PITANSEMA-MAS, a solid-state NMR method to measure heteronuclear dipolar couplings under MAS

A 2D NMR method is presented for the measurement of the dipole-dipole interaction between a proton and a low-frequency nuclear spin species in the solid state under the magic angle spinning. It employs the time averaged nutation concept to dramatically reduce the required radio frequency (rf) power on the low γ nuclear channel and spin exchange at the magic angle is used to suppress (1)H-(1)H dipolar interactions and chemical shifts. The flexibility in choosing the spinning speed, rf power and the scaling factor of the pulse sequence are of considerable importance for the structural studies of biological solids. The performance of the pulse sequence has been numerically and experimentally demonstrated on several solids.     

13C direct detected NMR increases the detectability of residual dipolar couplings

13C direct detection is becoming an increasingly efficient approach to identify signals of residues that escape detection in 1H detected experiments. Pulse sequences have been developed to obtain 1H partially recoupled experiments for the measurement of the 1JHalphaCalpha and 1JHN couplings with the same resolution available in conventional 1H detected experiments. A consistent set of backbone rdc obtained without any 1H-based experiment has been obtained and shown to be effective for protein solution structure determination.     

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.     

Determination of the structures of symmetric protein oligomers from NMR chemical shifts and residual dipolar couplings

Symmetric protein dimers, trimers, and higher-order cyclic oligomers play key roles in many biological processes. However, structural studies of oligomeric systems by solution NMR can be difficult due to slow tumbling of the system and the difficulty in identifying NOE interactions across protein interfaces. Here, we present an automated method (RosettaOligomers) for determining the solution structures of oligomeric systems using only chemical shifts, sparse NOEs, and domain orientation restraints from residual dipolar couplings (RDCs) without a need for a previously determined structure of the monomeric subunit. The method integrates previously developed Rosetta protocols for solving the structures of monomeric proteins using sparse NMR data and for predicting the structures of both nonintertwined and intertwined symmetric oligomers. We illustrated the performance of the method using a benchmark set of nine protein dimers, one trimer, and one tetramer with available experimental data and various interface topologies. The final converged structures are found to be in good agreement with both experimental data and previously published high-resolution structures. The new approach is more readily applicable to large oligomeric systems than conventional structure-determination protocols, which often require a large number of NOEs, and will likely become increasingly relevant as more high-molecular weight systems are studied by NMR.     

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.     

An alternative method for pucker determination in carbohydrates from residual dipolar couplings: a solution NMR study of the fructofuranosyl ring of sucrose

A simple solution NMR method is presented for pucker determination of five-membered rings using only residual dipolar couplings obtained in a single liquid crystalline medium, DMPC/DHPC bicelles (DMPC = dimyristoylphosphatidylcholine; DHPC = dihexanoylphosphatidylcholine). The method was applied to determine the pucker of the fructofuranosyl ring of sucrose. The results indicate a fructofuranosyl pucker phase in the 20 degrees - 70 degrees range. The pucker phases are in agreement with those from previous NMR and optical spectroscopic studies and, importantly, do not rely on empirically parametrized Karplus curves. Furthermore, the analysis implies more than one stable pucker phase and rapid ring interconversion in this range. The present results suggest that using residual dipolar couplings alone can reveal multiple conformations present in solution. Hence, when a sufficient number of residual dipolar coupling constants is measured, the outcome is a robust, reliable, and independent route for determining carbohydrate and nucleic acid structure by NMR spectroscopy.     

On the interpretation of residual dipolar couplings as reporters of molecular dynamics

The analysis of residual dipolar couplings from an ensemble of conformations to extract molecular dynamics is intricate. The very mechanism that is necessary to perturb overall molecular tumbling to generate nonvanishing residual dipolar couplings gives rise to convoluted data. The measured values are essentially weighted averages over conformations. However, the weights are not simply the populations of conformations. Consequently, the observed order parameter is not exactly the true measure of motion. In the case of paramagnetic alignment, the apparent order parameter is expected to depend on the number of torsions that separate the locus of interest from the paramagnetic site. In the case of alignment due to steric obstruction, the uneven selection of conformations by their differing Saupe order matrices leads to a bias in the residual dipolar couplings-probed molecular dynamics.     

Lanthanide-binding peptides for NMR measurements of residual dipolar couplings and paramagnetic effects from multiple angles

Lanthanide-binding peptide tags (LBTs) containing a single cysteine residue can be attached to proteins via a disulfide bond, presenting a flexible means of tagging proteins site-specifically with a lanthanide ion. Here we show that cysteine residues placed in different positions of the LBT can be used to expose the protein to different orientations of the magnetic susceptibility anisotropy (delta chi) tensor and to generate different molecular alignments in a magnetic field. Delta chi tensors determined by nuclear magnetic resonance (NMR) spectroscopy for LBT complexes with Yb3+, Tm3+, and Er3+ suggest a rational way of producing alignment tensors with different orientations. In addition, knowledge of the delta chi tensor of LBT allows modeling of the protein-LBT structures. Despite evidence for residual mobility of the LBTs with respect to the protein, the pseudocontact shifts and residual dipolar couplings displayed by proteins disulfide-bonded to LBTs are greater than those achievable with most other lanthanide binding tags.     

