Measurements of side-chain 13C-13C residual dipolar couplings in uniformly deuterated proteins

13C-only spectroscopy was used to measure multiple residual (13)C-(13)C dipolar couplings (RDCs) in uniformly deuterated and (13)C-labeled proteins. We demonstrate that (13)C-start and (13)C-observe spectra can be routinely used to measure an extensive set of the side-chain residual (13)C-(13)C dipolar couplings upon partial alignment of human ubiquitin in the presence of bacteriophages Pf1. We establish that, among different broadband polarization transfer schemes, the FLOPSY family can be used to exchange magnetization between a J coupled network of spins while largely decoupling dipolar interactions between these spins. An excellent correlation between measured RDCs and the 3D structure of the protein was observed, indicating a potential use of the (13)C-(13)C RDCs in the structure determination of perdeuterated proteins.     

Measurement of long-range 1H-1H dipolar couplings in weakly aligned proteins

Measurement of 1H-1H dipolar couplings in macromolecules, weakly oriented by a dilute liquid crystalline medium, is generally limited to the largest such interactions. By removing dipolar couplings to nearest neighbors, either by decoupling, deuteration, or both, more remote interactions become accessible. The approach is demonstrated for measurement of amide-amide interactions in the proteins calmodulin and ubiquitin and permits observation of direct dipolar couplings between protons up to 7 A apart. Quantitative evaluation of 1H-1H dipolar couplings measured in ubiquitin shows excellent agreement with its solution structure.     

Measurement of 13C-1H dipolar couplings in solids by using ultra-fast magic-angle spinning NMR spectroscopy with symmetry-based sequences

We show that (13)C-(1)H dipolar couplings in fully protonated organic solids can be measured by applying a Symmetry-based Resonance-Echo DOuble-Resonance (S-REDOR) experiment at ultra-fast Magic-Angle Spinning (MAS). The (13)C-(1)H dipolar couplings are recovered by using the R12 recoupling scheme, while the interference of (1)H-(1)H dipolar couplings are suppressed by the symmetry properties of this sequence and the use of high MAS frequency (65 kHz). The R12 method is especially advantageous for large (13)C-(1)H dipolar interactions, since the dipolar recoupling time can be incremented by steps as short as one rotor period. This allows a fine sampling for the rising part of the dipolar dephasing curve. We demonstrate experimentally that one-bond (13)C-(1)H dipolar coupling in the order of 22 kHz can be accurately determined. Furthermore, the proposed method allows a rapid evaluation of the dipolar coupling by fitting the S-REDOR dipolar dephasing curve with an analytical expression.     

Limited flexibility of lactose detected from residual dipolar couplings using molecular dynamics simulations and steric alignment methods

The conformational flexibility of lactose in solution has been investigated by residual dipolar couplings (RDCs). One-bond carbon-proton and proton-proton coupling constants have been measured in two oriented media and interpreted in combination with molecular dynamics simulations (MD). Two different approaches, known as PALES (Zweckstetter et al., J. Am. Chem. Soc. 2000, 122, 3791-3792) and TRAMITE (Azurmendi et al., J. Am. Chem. Soc. 2002, 124, 2426-2427), have been used to determine the alignment tensor from a shape-induced alignment model with the oriented medium. The steric alignment of the structures from several MD trajectories has provided ensemble averaged RDCs that have been compared with the experimental ones. The obtained results reveal the almost exclusive presence of a major low energy region defined as syn-phi/syn-psi (> 97%), for which sampling occurs in a dynamic manner. This result satisfactorily agrees with that determined by standard NOE-based methods.     

Determination of natural abundance 15N-1H and 13C-1H dipolar couplings of molecules in a strongly orienting media using two-dimensional inverse experiments

NMR spectra of molecules oriented in liquid crystals provide homo- and heteronuclear dipolar couplings and thereby the geometry of the molecules. Several inequivalent dilute spins such as 13C and 15N coupled to protons form different coupled spin systems in their natural abundance and appear as satellites in the proton spectra. Identification of transitions belonging to each spin system is essential to determine heteronuclear dipolar couplings, which is a formidable task. In the present study, using 15N-1H and 13C-1H HSQC, and HMQC experiments we have selectively detected spectra of each rare spin coupled to protons. The 15N-1H and 13C-1H dipolar couplings have been determined in the natural abundance of 13C and 15N for the molecules pyrazine, pyrimidine and pyridazine oriented in a thermotropic liquid crystal.     

