Overall structure and sugar dynamics of a DNA dodecamer from homo- and heteronuclear dipolar couplings and 31P chemical shift anisotropy

The solution structure of d(CGCGAATTCGCG)₂ has been determined on the basis of an exceptionally large set of residual dipolar couplings. In addition to the heteronuclear ¹³C-¹H and ¹⁵N-¹H and qualitative homonuclear ¹H-¹H dipolar couplings, previously measured in bicelle medium, more than 300 quantitative ¹H-¹H and 22 ³¹P-¹H dipolar restraints were obtained in liquid crystalline Pf1 medium, and 22 ³¹P chemical shift anisotropy restraints. High quality DNA structures can be obtained solely on the basis of these new restraints, and these structures are in close agreement with those calculated previously on the basis of ¹³C-¹H and ¹⁵N-¹H dipolar couplings. In the newly calculated structures, ³¹P-¹H dipolar and ³Jsub H3 P sub couplings and ³¹P CSA data restrain the phosphodiester backbone torsion angles. The final structure represents a quite regular B-form helix with a modest bending of 10°, which is essentially independent of whether or not electrostatic terms are used in the calculation. Combined, the number of homo- and heteronuclear dipolar couplings significantly exceeds the number of degrees of freedom in the system. Results indicate that the dipolar coupling data cannot be fit by a single structure, but are compatible with the presence of rapid equilibria between C2-endo and C3-endo deoxyribose puckers (sugar switching). The C2-H2/H2 dipolar couplings in B-form DNA are particularly sensitive to sugar pucker and yield the largest discrepancies when fit to a single structure. To resolve these discrepancies, we suggest a simplified dipolar coupling analysis that yields N/S equilibria for the ribose sugar puckers, which are in good agreement with previous analyses of NMR J_HH couplings, with a population of the minor C3-endo form higher for pyrimidines than for purines.     

Induced alignment and measurement of dipolar couplings of an SH2 domain through direct binding with filamentous phage

Large residual ¹⁵N-¹H dipolar couplings have been measured in a Src homology II domain aligned at Pf1 bacteriophage concentrations an order of magnitude lower than used for induction of a similar degree of alignment of nucleic acids and highly acidic proteins. An increase in ¹ H and ¹⁵N protein linewidths and a decrease in T₂ and T₁ relaxation time constants implicates a binding interaction between the protein and phage as the mechanism of alignment. However, the associated increased linewidth does not preclude the accurate measurement of large dipolar couplings in the aligned protein. A good correlation is observed between measured dipolar couplings and predicted values based on the high resolution NMR structure of the SH2 domain. The observation of binding-induced protein alignment promises to broaden the scope of alignment techniques by extending their applicability to proteins that are able to interact weakly with the alignment medium.     

Determination of protein global folds using backbone residual dipolar coupling and long-range NOE restraints

We report the determination of the global fold of human ubiquitin using protein backbone NMR residual dipolar coupling and long-range nuclear Overhauser effect (NOE) data as conformational restraints. Specifically, by use of a maximum of three backbone residual dipolar couplings per residue (N_i-H^N _i, N_i-C_i–1, H^N _i - C_i–1) in two tensor frames and only backbone H^N-H^N NOEs, a global fold of ubiquitin can be derived with a backbone root-mean-square deviation of 1.4 Å with respect to the crystal structure. This degree of accuracy is more than adequate for use in databases of structural motifs, and suggests a general approach for the determination of protein global folds using conformational restraints derived only from backbone atoms.     

Structure refinement of flexible proteins using dipolar couplings: Application to the protein p8MTCP1

The present study deals with the relevance of using mobility-averaged dipolar couplings for the structure refinement of flexible proteins. The 68-residue protein p8^MTCP1 has been chosen as model for this study. Its solution state consists mainly of three -helices. The two N-terminal helices are strapped in a well-determined -hairpin, whereas, due to an intrinsic mobility, the position of the third helix is less well defined in the NMR structure. To further characterize the degrees of freedom of this helix, we have measured the dipolar coupling constants in the backbone of p8^MTCP1 in a bicellar medium. We show here that including D _HN ^dip dipolar couplings in the structure calculation protocol improves the structure of the -hairpin but not the positioning of the third helix. This is due to the motional averaging of the dipolar couplings measured in the last helix. Performing two calculations with different force constants for the dipolar restraints highlights the inconstancy of these mobility-averaged dipolar couplings. Alternatively, prior to any structure calculations, comparing the values of the dipolar couplings measured in helix III to values back-calculated from an ideal helix demonstrates that they are atypical for a helix. This can be partly attributed to mobility effects since the inclusion of the ¹⁵N relaxation derived order parameter allows for a better fit.     

