Periodicity in residual dipolar couplings and nucleic acid structures

The periodicity in nucleic acid duplex structures is shown to be correlated to the periodicity in residual dipolar couplings (RDCs) in the form of an "RDC wave". This "RDC wave" is characteristic of the alignment of the duplex in the magnetic field, and hence fitting of the data allows the duplex global orientation (, Phi) to be extracted. Further, because the "RDC wave" is fit as a data set of a corresponding secondary structure element, the degeneracy problem is greatly reduced. Consequently, with the global orientation (, Phi) determined, local bond vector conformations are defined. The fit is demonstrated in the examples of the imino RDCs of the negative regulator of splicing RNA fragment (NRS23) and for the C1'H1' RDCs of the Dickerson dodecamer.     

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.     

Searching and optimizing structure ensembles for complex flexible sugars

NMR restrictions are suitable to specify the geometry of a molecule when a single well-defined global free energy minimum exists that is significantly lower than other local minima. Carbohydrates are quite flexible, and therefore, NMR observables do not always correlate with a single conformer but instead with an ensemble of low free energy conformers that can be accessed by thermal fluctuations. In this communication, we describe a novel procedure to identify and weight the contribution to the ensemble of local minima conformers based on comparison to residual dipolar couplings (RDCs) or other NMR observables, such as scalar couplings. A genetic algorithm is implemented to globally minimize the R factor comparing calculated RDCs to experiment. This is done by optimizing the weights of different conformers derived from the exhaustive local minima conformational search program, fast sugar structure prediction software (FSPS). We apply this framework to six human milk sugars, LND-1, LNF-1, LNF-2, LNF-3, LNnT, and LNT, and are able to determine corresponding population weights for the ensemble of conformers. Interestingly, our results indicate that in all cases the RDCs can be well represented by only a few most important conformers. This confirms that several, but not all of the glycosidic linkages in histo-blood group "epitopes" are quite rigid.     

Computer‐Assisted 3D Structure Elucidation of Natural Products using Residual Dipolar Couplings

An enhanced computer‐assisted procedure for the determination of the relative configuration of natural products, which starts from the molecular formula and uses a combination of conventional 1D and 2D NMR spectra, and residual dipolar couplings (RDCs), is reported. Having already the data acquired (1D/2D NMR and RDCs), the procedure begins with the determination of the molecular constitution using standard computer‐assisted structure elucidation (CASE) and is followed by fully automated determination of relative configuration through RDC analysis. In the case of moderately flexible molecules the simplest data‐explaining conformational model is selected by the use of the Akaike information criterion.     

Paramagnetic-based NMR restraints lift residual dipolar coupling degeneracy in multidomain detergent-solubilized membrane proteins

Residual dipolar couplings (RDCs) are widely used as orientation-dependent NMR restraints to improve the resolution of the NMR conformational ensemble of biomacromolecules and define the relative orientation of multidomain proteins and protein complexes. However, the interpretation of RDCs is complicated by the intrinsic degeneracy of analytical solutions and protein dynamics that lead to ill-defined orientations of the structural domains (ghost orientations). Here, we illustrate how restraints from paramagnetic relaxation enhancement (PRE) experiments lift the orientational ambiguity of multidomain membrane proteins solubilized in detergent micelles. We tested this approach on monomeric phospholamban (PLN), a 52-residue membrane protein, which is composed of two helical domains connected by a flexible loop. We show that the combination of classical solution NMR restraints (NOEs and dihedral angles) with RDC and PRE constraints resolves topological ambiguities, improving the convergence of the PLN structural ensemble and giving the depth of insertion of the protein within the micelle. The combination of RDCs with PREs will be necessary for improving the accuracy and precision of membrane protein conformational ensembles, where three-dimensional structures are dictated by interactions with the membrane-mimicking environment rather than compact tertiary folds common in globular proteins.     

Mapping the potential energy landscape of intrinsically disordered proteins at amino Acid resolution

Intrinsically disordered regions are predicted to exist in a significant fraction of proteins encoded in eukaryotic genomes. The high levels of conformational plasticity of this class of proteins endows them with unique capacities to act in functional modes not achievable by folded proteins, but also places their molecular characterization beyond the reach of classical structural biology. New techniques are therefore required to understand the relationship between primary sequence and biological function in this class of proteins. Although dependences of some NMR parameters such as chemical shifts (CSs) or residual dipolar couplings (RDCs) on structural propensity are known, so that sampling regimes are often inferred from experimental observation, there is currently no framework that allows for a statistical mapping of the available Ramachandran space of each amino acid in terms of conformational propensity. In this study we develop such an approach, combining highly efficient conformational sampling with ensemble selection to map the backbone conformational sampling of IDPs on a residue specific level. By systematically analyzing the ability of NMR data to map the conformational landscape of disordered proteins, we identify combinations of RDCs and CSs that can be used to raise conformational degeneracies inherent to different data types, and apply these approaches to characterize the conformational behavior of two intrinsically disordered proteins, the K18 domain from Tau protein and N(TAIL) from measles virus nucleoprotein. In both cases, we identify the enhanced populations of turn and helical regions in key regions of the proteins, as well as contiguous strands that show clear and enhanced polyproline II sampling.     

