Residual dipolar couplings in nucleic acid structure determination

Solution NMR spectroscopy of nucleic acids has been limited by the short-range nature of the nuclear Overhauser effect and scalar coupling restraints normally used in structure determination. The addition of residual dipolar couplings, obtained from slightly oriented mixtures, provides bond vector angles relative to a universal alignment tensor. The accurate determination of helix curvature, domain orientation and the stoichiometry of homomultimeric nucleic acid complexes is now possible.     

NMR experiments for the sign determination of homonuclear scalar and residual dipolar couplings

A modified version of the JHH-TOCSY experiment, `signed COSY', is presented that allows the determination of the sign of residual dipolar ¹H-¹H coupling constants with respect to the sign of one-bond ¹H-X coupling constants in linear three-spin systems X-¹H-¹H, where X = ¹³C or ¹⁵N. In contrast to the original JHH-TOCSY experiments, the signs of J _HH couplings may be determined for CH₂-CH₂ moieties and for uniformly ¹³C/¹⁵N-labelled samples. In addition, sensitivity is enhanced, diagonal peaks are suppressed and cross peaks are observed only between directly coupled protons, as in a COSY spectrum.     

Detecting protein kinase recognition modes of calmodulin by residual dipolar couplings in solution NMR

Calmodulin-regulated serine/threonine kinases (CaM kinases) play crucial roles in Ca2+-dependent signaling transduction pathways in eukaryotes. Despite having a similar overall molecular architecture of catalytic and regulatory domains, CaM kinases employ different binding modes for Ca2+/CaM recruitment which is required for their activation. Here we present a residual dipolar coupling (RDC)-based NMR approach to characterizing the molecular recognition of CaM with five different CaM kinases. Our analyses indicate that CaM kinase I and likely IV use the same CaM binding mode as myosin light chain kinase (1-14 motif), distinct from those of CaM kinase II (1-10 motif) and CaM kinase kinase (1-16- motif). This NMR approach provides an efficient experimental guide for homology modeling and structural characterization of CaM-target complexes.     

Rapid determination of protein folds using residual dipolar couplings

Over the next few years, various genome projects will sequence many new genes and yield many new gene products. Many of these products will have no known function and little, if any, sequence homology to existing proteins. There is reason to believe that a rapid determination of a protein fold, even at low resolution, can aid in the identification of function and expedite the determination of structure at higher resolution. Recently devised NMR methods of measuring residual dipolar couplings provide one route to the determination of a fold. They do this by allowing the alignment of previously identified secondary structural elements with respect to each other. When combined with constraints involving loops connecting elements or other short-range experimental distance information, a fold is produced. We illustrate this approach to protein fold determination on (15)N-labeled Eschericia coli acyl carrier protein using a limited set of (15)N-(1)H and (1)H-(1)H dipolar couplings. We also illustrate an approach using a more extended set of heteronuclear couplings on a related protein, (13)C, (15)N-labeled NodF protein from Rhizobium leguminosarum.     

Complete protein structure determination using backbone residual dipolar couplings and sidechain rotamer prediction

Residual dipolar couplings provide significant structural information for proteins in the solution state, which makes them attractive for the rapid determination of protein structures. While dipolar couplings contain inherent structural ambiguities, these can be reduced via an overlap similarity measure that insists that protein fragments assigned to overlapping regions of the sequence must have self-consistent structures. This allows us to determine a backbone fold (including the correct C–C bond orientations) using only residual dipolar coupling data from one ordering medium. The resulting backbone structures are of sufficient quality to allow for modeling of sidechain rotamer states using a rotamer prediction algorithm and a force field employing the Surface Generalized Born continuum solvation model. We demonstrate the applicability of the method using experimental data for ubiquitin. These results illustrate the synergies that are possible between protein structural database and molecular modeling methods and NMR spectroscopy, and we expect that the further development of these methods will lead to the extraction of high resolution structural information from minimal NMR data.     

