to suit the size of the TROSY effect, i.e., the degree of interference between dipolar and chemical shift anisotropy relaxation mechanisms. The methods edit 2D or 3D spectra into two separate subspectra corresponding to the two possible spin states of the ¹H^N spin during evolution of ¹³CO coherences. This allows ^2hJ_C′H to be measured in an E.COSY-type way while ^3hJ_C′N can be measured in the so-called quantitative way provided a reference spectrum is also recorded. A demonstration of the new methods is shown for the ¹⁵N,¹³C-labeled protein chymotrypsin inhibitor 2.     

CHHC and H–H magnetization exchange: Analysis by experimental solid-state NMR and 11-spin density-matrix simulations

A protocol is presented for correcting the effect of non-specific cross-polarization in CHHC solid-state MAS NMR experiments, thus allowing the recovery of the ¹H–¹H magnetization exchange functions from the mixing-time dependent buildup of experimental CHHC peak intensity. The presented protocol also incorporates a scaling procedure to take into account the effect of multiplicity of a CH₂ or CH₃ moiety. Experimental CHHC buildup curves are presented for l-tyrosine·HCl samples where either all or only one in 10 molecules are U–¹³C labeled. Good agreement between experiment and 11-spin SPINEVOLUTION simulation (including only isotropic ¹H chemical shifts) is demonstrated for the initial buildup (t_mix < 100 μs) of CHHC peak intensity corresponding to an intramolecular close (2.5 Å) H–H proximity. Differences in the initial CHHC buildup are observed between the one in 10 dilute and 100% samples for cases where there is a close intermolecular H–H proximity in addition to a close intramolecular H–H proximity. For the dilute sample, CHHC cross-peak intensities tended to significantly lower values for long mixing times (500 μs) as compared to the 100% sample. This difference is explained as being due to the dependence of the limiting total magnetization on the ratio N_obs/N_tot between the number of protons that are directly attached to a ¹³C nucleus and hence contribute significantly to the observed ¹³C CHHC NMR signal, and the total number of ¹H spins into the system. ¹H–¹H magnetization exchange curves extracted from CHHC spectra for the 100% l-tyrosine·HCl sample exhibit a clear sensitivity to the root sum squared dipolar coupling, with fast buildup being observed for the shortest intramolecular distances (2.5 Å) and slower, yet observable buildup for the longer intermolecular distances (up to 5 Å).     

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.     

Proton and Carbon - 13 NMR Assignments for Fluorinated and Nonfluorinated Aromatic Ketimines

The ¹H and ¹³C NMR spectra of three aromatic ketimines with varying degrees of fluoro substitution have been extensively studied using one and two dimensional techniques. COSY experiments were conducted to identify the protons on each of the aromatic groups, HETCORR experiments were then utilized to identify the corresponding carbon atoms, and then the order of the carbon atoms was established by long-range HETCORR (HRTCORRLR) results. These studies have allowed rigorous assignments to be made for most of the carbon and hydrogen atoms present in these compounds. Fluorine splittings were very helpful in the analyses. In the course of this study, the NMR absorbances (¹H and ¹³C) of related aldimines have also been assigned. This constitutes the first report on the assignments of ¹H and ¹³C absorbances for ketimines. The observed spectral properties suggest that the structure of aromatic ketimines is one in which the aromatic rings are in three different planes. Two of the aromatic rings, the N-substituted ring and the C-substituted ring in the cis configuration, are twisted substantially out of the plane with respect to the -C=N- bond. The remaining C-substituted aromatic ring, trans to the N-substituted ring, lies in the deshielding zone of the imine bond.     

13C NMR Chemical Shifts of Heterocycles: Empirical Substituent Effects in 5-Halomethylisoxazoles

Evaluation by empirically derived equations for the Substituent effect (α, β, γ, δ) on the ¹³C NMR chemical shifts for C-3, C-4. C-5 and halomethyl-substituent carbon (C-6) in isoxazoles 1-5 [where C-3 substituent (R¹) = H, alkyl or phenyl, C-4 Substituent (R²) = H, alkyl, and C-5 substituent (R³) = di-or trihalomethyl, methyl and H], taking as reference the compound la, is reported. From the calculated values for the α, β, γ, δ effects for each substituent it was possible to estimate the chemical shift of each carbon of the compounds 1–5. The ¹³ C chemical shifts of the C-3, C-4, C-5, C-6 of these compounds, can be estimated with good precision: 94% of the calculated chemical shifts are found to be within ±1.0ppm, and 100% are found to be within ±1.5ppm.     

Assignments of 1H and 13C NMR Spectral Data for Benzoylecgonine,a Cocaine Metabolite

The complete assignment of the ¹H and ¹³C NMR spectra of benzoylecgonine, a cocaine metabolite, was performed, with the aid of some 2D experiments such as gCOSY and gHSQC.     