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.     

Complete relative stereochemistry of multiple stereocenters using only residual dipolar couplings

Residual dipolar couplings (RDCs), in combination with molecular order matrix calculations, were used to unambiguously determine the complete relative stereochemistry of an organic compound with five stereocenters. Three simple one-dimensional experiments were utilized for the measurements of (13)C-(1)H, (13)C-(19)F, (19)F-(1)H, and (1)H-(1)H RDCs. The order matrix calculation was performed on each chiral isomer independently. The fits were evaluated by the comparison of the root-mean-square deviation (rmsd) of calculated and measured RDCs. The order tensor simulations based on two different sets of RDC data collected with phage and bicelles are consistent. The resulting stereochemical assignments of the stereocenters obtained from using only RDCs are in perfect agreement with those obtained from the single-crystal X-ray structure. Six RDCs are found to be necessary to run the simulation, and seven are the minimum to get an acceptable result for the investigated compound. It was also shown that (13)C-(1)H and (1)H-(1)H RDCs, which are the easiest to measure, are also the most important and information-rich data for the order matrix calculation. The effect of each RDC on the calculation depends on the location of the corresponding vector in the structure. The direct RDC of a stereocenter is important to the configuration determination, but the configuration of stereocenters devoid of protons can also be obtained from analysis of nearby RDCs.     

Determination of Configuration and Conformation of a Reserpine Derivative with Seven Stereogenic Centers Using Molecular Dynamics with RDC-Derived Tensorial Constraints**

NMR-based determination of the configuration of complex molecules containing many stereocenters is often not possible using traditional NOE data and coupling patterns. Making use of residual dipolar couplings (RDCs), we were able to determine the relative configuration of a natural product containing seven stereocenters, including a chiral amine lacking direct RDC data. To identify the correct relative configuration out of 32 possible ones, experimental RDCs were used in three different approaches for data interpretation: by fitting experimental data based singular value decomposition (SVD) using a single alignment tensor and either (i) a single conformer or (ii) multiple conformers, or alternatively (iii) using molecular dynamics simulations with tensorial orientational constraints (MDOC). Even though in all three approaches one and the same configuration could be selected and clear discrimination between possible configurations was achieved, the experimental data was not fully satisfied by the methods based on single tensor approaches. While these two approaches are faster, only MDOC is able to fully reproduce experimental results, as the obtained conformational ensemble adequately covers the conformational space necessary to describe the molecule with inherent flexibility.     

Multiple alignment tensors from a denatured protein

The structural content of the denatured state has yet to be fully characterized. In recent years, large residual dipolar couplings (RDCs) from denatured proteins have been observed under alignment conditions produced by bicelles and strained polyacrylamide gels. In this report, we describe efforts to extend our picture of the residual structure in denatured nuclease by measuring RDCs with multiple alignment tensors. Backbone amide 15N-1H RDCs were collected from 4 M urea for a total of eight RDC data sets. The RDCs were analyzed by singular value decomposition (SVD) to determine the number of independent alignment tensors present in the data. On the basis of the resultant singular values and propagated error estimates, it is clear that there are at least three independent alignment tensors. These three independent RDC datasets can be reconstituted as orthogonal linear combinations, (OLC)-RDC datasets, of the eight actually recorded. The first, second, and third OLC-RDC datasets are highly robust to the removal of any single experimental RDC dataset, establishing the presence of three independent alignment tensors, sampled well above the level of experimental uncertainty. The observation that the RDC data span three or more dimensions of the five-dimensional parameter space demonstrates that the ensemble average structure of denatured nuclease must be asymmetric with respect to these three orthogonal principal axes, which is not inconsistent with earlier work demonstrating that it has a nativelike topology.     

The use of residual dipolar coupling for conformational analysis of structurally related natural products

Determining the conformational preferences of molecules in solution remains a considerable challenge. Recently, the use of residual dipolar coupling (RDC) analysis has emerged as a key method to address this. Whilst to date the majority of the applications have focused on biomolecules including proteins and DNA, the use of RDCs for studying small molecules is gaining popularity. Having said that, the method continues to develop, and here, we describe an early case study of the quantification of conformer populations in small molecules using RDC analysis. Having been inspired to study conformational preferences by unexpected differences in the NMR spectra and the reactivity of related natural products, we showed that the use of more established techniques was unsatisfactory in explaining the experimental observations. The use of RDCs provided an improved understanding that, following use of methods to quantify conformer populations using RDCs, culminated in a rationalisation of the contrasting diastereoselectivities observed in a ketone reduction reaction. Copyright © 2015 John Wiley & Sons, Ltd.     