A structure refinement protocol combining NMR residual dipolar couplings and small angle scattering restraints

We present the implementation of a target function based on Small Angle Scattering data (Gabel et al. Eur Biophys J 35(4):313-327, 2006) into the Crystallography and NMR Systems (CNS) and demonstrate its utility in NMR structure calculations by simultaneous application of small angle scattering (SAS) and residual dipolar coupling (RDC) restraints. The efficiency and stability of the approach are demonstrated by reconstructing the structure of a two domain region of the 31 kDa nuclear export factor TAP (TIP-associated protein). Starting with the high resolution X-ray structures of the two individual TAP domains, the translational and orientational domain arrangement is refined simultaneously. We tested the stability of the protocol against variations of the SAS target parameters and the number of RDCs and their uncertainties. The activation of SAS restraints results in an improved translational clustering of the domain positions and lifts part of the fourfold degeneracy of their orientations (associated with a single alignment tensor). The resulting ensemble of structures reflects the conformational space that is consistent with the experimental SAS and RDC data. The SAS target function is computationally very efficient. SAS restraints can be activated at different levels of precision and only a limited SAS angular range is required. When combined with additional data from chemical shift perturbation, paramagnetic relaxation enhancement or mutational analysis the SAS refinement is an efficient approach for defining the topology of multi-domain and/or multimeric biomolecular complexes in solution based on available high resolution structures (NMR or X-ray) of the individual domains.     

Theoretical framework for NMR residual dipolar couplings in unfolded proteins

A theoretical framework for the prediction of nuclear magnetic resonance (NMR) residual dipolar couplings (RDCs) in unfolded proteins under weakly aligning conditions is presented. The unfolded polypeptide chain is modeled as a random flight chain while the alignment medium is represented by a set of regularly arranged obstacles. For the case of bicelles oriented perpendicular to the magnetic field, a closed-form analytical result is derived. With the obtained analytical expression the RDCs are readily accessible for any locus along the chain, for chains of differing length, and for varying bicelle concentrations. The two general features predicted by the model are (i) RDCs in the center segments of a polypeptide chain are larger than RDCs in the end segments, resulting in a bell-shaped sequential distribution of RDCs, and (ii) couplings are larger for shorter chains than for longer chains at a given bicelle concentration. Experimental data available from the literature confirm the first prediction of the model, providing a tool for recognizing fully unfolded polypeptide chains. With less certainty experimental data appear to support the second prediction as well. However, more systematic experimental studies are needed in order to validate or disprove the predictions of the model. The presented framework is an important step towards a solid theoretical foundation for the analysis of experimentally measured RDCs in unfolded proteins in the case of alignment media such as polyacrylamide gels and neutral bicelle systems which align biomacromolecules by a steric mechanism. Various improvements and generalizations are possible within the suggested approach.     

Protein backbone structure determination using only residual dipolar couplings from one ordering medium

Residual dipolar couplings provide significant structural information for proteins in the solution state, which makes them attractive for the rapid determination of protein folds. Unfortunately, dipolar couplings contain inherent structural ambiguities which make them difficult to use in the absence of additional information. In this paper, we describe an approach to the construction of protein backbone folds using experimental dipolar couplings based on a bounded tree search through a structural database. We filter out false positives via an overlap similarity measure that insists that protein fragments assigned to overlapping regions of the sequence must have self-consistent structures. This allows us to determine a backbone fold (including the correct C-C bond orientations) using only residual dipolar coupling data obtained from one ordering medium. We demonstrate the applicability of the method using experimental data for ubiquitin.     

