Determination of (1)H homonuclear scalar couplings in unlabeled carbohydrates

The scarcity of structural information on carbohydrates results from combined difficulties to crystallize and the limitations in NMR analysis. Current methods for determining basic NMR parameters such as (1)H homonuclear scalar couplings are very limited, especially for large molecules such as polysaccharides, oligonucleotides, and the carbohydrate part of glycoproteins. In this paper, a NMR experiment for the determination of endocyclic (1)H homonuclear scalar couplings ((3)J(HH)) in unlabeled carbohydrates is presented. In addition to scalar couplings, cross-correlated dipole-dipole relaxation rates were measured for large polysaccharides. The measurement of all endocyclic homonuclear coupling constants within monosaccharide units is presented for lactose, a model disaccharide, and for a natural-abundance 2 MDa bacterial polysaccharide excreted by Streptococcus thermophilus Sfi39.     

Simplifying the complex 1H NMR spectra of fluorine-substituted benzamides by spin system filtering and spin-state selection: multiple-quantum-single-quantum correlation

The proton NMR spectra of fluorine-substituted benzamides are very complex (Figure 1) due to severe overlap of (1)H resonances from the two aromatic rings, in addition to several short and long-range scalar couplings experienced by each proton. With no detectable scalar couplings between the inter-ring spins, the (1)H NMR spectra can be construed as an overlap of spectra from two independent phenyl rings. In the present study we demonstrate that it is possible to separate the individual spectrum for each aromatic ring by spin system filtering employing the multiple-quantum-single-quantum correlation methodology. Furthermore, the two spin states of fluorine are utilized to simplify the spectrum corresponding to each phenyl ring by the spin-state selection. The demonstrated technique reduces spectral complexity by a factor of 4, in addition to permitting the determination of long-range couplings of less than 0.2 Hz and the relative signs of heteronuclear couplings. The technique also aids the judicious choice of the spin-selective double-quantum-single-quantum J-resolved experiment to determine the long-range homonuclear couplings of smaller magnitudes.     

1D NMR Homodecoupled 1H Spectra with Scalar Coupling Constants from 2D NemoZS‐DIAG Experiments

A two‐dimensional liquid‐state NMR experiment cleanly separating chemical shifts and scalar couplings information is introduced. This DIAG experiment takes advantage of a drastic reduction of the spectral window in the indirect dimension to be quickly recorded and of a new non‐equidistant modulation of the selective pulse to improve the sensitivity of the broadband homodecoupling Zangger–Sterk sequence element by one order of magnitude. A simple automatic analysis results in 1D spectra displaying singlets and lists of the scalar couplings for first‐order multiplets. This facilitates the analysis of 1D spectra by resolving multiplets based on their differences in chemical shifts and coupling structures.     

J‐Edited Pure Shift NMR for the Facile Measurement of nJHH for Specific Protons

We report a novel 1D J‐edited pure shift NMR experiment (J‐PSHIFT) that was constructed from a pseudo 2D experiment for the direct measurement of proton–proton scalar couplings. The experiment gives homonuclear broad‐band ¹H‐decoupled ¹H NMR spectra, which provide a single peak for chemically distinct protons, and only retain the homonuclear‐scalar‐coupled doublet pattern at the chemical‐shift positions of the protons in the coupled network of a specific proton. This permits the direct and unambiguous measurement of the magnitudes of the couplings. The incorporation of a 1D selective correlation spectroscopy (COSY)/ total correlation spectroscopy (TOCSY) block in lieu of the initial selective pulse, results in the exclusive detection of the correlated spectrum of a specific proton.     