High-accuracy residual 1HN-13C and 1HN-1HN dipolar couplings in perdeuterated proteins

Truncation by the presence of many short-range residual dipolar couplings (RDCs) hinders the observation of long-range RDCs in weakly aligned biomacromolecules. Perdeuteration of proteins followed by reprotonation of labile hydrogen positions greatly alleviates this problem. Here we show that for small perdeuterated proteins, a large number (up to 10 in protein G) of long-range RDCs to 13C and 1HN can be observed from individual amide protons. The 1HN <--> 13C RDCs comprise correlations to 13Calpha, 13Cbeta, and 13C' nuclei of the same and the preceding amino acid, as well as 13C' nuclei of hydrogen-bonded amino acids. The accuracy of the coupling constants is very high and defines individual internuclear distances to within few picometers. Deviations between measured RDC values and values predicted from the 1.1 A crystal structure of protein G are mainly found in two surface-exposed loop regions. The deviations show a strong correlation to the B-factor of the crystal structure.     

Protein Backbone 1H(N)-13Calpha and 15N-13Calpha residual dipolar and J couplings: new constraints for NMR structure determination

A simple, sensitivity-enhanced experiment was devised for accurate measurement of backbone 15N-13Calpha and 1HN-13Calpha couplings in proteins. The measured residual dipolar couplings 2DHCA, 1DNCA, 3DHCA, and 2DNCA for protein GB1 display very good agreement with the refined NMR structure (PDB code: 3GB1). A Karplus-type relationship between the one-bond 1JNCA couplings and the backbone dihedral psi angles holds, and on the basis of the two-bond 2JNCA couplings a secondary structure index can be established.     

Simultaneous definition of high resolution protein structure and backbone conformational dynamics using NMR residual dipolar couplings.

Despite the importance of molecular dynamics for biological activity, most approaches to protein structure determination, whether based on crystallographic or solution studies, propose three-dimensional atomic representations of a single configuration that take no account of conformational fluctuation. Non-averaged anisotropic NMR interactions, such as residual dipolar couplings, that become measurable under conditions of weak alignment, provide sensitive probes of both molecular structure and dynamics. Residual dipolar couplings are becoming increasingly powerful for the study of proteins in solution. In this minireview we present their use for the simultaneous determination of protein structure and dynamics.     

(13)C-(1)H and (13)C-(13)C NMR J-couplings in (13)C-labeled N-acetyl-neuraminic acid: correlations with molecular structure

N-acetyl-neuraminic acid (Neu5Ac, 2) was prepared enzymatically containing single sites of (13)C-enrichment at C1, C2, and C3. Aqueous solutions of the three (13)C isotopomers were studied by (1)H and (13)C NMR spectroscopy at p(2)H 2 and pH 8 to obtain J(CH) and J(CC) values involving the labeled carbons. Experimental studies were complemented by DFT calculations of the same set of J-couplings in protonated and ionized structural mimics of 2 to determine how well theoretical predictions match the experimental findings in saccharides bearing ionizable functionality. Results show that: (a) (2)J(C2,H3ax/eq) values in 2 depend on anomeric configuration, thus complementing (3)J(C1,H3ax/eq) behavior, (b) J(CH) and J(CC) values involving C2 depend on anomeric configuration, the C1-C2 bond torsion, and solution pH, and (c) long-range (4)J(C2,H7) is sensitive to glycerol side-chain conformation. Intraring J(HH) and most (2)J(CH), (3)J(CH), (2)J(CC), and (3)J(CC) involving C1-C3 of 2 appear largely unaffected by the ionization state of the carboxyl group. In vacuo and solvated DFT calculations of geminal and vicinal J(CH) and J(CC) values are similar and reproduce the experimental data well, but better agreement with experiment was observed for (1)J(C1,C2) in the solvated calculations. The present work provides new information for future treatments of trans-glycoside couplings involving Neu5Ac residues by (a) providing new standard values of intraring J(CC) for coupling pathways that mimic those for trans-glycoside J(CC), (b) identifying potential effects of solution pH on trans-glycoside couplings inferred through the behavior of related intraring couplings, and (c) providing specific guidelines for more reliable DFT predictions of J(CH) and J(CC) values in ionizable saccharides.     

