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.     

Increasing the accuracy of solution NMR structures of membrane proteins by application of residual dipolar couplings. High-resolution structure of outer membrane protein A

The structure determination of membrane proteins is one of the most challenging applications of solution NMR spectroscopy. The paucity of distance information available from the highly deuterated proteins employed requires new approaches in structure determination. Here we demonstrate that significant improvement in the structure accuracy of the membrane protein OmpA can be achieved by refinement with residual dipolar couplings (RDCs). The application of charged polyacrylamide gels allowed us to obtain two alignments and accurately measure numerous heteronuclear dipolar couplings. Furthermore, we have demonstrated that using a large set of RDCs in the refinement can yield a structure with 1 A rms deviation to the backbone of the high-resolution crystal structure. Our simulations with various data sets indicate that dipolar couplings will be critical for obtaining accurate structures of membrane proteins.     

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.     

Dipolar waves as NMR maps of protein structure

The anisotropy of nuclear spin interactions results in a unique mapping of structure to the resonance frequencies and split tings observed in NMR spectra, however, the determination of molecular structure from experimentally measured spectral parameters is complicated by angular ambiguities resulting from the symmetry properties of dipole-dipole and chemical shift interactions. This issue can be addressed through the periodicity inherent in secondary structure elements, which can be used as an index of topology. Distinctive wheel-like patterns are observed in two-dimensional 1H-15N heteronuclear dipolar/15N chemical shift PISEMA (polarization inversion spin-exchange at the magic angle) spectra of helical membrane proteins in highly aligned lipid bilayer samples. One-dimensional dipolar waves are an extension of two-dimensional PISA (polarity index slant angle) wheels to map protein structure in NMR spectra of both highly and weakly aligned samples. Dipolar waves describe the periodic wavelike variations of the magnitudes of the static heteronuclear dipolar couplings as a function of residue number in the absence of chemical shift effects. Weakly aligned samples of proteins display these same effects, primarily as residual dipolar couplings (RDCs), in solution NMR spectra. The corresponding properties of the RDCs in solution NMR spectra of weakly aligned helices represent a convergence of solid-state and solution NMR approaches to structure determination.     

Self-consistency analysis of dipolar couplings in multiple alignments of ubiquitin

A self-consistency analysis of backbone N-H residual dipolar couplings of ubiquitin collected in 10 different media is described to assess the degree of structural and dynamic heterogeneous behavior across the media. The SECONDA method, which works with and without any structural or dynamic information about the molecular system, is based on a principal component analysis and is very sensitive to the presence of heterogeneities or experimental errors. It is found that the regular secondary structural elements behave highly homogeneously, while small heterogeneities are manifested in the loop region 51-63. Many residues that exhibit increased dynamics in NMR relaxation experiments are inert with respect to changes in the alignment.     

Simultaneous NMR study of protein structure and dynamics using conservative mutagenesis

A novel iterative procedure is described that allows both the orientation and dynamics of internuclear bond vectors to be determined from direct interpretation of NMR dipolar couplings, measured under at least three orthogonal alignment conditions. If five orthogonal alignments are available, the approach also yields information on the degree of motional anisotropy and the direction in which the largest amplitude internal motion of each bond vector takes place. The method is demonstrated for the backbone (15)N-(1)H, (13)C(alpha)-(1)H(alpha), and (13)C(alpha)-13C' interactions in the previously well-studied protein domain GB3, dissolved in a liquid crystalline suspension of filamentous phage Pf1. Alignment variation is achieved by using conservative mutations of charged surface residues. Results indicate remarkably uniform backbone dynamics, with amplitudes that agree well with those of previous (15)N relaxation studies for most residues involved in elements of secondary structure, but larger amplitude dynamics than those found by (15)N relaxation for residues in loop and turn regions. In agreement with a previous analysis of dipolar couplings, the N-H bonds in the second beta-strand, which is involved in antibody recognition, show elevated dynamics with largest amplitudes orthogonal to the chain direction.     