NMR studies of RNA dynamics and structural plasticity using NMR residual dipolar couplings

An increasing number of RNAs are being discovered that perform their functions by undergoing large changes in conformation in response to a variety of cellular signals, including recognition of proteins and small molecular targets, changes in temperature, and RNA synthesis itself. The measurement of NMR residual dipolar couplings (RDCs) in partially aligned systems is providing new insights into the structural plasticity of RNA through combined characterization of large-amplitude collective helix motions and local flexibility in noncanonical regions over a wide window of biologically relevant timescales (相似文献   

Towards structural genomics of RNA: rapid NMR resonance assignment and simultaneous RNA tertiary structure determination using residual dipolar couplings

We report a new residual dipolar couplings (RDCs) based NMR procedure for rapidly determining RNA tertiary structure demonstrated on a uniformly (15)N/(13)C-labeled 27 nt variant of the trans-activation response element (TAR) RNA from HIV-I. In this procedure, the time-consuming nuclear Overhauser enhancement (NOE)-based sequential assignment step is replaced by a fully automated RDC-based assignment strategy. This approach involves examination of all allowed sequence-specific resonance assignment permutations for best-fit agreement between measured RDCs and coordinates for sub-structures in a target RNA. Using idealized A-form geometries to model Watson-Crick helices and coordinates from a previous X-ray structure to model a hairpin loop in TAR, the best-fit RDC assignment solutions are determined very rapidly (相似文献   

Refinement of the solution structure of the heparin-binding domain of vascular endothelial growth factor using residual dipolar couplings

Previous NMR structural studies of the heparin-binding domain of vascular endothelial growth factor (VEGF₁₆₅) revealed a novel fold comprising two subdomains, each containing two disulfide bridges and a short two-stranded antiparallel -sheet. The mutual orientation of the two subdomains was poorly defined by the NMR data. Heteronuclear relaxation data suggested that this disorder resulted from a relative lack of experimental restraints due to the limited size of the interface, rather than inherent high-frequency flexibility. Refinement of the structure using ¹H^N-¹⁵N residual dipolar coupling restraints results in significantly improved definition of the relative subdomain orientations.     

Magnetic field induced residual dipolar couplings of imino groups in nucleic acids from measurements at a single magnetic field

For base-paired nucleic acids, variations in ¹ J _NH and the imino ¹H chemical shift are both dominated by hydrogen bond length. In the absence of molecular alignment, the ¹ J _NH coupling for the imino proton then can be approximated by ¹ J _NH = (1.21Hz/ppm)δ_H − 103.5 ± 0.6 Hz, where δ_H represents the chemical shift of the imino proton in ppm. This relation permits imino residual dipolar couplings (RDCs) resulting from magnetic susceptibility anisotropy (MSA) to be extracted from measurement of (¹ J _NH + RDC) splittings at a single magnetic field strength. Magnetic field-induced RDCs were measured for tRNA^Val and the alignment tensor determined from magnetic-field alignment of tRNA^Val agrees well with the tensor calculated by summation of the MSA tensors of the individual nucleobases. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Jinfa Ying, Alexander Grishaev and Michael P. Latham contributed equally to this work.     

Backbone assignment of proteins with known structure using residual dipolar couplings

A prerequisite for NMR studies of protein-ligand interactions or protein dynamics is the assignment of backbone resonances. Here we demonstrate that protein assignment can significantly be enhanced when experimental dipolar couplings (RDCs) are matched to values back-calculated from a known three-dimensional structure. In case of small proteins, the program MARS allows assignment of more than 90% of backbone resonances without the need for sequential connectivity information. For bigger proteins, we show that the combination of sequential connectivity information with RDC-matching enables more residues to be assigned reliably and backbone assignment to be more robust against missing data. Structural or dynamic deviations from the employed 3D coordinates do not lead to an increased error rate in RDC-supported assignment. RDC-enhanced assignment is particularly useful when chemical shifts and sequential connectivity only provide a few reliable assignments.     

The importance of being ordered: improving NMR structures using residual dipolar couplings

Residual dipolar couplings arise from small degrees of alignment of molecules in a magnetic field. Most biomolecules lack sufficient intrinsic magnetic susceptibility anisotropies for practical purposes; however, alignment can be achieved using dilute aqueous phospholipid mixtures, colloidal suspensions of rod-shaped viruses, complex phases of surfactant systems and strained gels. The stability of the liquid crystalline phases varies with respect to temperature range, pH variation and time and is critically dependent on sample composition and experimental conditions. The magnitude of the residual dipolar couplings depends upon the degree of ordering and allows the determination of the corresponding inter-nuclear vectors with respect to the molecule's alignment frame. Inclusion of dipolar constraints into NMR structure calculations leads to improved precision and accuracy of the resulting structures, especially in cases where the information content provided by traditional NOE constraints is limited. In addition, rapid evaluation of backbone protein folds and determination of the relative orientations of individual components in multi-molecular complexes have become feasible. Dipolar coupling based strategies may well emerge as the most critical developments, in establishing NMR as a valuable and competitive methodology in the structural genomics initiative.     