Measurement of long-range 1H-19F scalar coupling constants and their glycosidic torsion dependence in 5-fluoropyrimidine-substituted RNA

Long-range scalar 5J(H1',F) couplings were observed in 5-fluoropyrimidine-substituted RNA. We developed a novel S3E-19F-alpha,beta-edited NOESY experiment for quantitation of these long-range scalar 5J(H1',F) couplings, where the J-couplings can be extracted from inspection of intraresidual (H1',H6) NOE cross-peaks. Quantum chemical calculations were exploited to investigate the relation between scalar couplings and conformations around the glycosidic bond in oligonucleotides. The theoretical dependence of the observed 5J(H1',F) couplings on the torsion angle chi can be described by a generalized Karplus relationship. The corresponding density functional theory (DFT) analysis is outlined. Additional NMR experiments facilitating the resonance assignments of 5-fluoropyrimidine-substituted RNAs are described, and chemical shift changes due to altered shielding in the presence of fluorine-19 (19F) are presented.     

Qualitative and quantitative measurements of hydrogen bond mediated scalar couplings in acyclic 1,3-diols

[Structure: see text] OH...OH hydrogen bond mediated scalar couplings have been observed in acyclic syn- and anti-1,3-diols using a 2D 1H COSYLR NMR experiment and quantified with an uncertainty of +/-0.02 Hz with a selective-excitation spin-echo NMR experiment. A theoretical investigation confirmed the importance of the hydrogen bond in mediating the spin-spin coupling in these systems.     

Accurate heteronuclear J-coupling measurements in dilute spin systems using the multiple-quantum filtered J-resolved experiment

A new solid-state MAS NMR experiment is proposed to accurately measure heteronuclear (19)F-(207)Pb J-coupling constants, even though these couplings are not visible on high speed (19)F 1D MAS spectra; in particular, we demonstrate that the J-resolved experiment combined with scalar multiple-quantum filtering considerably improves the resolution of J-multiplet patterns for dilute spin systems.     

Spin echo NMR spectra without J modulation

The spin echo is the single most important building block in modern NMR spectroscopy, but echo modulation by scalar couplings J can severely complicate its use. We show for the first time that a general but unacknowledged solution to such complications already exists.     

Theoretical study of the scalar coupling constants across the noncovalent contacts in RNA base pairs: the cis- and trans-watson-crick/sugar edge base pair family

The structure and function of RNA molecules are substantially affected by non-Watson-Crick base pairs actively utilizing the 2'-hydroxyl group of ribose. Here we correlate scalar coupling constants across the noncovalent contacts calculated for the cis- and trans-WC/SE (Watson-Crick/sugar edge) RNA base pairs with the geometry of base to base and sugar to base hydrogen bond(s). 23 RNA base pairs from the 32 investigated were found in RNA crystal structures, and the calculated scalar couplings are therefore experimentally relevant with regard to the binding patterns occurring in this class of RNA base pairs. The intermolecular scalar couplings 1hJ(N,H), 2hJ(N,N), 2hJ(C,H), and 3hJ(C,N) were calculated for the N-H...N and N-H...O=C base to base contacts and various noncovalent links between the sugar hydroxyl and RNA base. Also, the intramolecular 1J(N,H) and 2J(C,H) couplings were calculated for the amino or imino group of RNA base and the ribose 2'-hydroxyl group involved in the noncovalent interactions. The calculated scalar couplings have implications for validation of local geometry, show specificity for the amino and imino groups of RNA base involved in the linkage, and can be used for discrimination between the cis- and trans-WC/SE base pairs. The RNA base pairs within an isosteric subclass of the WC/SE binding patterns can be further sorted according to the scalar couplings calculated across different local noncovalent contacts. The effect of explicit water inserted in the RNA base pairs on the magnitude of the scalar couplings was calculated, and the data for discrimination between the water-inserted and direct RNA base pairs are presented. The calculated NMR data are significant for structural interpretation of the scalar couplings in the noncanonical RNA base pairs.     