Simultaneous determination of 1H-1H and 1H-13C residual dipolar couplings in a chiral liquid crystal solvent using a natural abundance HSQC experiment

A high-resolution, phase-sensitive, natural abundance F2-coupled 1H-13C HSQC (F2HSQC) NMR experiment was developed to measure simultaneously both (n)D(HH) and 1D(CH) residual dipolar couplings (RDCs) of small molecules present in a chiral polypeptide liquid crystal solvent system composed of poly-gamma-benzyl-L-glutamate (PBLG) in CDCl3. Because this is an indirect-detection NMR experiment, the relatively small amount of sample (7.5 mg in this study) and short acquisition times (5 h) that are required make this HSQC experiment well suited for samples that are either limited in solubility or in quantity or require short analysis times. The F2HSQC experiment can be performed without any specialized equipment or sample modification and can enhance our ability to measure RDCs accurately and rapidly in polypeptide liquid crystal solvents.     

Comparison of aqueous molecular dynamics with NMR relaxation and residual dipolar couplings favors internal motion in a mannose oligosaccharide.

An investigation has been performed to assess how aqueous dynamical simulations of flexible molecules can be compared against NMR data. The methodology compares state-of-the-art NMR data (residual dipolar coupling, NOESY, and (13)C relaxation) to molecular dynamics simulations in water over several nanoseconds. In contrast to many previous applications of residual dipolar coupling in structure investigations of biomolecules, the approach described here uses molecular dynamics simulations to provide a dynamic representation of the molecule. A mannose pentasaccharide, alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->2)-D-Manp, was chosen as the model compound for this study. The presence of alpha-linked mannan is common to many glycopeptides, and therefore an understanding of the structure and the dynamics of this molecule is of both chemical and biological importance. This paper sets out to address the following questions. (1) Are the single structures which have been used to interpret residual dipolar couplings a useful representation of this molecule? (2) If dynamic flexibility is included in a representation of the molecule, can relaxation and residual dipolar coupling data then be simultaneously satisfied? (3) Do aqueous molecular dynamics simulations provide a reasonable representation of the dynamics present in the molecule and its interaction with water? In summary, two aqueous molecular dynamics simulations, each of 20 ns, were computed. They were started from two distant conformations and both converged to one flexible ensemble. The measured residual dipolar couplings were in agreement with predictions made by averaging the whole ensemble and from a specific single structure selected from the ensemble. However, the inclusion of internal motion was necessary to rationalize the relaxation data. Therefore, it is proposed that although residual dipolar couplings can be interpreted as a single-structure, this may not be a correct interpretation of molecular conformation in light of other experimental data. Second, the methodology described here shows that the ensembles from aqueous molecular dynamics can be effectively tested against experimental data sets. In the simulation, significant conformational motion was observed at each of the linkages, and no evidence for intramolecular hydrogen bonds at either alpha(1-->2) or alpha(1-->3) linkages was found. This is in contrast to simulations of other linkages, such as beta(1-->4), which are often predicted to maintain intramolecular hydrogen bonds and are coincidentally predicted to have less conformational freedom in solution.     

Simultaneous and accurate determination of one-bond (15)N-(13)C' and two-bond (1)H(N)--(13)C' dipolar couplings

Using the echo-anti-echo manipulation, the 15N-1HN cross-peaks split in the E.COSY spectrum by the 13CO couplings are separated into different, distinct regions in the HSQC spectrum. From this novel E.COSY 15N-1HN HSQC spectrum, the small one-bond 15N-13C' and two-bond 1HN-13C' residual dipolar couplings can be extracted easily and accurately. These dipolar couplings provide a set of important long-range constraints for protein structure determination.     

Direct prediction of residual dipolar couplings of small molecules in a stretched gel by stochastic molecular dynamics simulations

Residual dipolar couplings are highly useful NMR parameters for calculating and refining molecular structures, dynamics, and interactions. For some applications, however, it is inevitable that the preferred orientation of a molecule in an alignment medium is calculated a priori. Several methods have been developed to predict molecular orientations and residual dipolar couplings. Being beneficial for macromolecules and selected small‐molecule applications, such approaches lack sufficient accuracy for a large number of organic compounds for which the fine structure and eventually the flexibility of all involved molecules have to be considered or are limited to specific, well‐studied liquid crystals. We introduce a simplified model for detailed all‐atom molecular dynamics calculations with a polymer strand lined up along the principal axis as a new approach to simulate the preferred orientation of small to medium‐sized solutes in polymer‐based, gel‐type alignment media. As is shown by a first example of strychnine in a polystyrene/CDCl₃ gel, the simulations potentially enable the accurate prediction of residual dipolar couplings taking into account structural details and dynamic averaging effects of both the polymer and the solute. Copyright © 2015 John Wiley & Sons, Ltd.     