Molecular conformations in a phospholipid bilayer extracted from dipolar couplings: a computer simulation study

This paper describes an analysis of NMR dipolar couplings in a bilayer formed by dimyristoylphosphatidylcholine (DMPC). The couplings are calculated from a trajectory generated in a molecular dynamics (MD) simulation based on a realistic atom-atom interaction potential. The analysis is carried out employing a recently developed approach that focuses on the construction of the conformational distribution function. This approach is a combination of two models, the additive potential (AP) model and the maximum entropy (ME) method, and is therefore called APME. In contrast to the AP model, the APME procedure does not require an intuition-based choice of the functional form of the torsional potential and is, unlike the ME method, applicable to weakly ordered systems. The conformational distribution function for the glycerol moiety of the DMPC molecule derived from the APME analysis of the dipolar couplings is in reasonable agreement with the "true" distributions calculated from the trajectory. Analyses of dipolar couplings derived from MD trajectories can, in general, serve as guidelines for experimental investigations of bilayers and other complex biological systems.     

1H-1H dipolar couplings provide a unique probe of RNA backbone structure

A NMR method is described that permits simultaneous measurement of the geminal 2JH1H2 + 2DH1H2 splitting and the sum of the 1JCH1 + 1DCH1 + 1JCH2 + 1DCH2 couplings for methylene groups, where 2DH1H2 and 1DCH are residual dipolar couplings, occurring when molecules are weakly oriented relative to the magnetic field. By suppressing either the upfield or downfield half of the 1H-1H geminal doublet, the experiment yields improved resolution relative to regular two-dimensional 1H-13C correlation spectra, making it applicable to systems of considerable complexity. The method is demonstrated for measurement of all 2DH5'H5' couplings in a 24-nucleotide 13C-enriched RNA stem loop structure, weakly aligned in liquid crystalline Pf1. The method is equally applicable to methylene groups in 13C-labeled proteins and to natural abundance samples of smaller molecules.     

Composite alignment media for the measurement of independent sets of NMR residual dipolar couplings

The measurement of independent sets of NMR residual dipolar couplings (RDCs) in multiple alignment media can provide a detailed view of biomolecular structure and dynamics, yet remains experimentally challenging. It is demonstrated here that independent sets of RDCs can be measured for ubiquitin using just a single alignment medium composed of aligned bacteriophage Pf1 particles embedded in a strained polyacrylamide gel matrix. Using this composite medium, molecular alignment can be modulated by varying the angle between the directors of ordering for the Pf1 and strained gel matrix, or by varying the ionic strength or concentration of the Pf1 particles. This approach offers significant advantages in that greater experimental control can be exercised over the acquisition of multi-alignment RDC data while a homogeneous chemical environment is maintained across all of the measured RDC data.     

On the origin of residual dipolar couplings from denatured proteins

Effects of steric obstruction on random flight chains are examined. Spatial probability distributions are elaborated to calculate residual dipolar couplings and residual chemical shift anisotropy, parameters that are acquired by NMR spectroscopy from solutes dissolved in dilute liquid crystals. Calculations yield chain length and residue position-dependent values in good agreement with simulations to provide understanding of recently acquired data from denatured proteins.     

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.     

A novel approach to the retrieval of structural and dynamic information from residual dipolar couplings using several oriented media in biomolecular NMR spectroscopy

The interpretation of residual dipolar couplings in terms of molecular properties of interest is complicated because of difficulties in separating structural and dynamic effects as well as the need to estimate alignment tensor parameters a priori. An approach is introduced here that allows many of these difficulties to be circumvented when data are acquired in multiple alignment media. The method allows the simultaneous extraction of both structural and dynamic information directly from the residual dipolar coupling data, in favorable cases even in the complete absence of prior structural knowledge. Application to the protein ubiquitin indicates greater amplitudes of internal motion than expected from traditional (15)N spin relaxation analysis.     

Intrinsic information content of NMR dipolar couplings: a conformational investigation of 1,3-butadiene in a nematic phase.

The conformational equilibrium of 1,3-butadiene in a condensed fluid phase is investigated by liquid-crystal NMR spectroscopy. The full set of D(HH) and D(CH) dipolar couplings is determined from the analysis of the (1)H spectra of the three 1,3-butadiene most-abundant isotopomers (i.e. the all (12)C and the two single-labeled (13)C isotopomers) for a total of 21 independent dipolar couplings. A very good starting set of spectral parameters for the analysis of the (1)H spectrum is determined in a semiautomated way by the analysis of the (N-1) (specifically, N=6, the number of 1/2 spin nuclei in the spin system) quantum refocused (5QR), and not (5Q), spectra. As an alternative approach, a Monte Carlo (MC) numerical simulation, capable of predicting the solute ordering, is tested to simulate the 5QR spectrum. The set of D(ij) couplings is very good, proving that the MC method can represent a novel, valid alternative to the existing spectral simplification procedures. The experimentally determined dipolar-coupling data set is fully compatible with the 1,3-butadiene conformational distribution reported in the literature for isolated molecules, indicating the presence of about 99 % of s-trans conformer. With regards to the remaining 1 %, in spite of the direct and very strong dependence of the observables on the molecular structure, it was not possible to discriminate between the planar s-cis and s-gauche forms, both of which produce a very good fit of the dipolar couplings. Vibrational corrections, up to the anharmonic term, were applied; the calculated geometrical parameters are in good- although not exact-agreement with those reported in the literature from experimental and theoretical investigations. This result can be considered as supporting the methodology used for obtaining the structure and conformational distribution of a flexible molecule in a liquid phase.