Protein structural motif recognition via NMR residual dipolar couplings.

NMR residual dipolar couplings have great potential to provide rapid structural information for proteins in the solution state. This information even at low resolution may be used to advantage in proteomics projects that seek to annotate large numbers of gene products for entire genomes. In this paper, we describe a novel approach to the structural interpretation of dipolar couplings which is based on structural motif pattern recognition, where a predefined gapless structural template for a motif is used to search a set of residual dipolar couplings for good matches. We demonstrate the applicability of the method using synthetic and experimental data. We also provide an analysis of the statistical power of the method and the effects of order tensor frame orientation, motif size, and structural complexity on motif detection. Finally, we discuss remaining problems that must be overcome before the method can be used routinely to identify protein homologies.     

A dipolar coupling based strategy for simultaneous resonance assignment and structure determination of protein backbones.

A new approach for simultaneous protein backbone resonance assignment and structure determination by NMR is introduced. This approach relies on recent advances in high-resolution NMR spectroscopy that allow observation of anisotropic interactions, such as dipolar couplings, from proteins partially aligned in field ordered media. Residual dipolar couplings are used for both geometric information and a filter in the assembly of residues in a sequential manner. Experimental data were collected in less than one week on a small redox protein, rubredoxin, that was 15N enriched but not enriched above 1% natural abundance in 13C. Given the acceleration possible with partial 13C enrichment, the protocol described should provide a very rapid route to protein structure determination. This is critical for the structural genomics initiative where protein expression and structural determination in a high-throughput manner will be needed.     

Limits on variations in protein backbone dynamics from precise measurements of scalar couplings

3JHN,Halpha, 3JHN,Cbeta, and 3JHN,C' couplings, all related to the backbone torsion angle phi, were measured for the third immunoglobulin binding domain of protein G, or GB3. Measurements were carried out using both previously published methods and novel sequences based on the multiple-quantum principle, which limit attenuation of experimental couplings caused by finite lifetimes of the spin states of passive spins. High reproducibility between the multiple-quantum and conventional approaches confirms the accuracy of the measurements. With few exceptions, close agreement between 3JHN,Halpha, 3JHN,Cbeta, and 3JHN,C' and values predicted by their respective Karplus equations is observed. For the three types of couplings, up to 20% better agreement is obtained when fitting the experimental couplings to a dynamic ensemble NMR structure, which has a phi angle root-mean-square spread of 9 +/- 4 degrees and was previously calculated on the basis of a very extensive set of residual dipolar couplings, than for any single static NMR structure. Fits of 3J couplings to a 1.1-A X-ray structure, with hydrogens added in idealized positions, are 40-90% worse. Approximately half of the improvement when fitting to the NMR structures relates to the amide proton deviating from its idealized, in-peptide-plane position, indicating that the positioning of hydrogens relative to the backbone atoms is one of the factors limiting the accuracy at which the backbone torsion angle phi can be extracted from 3J couplings. Introducing an additional, residue-specific variable for the amplitude of phi angle fluctuations does not yield a statistically significant improvement when fitting to a set of dynamic Karplus curves, pointing to a homogeneous behavior of these amplitudes.     

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.     

Structural and dynamic analysis of residual dipolar coupling data for proteins.

The measurement of residual dipolar couplings in weakly aligned proteins can potentially provide unique information on their structure and dynamics in the solution state. The challenge is to extract the information of interest from the measurements, which normally reflect a convolution of the structural and dynamic properties. We discuss here a formalism which allows a first order separation of their effects, and thus, a simultaneous extraction of structural and motional parameters from residual dipolar coupling data. We introduce some terminology, namely a generalized degree of order, which is necessary for a meaningful discussion of the effects of motion on residual dipolar coupling measurements. We also illustrate this new methodology using an extensive set of residual dipolar coupling measurements made on (15)N,(13)C-labeled human ubiquitin solvated in a dilute bicelle solution. Our results support a solution structure of ubiquitin which on average agrees well with the X-ray structure (Vijay-Kumar, et al., J. Mol. Biol. 1987, 194, 531--544) for the protein core. However, the data are also consistent with a dynamic model of ubiquitin, exhibiting variable amplitudes, and anisotropy, of internal motions. This work suggests the possibility of primary use of residual dipolar couplings in characterizing both structure and anisotropic internal motions of proteins in the solution state.     