Measurement and application of 1H-19F dipolar couplings in the structure determination of 2′-fluorolabeled RNA

Residual dipolar couplings can provide powerful restraints for determination and refinement of the solution structure of macromolecules. The application of these couplings in nucleic acid structure elucidation can have an especially dramatic impact, since they provide long-range restraints, typically absent in NOE and J-coupling measurements. Here we describe sensitive X-filtered-E.COSY-type methods designed to measure both the sign and magnitude of long-range ¹H-¹⁹F dipolar couplings in selectively fluorine labeled RNA oligonucleotides oriented in solution by a liquid crystalline medium. The techniques for measuring ¹H-¹⁹F dipolar couplings are demonstrated on a 21-mer RNA hairpin, which has been specifically labeled with fluorine at the 2-hydroxyl position of three ribose sugars. Experimentally measured ¹H-¹⁹F dipolar couplings for the 2-deoxy-2-fluoro-sugars located in the helical region of the RNA hairpin were found to be in excellent agreement with values predicted using canonical A-form helical geometry, demonstrating that these couplings can provide accurate restraints for the refinement of RNA structures determined by NMR.     

A method for incorporating dipolar couplings into structure calculations in cases of (near) axial symmetry of alignment

A method for incorporating dipolar coupling restraints into structure calculations is described which follows closely on methodology that has been recently presented for orienting peptide planes using dipolar couplings [Mueller et al. (2000) J. Mol. Biol., 300, 197–212] and is specifically developed for use in cases of an axially symmetric alignment tensor. Modeling studies on an all -helical protein, farnesyl diphosphate synthase, establish the utility of the approach. A global fold of the 370-residue maltose binding protein in complex with -cyclodextrin is obtained from experimentally derived restraints. The average pairwise rmsd values between the N- and C-terminal domains in this NMR structure and the corresponding regions in the X-ray structure of the protein are 2.8 and 3.1 Å, respectively.     

Force Field Dependence of NMR-Based,Restrained Molecular Dynamics DNA Structure Calculations Including an Analysis of the Influence of Residual Dipolar Coupling Restraints

Abstract

Restrained molecular dynamics is widely used to calculate DNA structures from NMR data. Here, results of an in silico experiment show that the force field can be significant compared to the NMR restraints in driving the final structures to converge. Specifically, we observed that i) the influence of the force field leads to artificially tight convergence within final families of structures and ii) the precision and character of resulting structures depend on the choice of force field used in the calculations. A canonical B-DNA model was used as a target structure. Distances, dihedral angles, and simulated residual dipolar couplings were measured in the target structure and used as restraints. X-PLOR and Discover, which use force fields developed for CHARMM and AMBER programs, respectively, were tested and found to produce different final structures despite the use of identical distance and dihedral restraints. Incorporation of residual dipolar coupling restraints in X-PLOR improves convergence with the target structure and between families of structures indicating that the force field dependence can potentially be overcome if residual dipolar coupling restraints are employed.     

A target function for quaternary structural refinement from small angle scattering and NMR orientational restraints

We present a novel target function based on atomic coordinates that permits quaternary structural refinement of multi-domain protein–protein or protein–RNA complexes. It requires that the high-resolution structures of the individual domains are known and that small angle scattering (SAS) data as well as NMR orientational restraints from residual dipolar couplings (RDCs) of the complex are available. We show that, when used in combination, the translational and rotational restraints contained in SAS intensities and RDCs, respectively, define a target potential function that permits to determine the overall topology of complexes made up of domains with low internal symmetry. We apply the target function on a modestly anisotropic model system, the Barnase/Barstar complex, and discuss factors that influence the structural refinement such as data errors and the geometrical properties of the individual domains.     

A Refined Model for the Structure of Acireductone Dioxygenase from Klebsiella ATCC 8724 Incorporating Residual Dipolar Couplings

Acireductone dioxygenase (ARD) from Klebsiella ATCC 8724 is a metalloenzyme that is capable of catalyzing different reactions with the same substrates (acireductone and O₂) depending upon the metal bound in the active site. A model for the solution structure of the paramagnetic Ni²⁺-containing ARD has been refined using residual dipolar couplings (RDCs) measured in two media. Additional dihedral restraints based on chemical shift (TALOS) were included in the refinement, and backbone structure in the vicinity of the active site was modeled from a crystallographic structure of the mouse homolog of ARD. The incorporation of residual dipolar couplings into the structural refinement alters the relative orientations of several structural features significantly, and improves local secondary structure determination. Comparisons between the solution structures obtained with and without RDCs are made, and structural similarities and differences between mouse and bacterial enzymes are described. Finally, the biological significance of these differences is considered.     