2H NMR of Deuterofullerites C60D x

Deuterofullerites C₆₀D _x have been studied by means of ²H NMR spectroscopy. It has been established that there are two types of carbon–deuterium bindings in the samples under study: tip C–D with quadrupole constant coupling (QCC) 171 kHz and bridge –C...D...C– with QCC 56 kHz. It is possible that the latter bond is a result of the rigidity of the lattice, which is unusual for fullerene compounds.     

17O and 13C NMR Studies of Isobenzopyrylium Salts

¹³C and ¹⁷O NMR chemical shifts for a series of isobenzopyrylium salts are reported. The oxygen signal range from 300 to 270 ppm as a double bonded carboxylic oxygen, From the ¹⁷O and ¹³C data valuable informations on the conjugative and substituent effects of isobenzopyrylium salts were obtained.     

13C NMR Studies on 3-Aryl-4-Cyanosydnones (II). NMR Spectroscopy and the Chain-Conjugated Structure of Sydnones

Carbon-13 NMR spectra of 3-aryl-4-cyanosydnones have been analyzed to serve as an electronic structure probe of sydnones. 2-Dimensional INADEQUATE experiments and spin-lattice relaxation time measurements were employed to ascertain exact assignments. Deshielding of the phenyl carbon para to the sydnone ring manifests that N(3) bears positive charge. The electronic-rich property of C(4) is confirmed by shift data of cyano carbons, which have been found by far the most shielded cyano carbons in nitrile-containing compounds yet reported. Shift variations of C(5) resulting from the substitution at C(4) and supplementary oxygen-17 chemical shift data provide evidence in favor of the chain-conjugated structure.     

13C NMR in the Structural Assignment of Fused Furoxans

The chemical shifts and multiplicities of the two bridgehead carbons in the ¹³C NMR spectra of various fused furoxans are snown to provide a general method for assigning structure in these tautomeric systems.     

13C NMR Study of the Natural Glycosides Vicine and Convicine

¹³C NMR parameters have been obtained for vicine and convicine in DMSO, D₂O/DMSO and D₂O. Complete assignment of the spectra has been achieved. Interpretation of spin-lattice relaxation rates and heteronu-clear NOES has yielded evidence of intramolecular structuring in the case of vicine and not in that of ronvirine and also of a complex network of solute-solvent interactions.     

Complete Analysis of the 1H and 13C NMR Spectra of 5-Isopropylsulfonyl-2-Norbornenes Through the Application of Two-Dimensional NMR Techniques

Total assignment of ¹³C and ¹H NMR spectra of the 5-isopropylsulfonyl-2-norbornenes 2 was achieved using the concerted application of two-dimensional homonuclear and heteronuclear chemical shift correlations. The stereochemistry of both the diastereoisomers endo 2a and exo 2b have been established using the magnitude of the proton coupling constants.     

A C solid-state NMR analysis of vitamin D compounds

¹³C cross-polarization/magic-angle spinning (CP/MAS) solid-state NMR spectroscopy has been employed to analyze four vitamin D compounds, namely vitamin D3 (D3), vitamin D2 (D2), and the precursors ergosterol (Erg) and 7-dehydrocholesterol (7DHC). The ¹³C NMR spectrum of D3 displays a doublet pattern for each of the carbon atoms, while that of Erg contains both singlet and doublet patterns. In the cases of 7DHC and D2, the ¹³C spectra display various multiplet patterns, viz. singlets, doublets, triplets, and quartets. To overcome the signal overlap between the ¹³C resonances of protonated and unprotonated carbons, we have subjected these vitamin D compounds to 1D ¹H-filtered ¹³C CP/MAS and {¹H}/¹³C heteronuclear correlation (Hetcor) NMR experiments. As a result, assisted by solution NMR data, all of the ¹³C resonances have been successfully assigned to the respective carbon atoms of these vitamin D compounds. The ¹³C multiplets are interpreted due to the presence of s-cis and s-trans configurations in the α- and β-molecular conformers, consistent with computer molecular modeling determined by molecular dynamics and energy minimization calculations. To further characterize the ring conformations in D3, we have successfully extracted chemical shift tensor elements for the ¹³C doublets. It is demonstrated that ¹³C solid-state NMR spectroscopy provides a robust and high sensitive means of characterizing molecular conformations in vitamin D compounds.     

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.     

1H AND 13C NMR Assignments of Artheethers

The ¹H and ¹³C nmr assignments for all hydrogen and carbon atoms were made for β and α arteethers (5 and 6) based on chemical shift theory and 2D-nmr techniques (COSY and HETCOR).