NMR Structure Determination of Saccharose and Raffinose by Means of Homo‐ and Heteronuclear Dipolar Couplings

Residual dipolar couplings have dramatically improved the accuracy and precision of high‐resolution NMR structures during the last years. This was first demonstrated for proteins. In this article, we describe, with raffinose and saccharose as examples, that dipolar couplings improve the precision of structures of carbohydrates for which usually very few structural parameters are available. The relative orientation as well as the dynamics of the monosaccharide moieties with respect to each other can be determined with the help of ¹³C,¹H and ¹H,¹H dipolar couplings, which can easily be measured. Significant differences between the solution and the X‐ray crystal structure exist. These results indicate that residual dipolar‐coupling data may provide a more complete and dynamic model of carbohydrates in particular, and small molecules in general.     

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.     

Docking of protein-protein complexes on the basis of highly ambiguous intermolecular distance restraints derived from 1H/15N chemical shift mapping and backbone 15N-1H residual dipolar couplings using conjoined rigid body/torsion angle dynamics

A simple and reliable method for docking protein-protein complexes using (1)H(N)/(15)N chemical shift mapping and backbone (15)N-(1)H residual dipolar couplings is presented and illustrated with three complexes (EIN-HPr, IIA(Glc)-HPr, and IIA(Mtl)-HPr) of known structure. The (1)H(N)/(15)N chemical shift mapping data are transformed into a set of highly ambiguous, intermolecular distance restraints (comprising between 400 and 3000 individual distances) with translational and some degree of orientational information content, while the dipolar couplings provide information on relative protein-protein orientation. The optimization protocol employs conjoined rigid body/torsion angle dynamics in simulated annealing calculations. The target function also comprises three nonbonded interactions terms: a van der Waals repulsion term to prevent atomic overlap, a radius of gyration term (E(rgyr)) to avoid expansion at the protein-protein interface, and a torsion angle database potential of mean force to bias interfacial side chain conformations toward physically allowed rotamers. For the EIN-HPr and IIA(Glc)-HPr complexes, all structures satisfying the experimental restraints (i.e., both the ambiguous intermolecular distance restraints and the dipolar couplings) converge to a single cluster with mean backbone coordinate accuracies of 0.7-1.5 A. For the IIA(Mtl)-HPr complex, twofold degeneracy remains, and the structures cluster into two distinct solutions differing by a 180 degrees rotation about the z axis of the alignment tensor. The correct and incorrect solutions which have mean backbone coordinate accuracies of approximately 0.5 and approximately 10.5 A, respectively, can readily be distinguished using a variety of criteria: (a) examination of the overall (1)H(N)/(15)N chemical shift perturbation map (because the incorrect cluster predicts the presence of residues at the interface that experience only minimal chemical shift perturbations; this information is readily incorporated into the calculations in the form of ambiguous intermolecular repulsion restraints); (b) back-calculation of dipolar couplings on the basis of molecular shape; or (c) the E(rgyr) distribution which, because of its global nature, directly reflects the interfacial packing quality. This methodology should be particularly useful for high throughput, NMR-based, structural proteomics.     