Long‐Range Residual Dipolar Couplings: A Tool for Determining the Configuration of Small Molecules

Together with NOE and J coupling, one‐bond residual dipolar coupling (RDC), which reports on the three‐dimensional orientation of an internuclear vector in the molecular frame, plays an important role in the conformation and configuration analysis of small molecules in solution by NMR spectroscopy. When the molecule has few C? H bonds, or too many bonds are in parallel, the available RDCs may not be sufficient to obtain the alignment tensor used for structure elucidation. Long‐range RDCs that connect nuclei over multiple bonds are normally not parallel to the single bonds and therefore complement one‐bond RDCs. Herein we present a method for extracting the long‐range RDC of a chosen proton or group of protons to all remotely connected carbon atoms, including non‐protonated carbon atoms. Alignment tensors fitted directly to the total long‐range couplings (T=J+D) enabled straightforward analysis of both the long‐range and one‐bond RDCs for strychnine.     

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.     

Calculation of residual dipolar couplings from disordered state ensembles using local alignment

Residual dipolar couplings (RDCs) have been observed in disordered states of several proteins. While their nonuniform values were initially surprising, it has been shown that reasonable approximation of experimental RDCs can be obtained using simple statistical coil models and assuming global alignment of each structure, provided that many thousands of conformers are averaged. Here we show that, by using short local alignment tensors, we can achieve good agreement between experimental and simulated RDCs with far fewer structures than required when using global alignment. This makes the possibility of using RDCs as direct restraints in structural calculations of disordered proteins much more feasible. In addition, it provides insight into the nature of RDCs in disordered states, suggesting that they are primarily reporting on local structure.     

Residual Dipolar-Coupling-Based Conformational Comparison of Noncovalent Ubiquitin Homodimer with Covalently Linked Diubiquitin

Although the conformation of the polymer chain of Ubiquitin (Ub) mainly depends on the type of isopeptide linkage connecting two Ub molecules, the non-covalent (noncovalent) interaction between two Ub molecules within the chain could also tune their conformational preference. Here, we studied the conformation of noncovalently formed Ub dimers in solution using residual dipolar couplings (RDCs). Comparing the RDC derived alignment tensor of the noncovalently formed dimer with the two most abundant (K11 and K48) covalent linked Ub dimers revealed that the conformation of K11 linked and noncovalent Ub dimers were similar. Between the various NMR and crystal structures of K11 linked Ub dimers, RDC tensor analysis showed that the structure of K11 linked dimer crystalized at neutral pH is similar to noncovalent dimer. Analogous to the experimental study, the comparison of predicted order matrix of various covalent Ub dimers with that of the experimentally determined order matrix of noncovalent Ub dimer also suggests that the conformation of K11 linked dimers crystalized at neutral pH is similar to the noncovalent dimer.     

De novo determination of protein backbone structure from residual dipolar couplings using Rosetta

As genome-sequencing projects rapidly increase the database of protein sequences, the gap between known sequences and known structures continues to grow exponentially, increasing the demand to accelerate structure determination methods. Residual dipolar couplings (RDCs) are an attractive source of experimental restraints for NMR structure determination, particularly rapid, high-throughput methods, because they yield both local and long-range orientational information and can be easily measured and assigned once the backbone resonances of a protein have been assigned. While very extensive RDC data sets have been used to determine the structure of ubiquitin, it is unclear to what extent such methods will generalize to larger proteins with less complete data sets. Here we incorporate experimental RDC restraints into Rosetta, an ab initio structure prediction method, and demonstrate that the combined algorithm provides a general method for de novo determination of a variety of protein folds from RDC data. Backbone structures for multiple proteins up to approximately 125 residues in length and spanning a range of topological complexities are rapidly and reproducibly generated using data sets that are insufficient in isolation to uniquely determine the protein fold de novo, although ambiguities and errors are observed for proteins with symmetry about an axis of the alignment tensor. The models generated are not high-resolution structures completely defined by experimental data but are sufficiently accurate to accelerate traditional high-resolution NMR structure determination and provide structure-based functional insights.     

RDC‐enhanced structure calculation of a β‐heptapeptide in methanol

Residual dipolar couplings (RDCs) are a rich source of structural information that goes beyond the range covered by the nuclear Overhauser effect or scalar coupling constants. They can only be measured in partially oriented samples. RDC studies of peptides in organic solvents have so far been focused on samples in chloroform or DMSO. Here, we show that stretched poly(vinyl acetate) can be used for the partial alignment of a linear β‐peptide with proteinogenic side chains in methanol. ¹D_CH, ¹D_NH, and ²D_HH RDCs were collected with this sample and included as restraints in a simulated annealing calculation. Incorporation of RDCs in the structure calculation process improves the long‐range definition in the backbone of the resulting 3₁₄‐helix and uncovers side‐chain mobility. Experimental side‐chain RDCs of the central leucine and valine residues are in good agreement with predicted values from a local three‐state model. Copyright © 2016 John Wiley & Sons, Ltd.