High-resolution (19)F MAS NMR spectroscopy: structural disorder and unusual J couplings in a fluorinated hydroxy-silicate

High-resolution (19)F magic angle spinning (MAS) NMR spectroscopy is used to study disorder and bonding in a crystalline solid. (19)F MAS NMR reveals four distinct F sites in a 50% fluorine-substituted deuterated hydrous magnesium silicate (clinohumite, 4Mg(2)SiO(4)·Mg(OD(1-x)F(x))(2) with x = 0.5), indicating extensive structural disorder. The four (19)F peaks can be assigned using density functional theory (DFT) calculations of NMR parameters for a number of structural models with a range of possible local F environments generated by F(-)/OH(-) substitution. These assignments are supported by two-dimensional (19)F double-quantum MAS NMR experiments that correlate F sites based on either spatial proximity (via dipolar couplings) or through-bond connectivity (via scalar, or J, couplings). The observation of (19)F-(19)F J couplings is unexpected as the fluorines coordinate Mg atoms and the Mg-F interaction is normally considered to be ionic in character (i.e., there is no formal F-Mg-F covalent bonding arrangement). However, DFT calculations predict significant (19)F-(19)F J couplings, and these are in good agreement with the splittings observed in a (19)F J-resolved MAS NMR experiment. The existence of these J couplings is discussed in relation to both the nature of bonding in the solid state and the occurrence of so-called "through-space" (19)F-(19)F J couplings in solution. Finally, we note that we have found similar structural disorder and spin-spin interactions in both synthetic and naturally occurring clinohumite samples.     

Observation of CH⋅⋅⋅π Interactions between Methyl and Carbonyl Groups in Proteins

Protein structure and function is dependent on myriad noncovalent interactions. Direct detection and characterization of these weak interactions in large biomolecules, such as proteins, is experimentally challenging. Herein, we report the first observation and measurement of long-range “through-space” scalar couplings between methyl and backbone carbonyl groups in proteins. These J couplings are indicative of the presence of noncovalent C−H⋅⋅⋅π hydrogen-bond-like interactions involving the amide π network. Experimentally detected scalar couplings were corroborated by a natural bond orbital analysis, which revealed the orbital nature of the interaction and the origins of the through-space J couplings. The experimental observation of this type of CH⋅⋅⋅π interaction adds a new dimension to the study of protein structure, function, and dynamics by NMR spectroscopy.     

Observation of CH⋅⋅⋅π Interactions between Methyl and Carbonyl Groups in Proteins

Protein structure and function is dependent on myriad noncovalent interactions. Direct detection and characterization of these weak interactions in large biomolecules, such as proteins, is experimentally challenging. Herein, we report the first observation and measurement of long‐range “through‐space” scalar couplings between methyl and backbone carbonyl groups in proteins. These J couplings are indicative of the presence of noncovalent C−H⋅⋅⋅π hydrogen‐bond‐like interactions involving the amide π network. Experimentally detected scalar couplings were corroborated by a natural bond orbital analysis, which revealed the orbital nature of the interaction and the origins of the through‐space J couplings. The experimental observation of this type of CH⋅⋅⋅π interaction adds a new dimension to the study of protein structure, function, and dynamics by NMR spectroscopy.     

Determination of the geometry of acetoxyendiynes and acetoxyenynes by NMR heteronuclear 13C?1H scalar couplings and 13C NMR chemical shifts. Structural assignment of the oxylipin natural products peyssonenynes A and B

Solution NMR methods were used for the structural characterization of the acetoxyendiyne E/Z configuration of the marine natural products peyssonenynes A and B and their synthetic analogs derived from palmitic acid. The scarcity of protons in the proximity of the olefin precluded the determination of the double bond geometry using ¹H NMR methods that rely on proton–proton scalar couplings or experiments such as NOESY or ROESY. Long range ¹H? ¹³C heteronuclear scalar couplings, ⁿJ_CH, measured with the 2D excitation sculptured indirect detection experiment (EXSIDE) proved useful and highly reliable for the analysis of the enol acetate geometry. In addition, it was found that the chemical shift of some carbon atoms in the proximity of the olefin was also sensitive to the double bond configuration of these molecules providing an even simpler way to determine their geometry. This protocol showed its robustness by similar analysis of simpler silyl‐protected acetoxyenynes derived from fatty acids. These NMR experimental results and stereochemical predictions were rationalized by DFT calculations. Copyright © 2010 John Wiley & Sons, Ltd.     