Weak long-range correlated motions in a surface patch of ubiquitin involved in molecular recognition

Long-range correlated motions in proteins are candidate mechanisms for processes that require information transfer across protein structures, such as allostery and signal transduction. However, the observation of backbone correlations between distant residues has remained elusive, and only local correlations have been revealed using residual dipolar couplings measured by NMR spectroscopy. In this work, we experimentally identified and characterized collective motions spanning four β-strands separated by up to 15 ? in ubiquitin. The observed correlations link molecular recognition sites and result from concerted conformational changes that are in part mediated by the hydrogen-bonding network.     

Orientation of deoxyhemoglobin at high magnetic fields: structural insights from RDCs in solution

Human normal adult hemoglobin (Hb A) is a tetrameric protein molecule of ~64 kDa consisting of two identical -chains and two identical -chains of 141 and 146 amino acid residues each and four bound heme moieties. In the oxygen-free form of Hb A, also known as deoxyhemoglobin A (deoxy-Hb A), the hemes are paramagnetic with S = 2. We have measured the one-bond spin-spin couplings (1JNH + 1DNH) on (15N,2H)-labeled deoxy-Hb A in solution as a function of magnetic field strengths from 11.7 to 21.1 T and found that these couplings are linearly proportional to the square of the magnetic field. This field dependence provides an opportunity to extract the residual dipolar couplings (RDCs, 1DNH) and, thus, to compare predictions about the solution structure of deoxy-Hb A to crystal structures for this molecule. Such comparison is essential for our understanding of the structure, dynamics, and function of this allosteric protein under conditions close to the physiological state. This report illustrates the usefulness of using the magnetic-field dependent RDCs to determine the solution structure of a large paramagnetic protein. This method is especially valuable for those proteins whose structures must be determined in an oxygen-free environment.     

SESAME-HSQC for simultaneous measurement of NH and CH scalar and residual dipolar couplings

We present a novel pulse sequence, SESAME-HSQC, for the simultaneous measurement of several NH and CH scalar and residual dipolar couplings in double labeled proteins. The proposed Spin-statE Selective All Multiplicity Edited (SESAME)-HSQC combines gradient selected and sensitivity enhanced (15)N- and constant-time (13)C-HSQC experiments with the recently introduced spin-state selective method (Nolis et al., J. Magn. Reson. 180 (2006) 39-50) for measuring couplings simultaneously at amide and aliphatic regions. Excellent resolution and high sensitivity is warranted by removing all coupling interactions during the indirectly detected t(1) period, and by employing pulsed field gradients for coherence selection and utilizing coherence order selective spin-state selection. The scalar and residual dipolar couplings can be readily measured from a two-dimensional (15)N/(13)C-HSQC spectrum without additional spectral crowding. SESAME-HSQC can be used for epitope mapping by observing chemical shift changes in both amide and aliphatic regions. Simultaneously, potential conversion in protein conformation can be probed by analyzing changes in residual dipolar couplings induced by ligand binding. The pulse sequence is experimentally verified with a sample of (15)N/(13)C enriched human ubiquitin. The internuclear vector directions determined from the residual dipolar couplings are found to be in excellent correlation with those predicted from ubiquitin's refined solution structure.     

Protein backbone structure determination using RDC: An inverse kinematics approach with fast and exact solutions

Residual dipolar couplings (RDC) of proteins dissolved in anisotropic media promise to speed up the determination of protein structures. We consider the backbone as a robotic mechanism and formulate inverse kinematics problems using RDC restraints from two media. The φ, ψ of each secondary structure element (SSE) are computed from oriented vectors in consecutive peptide planes. We search for the optimum conformation joining the solutions of two independent backbone halves. The matrix transforming the vector Z of a global frame from one SSE into the other determines their orientation. Three distance constraints between two oriented SSE determine their relative position by solving nine polynomial equations. The benefit of this method is that complete and accurate solutions are obtained overcoming the local minima problems of heuristic procedures. The algorithm is implemented on MAPLE using the least number of experimental data; the runtimes take an order of seconds on a common PC. © 2013 Wiley Periodicals, Inc.     

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.