Side chain orientation from methyl 1H-1H residual dipolar couplings measured in highly deuterated proteins

High-level deuteration is a prerequisite for the study of high molecular weight systems using liquid-state NMR. Here, we present new experiments for the measurement of proton-proton dipolar couplings in CH(2)D methyl groups of (13)C labeled, highly deuterated (70-80%) proteins. (1)H-(1)H residual dipolar couplings (RDCs) have been measured in two alignment media for 57 out of 70 possible methyl containing residues in the 167-residue flavodoxin-like domain of the E. coli sulfite reductase. These data yield information on the orientation of the methyl symmetry axis with respect to the molecular alignment frame. The alignment tensor characteristics were obtained very accurately from a set of backbone RDCs measured on the same protein sample. To demonstrate that accurate structural information is obtained from these data, the measured methyl RDCs for Valine residues are analyzed in terms of chi(1) torsion angles and stereospecific assignment of the prochiral methyl groups. On the basis of the previously determined backbone solution structure of this protein, the methyl RDC data proved sufficient to determine the chi(1) torsion angles in seven out of nine valines, assuming a single-rotamer model. Methyl RDCs are complementary to other NMR data, for example, methyl-methyl NOE, to determine side chain conformation in high molecular weight systems.     

13C-labeled platinum(IV)-carbohydrate complexes: structure determination based on (1)H-(1)H, (13)C-(1)H, and (13)C-(13)C spin-spin coupling constants

The reaction of D-mannose and D-allose with [PtMe(3)(Me(2)CO)(3)]BF(4) 1 in acetone affords complexes [PtMe(3)L]BF(4) 5 and 6 (5, L = alpha-D-mannofuranose; 6, L = beta-D-allofuranose). The coordination mode and conformation of the carbohydrate ligands in 5 and 6 in acetone-d(6) have been determined from an analysis of J(HH), J(CH), and J(CC) in complexes formed using site-specific (13)C-labeled D-mannose and D-allose. These coupling data are compared to those measured in (13)C-labeled complex [PtMe(3)L]BF(4) 2 (L = 1, 2-O-isopropylidene-alpha-D-glucofuranose) and 1, 2-O-isopropylidene-alpha-D-glucofuranose 3, whose solid-state structures are known, and in (13)C-labeled 1,2;5, 6-di-O-isopropylidene-alpha-D-glucofuranose 4. The preferred furanose ring conformations in 2 and 5 are very similar ((3)E/E(4) and E(4)/(o)E/E(1), respectively; eastern hemisphere of the pseudorotational itinerary), with platinum coordination involving O3, O5, and O6 of the saccharide. In contrast, the furanose ring of 6 prefers an (4)E/E(o)/(1)E geometry (western hemisphere of the pseudorotational itinerary) resulting from altered complexation involving O1, O5, and O6. Couplings within the exocyclic fragments of 2, 5, and 6 also support the existence of two different platinum coordination modes. In addition to establishing the structures and conformations of 2, 5, and 6 in solution, one-, two-, and three-bond J(CH) and J(CC) observed in these complexes provide new insights into the effect of structure and conformation on the magnitudes of these couplings in saccharides. Weak platinum(IV) complexation with the carbohydrate conformationally restricts the furanose and exocyclic fragment without introducing undesirable structural strain, thereby allowing more reliable correlations between structure and coupling magnitude.     

De novo determination of bond orientations and order parameters from residual dipolar couplings with high accuracy

We report the de novo determination of 15N-1H bond orientations and motional order parameters for the protein ubiquitin with high accuracy based solely on NMR residual dipolar coupling measurements made in six distinct alignment media. The resulting bond orientations are in agreement with RDC-refined orientations of either solid or solution state coordinates to within approximately 2 degrees , which is also the estimated precision of the resulting orientations. The squared generalized order parameters, which reflect amplitudes of motion spanning the picosecond to millisecond time scales, exhibit a correlation with picosecond time scale order parameters derived from conventional NMR 15N spin relaxation methods. Provided that RDC measurements can be obtained using many different alignment media, this approach (called direct interpretation of dipolar couplings) may significantly impact the attainable accuracy and the molecular weight range accessible to NMR structure determination in the solution state, as well as provide a route for the study of motions occurring on the nanosecond to microsecond time scales, which have been traditionally difficult to study at atomic resolution.