CHARMM36 all‐atom additive protein force field: Validation based on comparison to NMR data

Protein structure and dynamics can be characterized on the atomistic level with both nuclear magnetic resonance (NMR) experiments and molecular dynamics (MD) simulations. Here, we quantify the ability of the recently presented CHARMM36 (C36) force field (FF) to reproduce various NMR observables using MD simulations. The studied NMR properties include backbone scalar couplings across hydrogen bonds, residual dipolar couplings (RDCs) and relaxation order parameter, as well as scalar couplings, RDCs, and order parameters for side‐chain amino‐ and methyl‐containing groups. It is shown that the C36 FF leads to better correlation with experimental data compared to the CHARMM22/CMAP FF and suggest using C36 in protein simulations. Although both CHARMM FFs contains the same nonbond parameters, our results show how the changes in the internal parameters associated with the peptide backbone via CMAP and the χ₁ and χ₂ dihedral parameters leads to improved treatment of the analyzed nonbond interactions. This highlights the importance of proper treatment of the internal covalent components in modeling nonbond interactions with molecular mechanics FFs. © 2013 Wiley Periodicals, Inc.     

Multiple refocusing in NMR spectroscopy: compensation of pulse imperfections by scalar couplings.

When applying multiple refocusing pulses to characterize the cross-correlated relaxation of heteronuclear multiple quantum coherence 2NxHx in biomolecules, the unavoidable effects of pulse imperfections are compensated by the scalar couplings between nitrogen atoms and protons. The experiment, which is useful as a tool for studying slow internal dynamics of biomolecules, greatly benefits from this compensation. The underlying effect is a manifestation of an interchange between three noncommuting components of the density operator. One perturbing Hamiltonian is counteracted by another, which leads to a nearly complete suppression of the perturbation. The effect proves to be an example of a hitherto unknown phenomenon in NMR spectroscopy.     

Measurement of slow (micros-ms) time scale dynamics in protein side chains by (15)N relaxation dispersion NMR spectroscopy: application to Asn and Gln residues in a cavity mutant of T4 lysozyme.

A new NMR experiment is presented for the measurement of micros-ms time scale dynamics of Asn and Gln side chains in proteins. Exchange contributions to the (15)N line widths of side chain residues are determined via a relaxation dispersion experiment in which the effective nitrogen transverse relaxation rate is measured as a function of the number of refocusing pulses in constant-time, variable spacing CPMG intervals. The evolution of magnetization from scalar couplings and dipole-dipole cross-correlations, which has limited studies of exchange in multi-spin systems in the past, does not affect the extraction of accurate exchange parameters from relaxation profiles of NH(2) groups obtained in the present experiment. The utility of the method is demonstrated with an application to a Leu --> Ala cavity mutant of T4 lysozyme, L99A. It is shown that many of the side chain amide groups of Asn and Gln residues in the C-terminal domain of the protein are affected by a chemical exchange process which may be important in facilitating the rapid binding of hydrophobic ligands to the cavity.     

A Systematic Investigation of Long-Range Couplings in the 1H NMR Spectra of Dimeric Indanones

 During the synthesis of methyl substituted indanones by intramolecular cyclization of 3-phenyl-propionic acids, dimerization led to by-products which can be considered as dimeric indanones. The proton NMR spectra of these compounds exhibit pronounced scalar couplings over up to seven bonds. A series of structures of the above type were investigated, and their NMR spectroscopic behaviour is discussed.     