Transverse relaxation optimised spin-state selective NMR experiments for measurement of residual dipolar couplings

Three transverse relaxation optimised NMR experiments (TROSY) for the measurement of scalar and dipolar couplings suitable for proteins dissolved in aqueous iso- and anisotropic solutions are described. The triple-spin-state-selective experiments yield couplings between ¹H^N-¹³C, ¹⁵N-¹³C, ¹H^N-¹³C _i–1, ¹⁵N-¹³C _i–1, ¹H^N-¹³C_i–1, ¹⁵N-¹³C_i–1, and ¹³C_i–1-¹³C _i–1 without introducing nonessential spectral crowding compared with an ordinary two-dimensional ¹⁵N-¹H correlation spectrum and without requiring explicit knowledge of carbon assignments. This set of /-J-TROSY experiments is most useful for perdeuterated proteins in studies of structure–activity relationships by NMR to observe, in addition to epitopes for ligands, also conformational changes induced by binding of ligands.     

Impact of static and dynamic A-form heterogeneity on the determination of RNA global structural dynamics using NMR residual dipolar couplings

We examined how static and dynamic deviations from the idealized A-form helix propagate into errors in the principal order tensor parameters determined using residual dipolar couplings (rdcs). A 20-ns molecular dynamics (MD) simulation of the HIV-1 transactivation response element (TAR) RNA together with a survey of spin relaxation studies of RNA dynamics reveals that pico-to-nanosecond local motions in non-terminal Watson-Crick base-pairs will uniformly attenuate base and sugar one bond rdcs by approximately 7%. Gaussian distributions were generated for base and sugar torsion angles through statistical comparison of 40 RNA X-ray structures solved to <3.0 A resolution. For a typical number (>or=11) of one bond C-H base and sugar rdcs, these structural deviations together with rdc uncertainty (1.5 Hz) lead to average errors in the magnitude and orientation of the principal axis of order that are <9% and <4 degrees, respectively. The errors decrease to <5% and <4 degrees for >or=17 rdcs. A protocol that allows for estimation of error in A-form order tensors due to both angular deviations and rdc uncertainty (Aform-RDC) is validated using theoretical simulations and used to analyze rdcs measured previously in TAR in the free state and bound to four distinct ligands. Results confirm earlier findings that the two TAR helices undergo large changes in both their mean relative orientation and dynamics upon binding to different targets.     

Sensitivity of NMR residual dipolar couplings to perturbations in folded and denatured staphylococcal nuclease

The invariance of NMR residual dipolar couplings (RDCs) in denatured forms of staphylococcal nuclease to changes in denaturant concentration or amino acid sequence has previously been attributed to the robustness of long-range structure in the denatured state. Here we compare RDCs of the wild-type nuclease with those of a fragment that retains a folded OB-fold subdomain structure despite missing the last 47 of 149 residues. The RDCs of the intact protein and of the truncation fragment are substantially different under conditions that favor folded structure. By contrast, there is a strong correlation between the RDCs of the full-length protein and the fragment under denaturing conditions (6 M urea). The RDCs of the folded and unfolded forms of the proteins are uncorrelated. Our results suggest that RDCs are more sensitive to structural changes in folded than unfolded proteins. We propose that the greater susceptibility of RDCs in folded states is a consequence of the close packing of the polypeptide chain under native conditions. By contrast, the invariance of RDCs in denatured states is more consistent with a disruption of cooperative structure than with the retention of a unique long-range folding topology.