Refined structure of a flexible heptasaccharide using 1H-13C and 1H-1H NMR residual dipolar couplings in concert with NOE and long range scalar coupling constants

The heptasaccharide isolated from the cell wall polysaccharide of Streptococcus mitis J22 serves as an important model for the dynamics and conformation of complex polysaccharides, illustrating the nature of flexibility with rigid epitopes joined by flexible hinges. One-bond C-H residual dipolar couplings (¹D_CH) and long-range H-H residual dipolar couplings (ⁿD_HH) were measured for the heptasaccharide in a cetylpyridinium chloride/hexanol/brine lamellar liquid crystal medium. A method is proposed to determine the ⁿD_HH in natural abundance based on a ¹³C resolved ¹H TOCSY pulse sequence previously published to determine the homonuclear scalar couplings. Different methods for interpretation of the ¹D_CH and the ⁿD_HH residual dipolar coupling data obtained were compared and combined with the NOE and long-range H,C and C,C scalar couplings available for this heptasaccharide. A flexible model of the heptasaccharide was determined in which two structurally well-defined regions involving four and two sugar residues, respectively are joined by a flexible hinge which involves two 16 glycosidic linkages.     

Improvement of hydrogen bond geometry in protein NMR structures by residual dipolar couplings – an assessment of the interrelation of NMR restraints

We have examined how the hydrogen bond geometry in three different proteins is affected when structural restraints based on measurements of residual dipolar couplings are included in the structure calculations. The study shows, that including restraints based solely on (1)H(N)-(15)N residual dipolar couplings has pronounced impact on the backbone rmsd and Ramachandran plot but does not improve the hydrogen bond geometry. In the case of chymotrypsin inhibitor 2 the addition of (13)CO-(13)C(alpha) and (15)N-(13)CO one bond dipolar couplings as restraints in the structure calculations improved the hydrogen bond geometry to a quality comparable to that obtained in the 1.8 A resolution X-ray structure of this protein. A systematic restraint study was performed, in which four types of restraints, residual dipolar couplings, hydrogen bonds, TALOS angles and NOEs, were allowed in two states. This study revealed the importance of using several types of residual dipolar couplings to get good hydrogen bond geometry. The study also showed that using a small set of NOEs derived only from the amide protons, together with a full set of residual dipolar couplings resulted in structures of very high quality. When reducing the NOE set, it is mainly the side-chain to side-chain NOEs that are removed. Despite of this the effect on the side-chain packing is very small when a reduced NOE set is used, which implies that the over all fold of a protein structure is mainly determined by correct folding of the backbone.     

Determination of protein rotational correlation time from NMR relaxation data at various solvent viscosities

An accurate determination of the overall rotation of a protein plays a crucial role in the investigation of its internal motions by NMR. In the present work, an innovative approach to the determination of the protein rotational correlation time _R from the heteronuclear relaxation data is proposed. The approach is based on a joint fit of relaxation data acquired at several viscosities of a protein solution. The method has been tested on computer simulated relaxation data as compared to the traditional _R determination method from T₁/T₂ ratio. The approach has been applied to ribonuclease barnase from Bacillus amyloliquefaciens dissolved in an aqueous solution and deuterated glycerol as a viscous component. The resulting rotational correlation time of 5.56 ± 0.01 ns and other rotational diffusion tensor parameters are in good agreement with those determined from T₁/T₂ ratio.     

Measurement of residual dipolar couplings from 1Hα to 13Cα and 15N using a simple HNCA-based experiment

Novel NMR pulse schemes for simultaneous measurement of ¹ D _CHand ² D _NHresidual dipolar couplings in proteins is presented. We show that ² D _NHcoupling can be very useful for protein structure determination. The ² D _NHcoupling can be measured from ¹⁵N dimension with good accuracy on a slowly relaxing TROSY resonance, utilizing HNCA-TROSY-based experiments, which concomitantly supply large ¹ D _CHcoupling. The dynamic range of ² D _NHcoupling is comparable to ¹ D _NC coupling, but instead, it also serves non-redundant information on the course of protein backbone, thanks to rotational degree of freedom with respect to peptide bond. The HNCA-TROSY-based experiments are optimal for measuring residual dipolar couplings at high magnetic fields owing to absence of rapid transverse relaxation of carbonyl carbon. The reliability of the proposed approach was tested on ¹⁵N/¹³C human ubiquitin. A very good correlation with ubiquitin solution as well as crystal structure, for both ¹ D _CHand ² D _NHcouplings, was obtained.     