Measurement of Large Dipolar Couplings of a Liquid Crystal with Terminal Phenyl Rings and Estimation of the Order Parameters

NMR spectroscopy is a powerful means of studying liquid‐crystalline systems at atomic resolutions. Of the many parameters that can provide information on the dynamics and order of the systems, ¹H–¹³C dipolar couplings are an important means of obtaining such information. Depending on the details of the molecular structure and the magnitude of the order parameters, the dipolar couplings can vary over a wide range of values. Thus the method employed to estimate the dipolar couplings should be capable of estimating both large and small dipolar couplings at the same time. For this purpose, we consider here a two‐dimensional NMR experiment that works similar to the insensitive nuclei enhanced by polarization transfer (INEPT) experiment in solution. With the incorporation of a modification proposed earlier for experiments with low radio frequency power, the scheme is observed to enable a wide range of dipolar couplings to be estimated at the same time. We utilized this approach to obtain dipolar couplings in a liquid crystal with phenyl rings attached to either end of the molecule, and estimated its local order parameters.     

Frequency selective heteronuclear dipolar recoupling in rotating solids: accurate (13)C-(15)N distance measurements in uniformly (13)C,(15)N-labeled peptides

We describe a magic-angle spinning NMR experiment for selective (13)C-(15)N distance measurements in uniformly (13)C,(15)N-labeled solids, where multiple (13)C-(15)N and (13)C-(13)C interactions complicate the accurate measurement of structurally interesting, weak (13)C-(15)N dipolar couplings. The new experiment, termed FSR (frequency selective REDOR), combines the REDOR pulse sequence with a frequency selective spin-echo to recouple a single (13)C-(15)N dipolar interaction in a multiple spin system. Concurrently the remaining (13)C-(15)N dipolar couplings and all (13)C-(13)C scalar couplings to the selected (13)C are suppressed. The (13)C-(15)N coupling of interest is extracted by a least-squares fit of the experimentally observed modulation of the (13)C spin-echo intensity to the analytical expression describing the dipolar dephasing in an isolated heteronuclear spin pair under conventional REDOR. The experiment is demonstrated in three uniformly (13)C,(15)N-labeled model systems: asparagine, N-acetyl-L-Val-L-Leu and N-formyl-L-Met-L-Leu-L-Phe; in N-formyl-[U-(13)C,(15)N]L-Met-L-Leu-L-Phe we have determined a total of 16 internuclear distances in the 2.5-6 A range.     

J-GFT NMR for precise measurement of mutually correlated nuclear spin-spin couplings

G-matrix Fourier transform (GFT) NMR spectroscopy is presented for accurate and precise measurement of chemical shifts and nuclear spin-spin couplings correlated according to spin system. The new approach, named "J-GFT NMR", is based on a largely extended GFT NMR formalism and promises to have a broad impact on projection NMR spectroscopy. Specifically, constant-time J-GFT (6,2)D (HA-CA-CO)-N-HN was implemented for simultaneous measurement of five mutually correlated NMR parameters, that is, 15N backbone chemical shifts and the four one-bond spin-spin couplings 13Calpha-1Halpha, 13Calpha-13C', 15N-13C', and 15N-1HNu. The experiment was applied for measuring residual dipolar couplings (RDCs) in an 8 kDa protein Z-domain aligned with Pf1 phages. Comparison with RDC values extracted from conventional NMR experiments reveals that RDCs are measured with high precision and accuracy, which is attributable to the facts that (i) the use of constant time evolution ensures that signals do not broaden whenever multiple RDCs are jointly measured in a single dimension and (ii) RDCs are multiply encoded in the multiplets arising from the joint sampling. This corresponds to measuring the couplings multiple times in a statistically independent manner. A key feature of J-GFT NMR, i.e., the correlation of couplings according to spin systems without reference to sequential resonance assignments, promises to be particularly valuable for rapid identification of backbone conformation and classification of protein fold families on the basis of statistical analysis of dipolar couplings.