Refinement of protein structure against non-redundant carbonyl <Superscript>13</Superscript>C NMR relaxation

Carbonyl ¹³C′ relaxation is dominated by the contribution from the ¹³C′ chemical shift anisotropy (CSA). The relaxation rates provide useful and non-redundant structural information in addition to dynamic parameters. It is straightforward to acquire, and offers complimentary structural information to the ¹⁵N relaxation data. Furthermore, the non-axial nature of the ¹³C′ CSA tensor results in a T₁/T₂ value that depends on an additional angular variable even when the diffusion tensor of the protein molecule is axially symmetric. This dependence on an extra degree of freedom provides new geometrical information that is not available from the NH dipolar relaxation. A protocol that incorporates such structural restraints into NMR structure calculation was developed within the program Xplor-NIH. Its application was illustrated with the yeast Fis1 NMR structure. Refinement against the ¹³C′ T₁/T₂ improved the overall quality of the structure, as evaluated by cross-validation against the residual dipolar coupling as well as the ¹⁵N relaxation data. In addition, possible variations of the CSA tensor were addressed. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

Estimating the Accuracy of Protein Structures using Residual Dipolar Couplings

It has been commonly recognized that residual dipolar coupling data provide a measure of quality for protein structures. To quantify this observation, a database of 100 single-domain proteins has been compiled where each protein was represented by two independently solved structures. Backbone ¹H–¹⁵N dipolar couplings were simulated for the target structures and then fitted to the model structures. The fits were characterized by an R-factor which was corrected for the effects of non-uniform distribution of dipolar vectors on a unit sphere. The analyses show that favorable values virtually guarantee high accuracy of the model structure (where accuracy is defined as the backbone coordinate rms deviation). On the other hand, unfavorable values do not necessarily suggest low accuracy. Based on the simulated data, a simple empirical formula is proposed to estimate the accuracy of protein structures. The method is illustrated with a number of examples, including PDZ2 domain of human phosphatase hPTP1E. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Direct measurement of 1H-1H dipolar couplings in proteins: A complement to traditional NOE measurements

An intensity-based constant-time COSY (CT-COSY) method is described for measuring ¹H-¹H residual dipolar couplings of proteins in weakly aligned media. For small proteins, the overall sensitivity of this experiment is comparable to the NOESY experiment. In cases where the ¹H-¹H distances are defined by secondary structure, such as ¹H-¹H^N and ¹H^N-¹H^N sequential distances in -helices and -sheets, these measurements provide useful orientational constraints for protein structure determination. This experiment can also be used to provide distance information similar to that obtained from NOE connectivities once the angular dependence is removed. Because the measurements are direct and non-coherent processes, such as spin diffusion, do not enter, the measurements can be more reliable. The 1/r ³ distance dependence of directly observed dipolar couplings, as compared with the 1/r ⁶ distance dependence of NOEs, also can provide longer range distance information at favorable angles. A simple 3D, ¹⁵N resolved version of the pulse sequence extends the method to provide the improved resolution required for application to larger biomolecules.     

Measurement of 1H3′-31P dipolar couplings in a DNA oligonucleotide by constant-time NOESY difference spectroscopy

The ratios of cross peak intensities in a selective constant-time NOESY experiment, recorded with and without ³¹P decoupling, yield values for the sum of the H3-P scalar and dipolar couplings. The selective refocusing of H3 resonances in this experiment results in excellent resolution and sensitivity, even in the liquid crystalline phase where the ¹H spectrum is broadened by unresolved homonuclear dipolar couplings. The vicinal H3-P scalar and dipolar couplings in the DNA oligomer d(CGCGAATTCGCG)₂ were measured in both isotropic solution, and in a liquid crystalline phase. Isotropic values are in good agreement with values reported previously. Dipolar couplings are in excellent agreement with the NMR structure for this dodecamer, and to a somewhat lesser extent with the X-ray structures.