Measurement of Scaled Residual Dipolar Couplings in Proteins Using Variable-angle Sample Spinning

NMR spectra of ubiquitin in the presence of bicelles at a concentration of 25% w/v have been recorded under sample spinning conditions for different angles of rotation. For an axis of rotation equal to the magic angle, the (1)H/(15)N HSQC recorded without any (1)H decoupling in the indirect dimension corresponds to the classical spectrum obtained on a protein in an isotropic solution and allows the measurement of scalar J-couplings (1) J (NH). For an angle of rotation smaller than the magic angle, the bicelles orient with their normal perpendicular to the spinning axis, whereas for an angle of rotation greater than the magic angle the bicelles orient with their normal along the spinning axis. This bicelle alignment creates anisotropic conditions that give rise to the observation of residual dipolar couplings in ubiquitin. The magnitude of these dipolar couplings depends directly on the angle that the rotor makes with the main magnetic field. By changing this angle in a controlled manner, residual dipolar couplings can be either scaled up or down thus offering the possibility to study simultaneously a wide range of dipolar couplings in the same sample.     

A liquid crystalline medium for measuring residual dipolar couplings over a wide range of temperatures

A mixture of dilauroyl phosphatidylcholine (DLPC) and 3-(cholamidopropyl)dimethylammonio-2-hydroxyl-1-propane sulfonate (CHAPSO) in water forms disc shaped bicelles that become ordered at high magnetic fields over a wide range of temperatures. As illustrated for the FK506 binding protein (FKBP), large residual dipolar couplings can be measured for proteins dissolved in low concentrations (5% w/v) of a DLPC/CHAPSO medium at a molar ratio of 4.2:1. This system is especially useful for measuring residual dipolar couplings for molecules that are only stable at low temperatures.     

Characterization of surfactant liquid crystal phases suitable for molecular alignment and measurement of dipolar couplings

Media employed for imparting partial alignment onto solute molecules have recently attracted considerable attention, since they permit the measurement of NMR parameters for solute biomolecules commonly associated with solid state NMR. Here we characterize a medium which is based on a quasi-ternary surfactant system comprising cetylpyridinium bromide/hexanol/sodium bromide. We demonstrate that dilute solutions of this system can exist in liquid crystalline phases which orient in the magnetic field and allow the measurement of residual dipolar couplings under a variety of conditions. The present system is extremely versatile and robust, tolerating different buffer conditions, temperature ranges and concentrations.     

A novel interactive tool for rigid-body modeling of multi-domain macromolecules using residual dipolar couplings

Residual dipolar couplings (RDC), measured by dissolving proteins in dilute liquid crystal media, or by studying naturally paramagnetic molecules, have rapidly become established as routine measurements in the investigation of the structure of macromolecules by NMR. One of the most obvious applications of the previously inaccessible long-range angular information afforded by RDC is the accurate definition of domain orientation in multi-module macromolecules or complexes. In this paper we describe a novel program developed to allow the determination of alignment tensor parameters for individual or multiple domains in macromolecules from residual dipolar couplings and to facilitate their manipulation to construct low-resolution models of macromolecular structure. For multi-domain systems the program determines the relative orientation of individual structured domains, and provides graphical user-driven rigid-body modeling of the different modules relative to the common tensorial frame. Translational freedom in the common frame, and equivalent rotations about the diagonalized (x,y,z) axes are used to position the different modules in the common frame to find a model in best agreement with experimentally measured couplings alone or in combination with additional experimental or covalent information.     

Characterization of magnetically oriented phospholipid micelles for measurement of dipolar couplings in macromolecules

Weak alignment of solute molecules with the magnetic field can be achieved in a dilute liquid crystalline medium, consisting of an aqueous mixture of dimyristoyl-phosphatidylcholine (DMPC) and dihexanoyl-phosphatidylcholine (DHPC). For a certain range of molar ratios, DMPC and DHPC can form large, disc-shaped particles, commonly referred to as bicelles (Sanders and Schwonek, 1992), which cooperatively align in the magnetic field and induce a small degree of alignment on asymmetrically shaped solute molecules. As a result, dipolar couplings between pairs of ¹H, ¹³C or ¹⁵N nuclei are no longer averaged to zero by rotational diffusion and they can be readily measured, providing valuable structural information. The stability of these liquid crystals and the degree of alignment of the solute molecules depend strongly on experimental variables such as the DMPC:DHPC ratio and concentration, the preparation protocol of the DMPC/DHPC mixtures, as well as salt, temperature, and pH. The lower temperature limit for which the liquid crystalline phase is stable can be reduced to 20 °C by using a ternary mixture of DHPC, DMPC, and 1-myristoyl-2-myristoleoyl-sn-glycero-3-phosphocholine, or a binary mixture of DHPC and ditridecanoyl-phosphatidylcholine. These issues are discussed, with an emphasis on the use of the medium for obtaining weak alignment of biological macromolecules.     

Study of conformational rearrangement and refinement of structural homology models by the use of heteronuclear dipolar couplings

For an increasing fraction of proteins whose structures are being studied, sequence homology to known structures permits building of low resolution structural models. It is demonstrated that dipolar couplings, measured in a liquid crystalline medium, not only can validate such structural models, but also refine them. Here, experimental ¹H-¹⁵N, ¹H-¹³C, and ¹³C-¹³C dipolar couplings are shown to decrease the backbone rmsd between various homology models of calmodulin (CaM) and its crystal structure. Starting from a model of the Ca²⁺-saturated C-terminal domain of CaM, built from the structure of Ca²⁺-free recoverin on the basis of remote sequence homology, dipolar couplings are used to decrease the rmsd between the model and the crystal structure from 5.0 to 1.25 Å. A better starting model, built from the crystal structure of Ca²⁺-saturated parvalbumin, decreases in rmsd from 1.25 to 0.93 Å. Similarly, starting from the structure of the Ca²⁺-ligated CaM N-terminal domain, experimental dipolar couplings measured for the Ca²⁺-free form decrease the backbone rmsd relative to the refined solution structure of apo-CaM from 4.2 to 1.0 Å.     

Multiplet component separation for measurement of methyl 13C-1H dipolar couplings in weakly aligned proteins

A simple spectral editing procedure is described that generates separate subspectra for the methyl ¹³C-¹H₃ multiplet components of ¹H-¹³C HSQC spectra. The editing procedure relies on co-addition of in-phase and antiphase spectra and yields ¹H-coupled constant-time HSQC subspectra for the methyl region that have the simplicity of the regular decoupled CT-HSQC spectrum. Resulting spectra permit rapid and reliable measurement of ¹H-¹³C J and dipolar couplings. The editing procedure is illustrated for a Ca²⁺-calmodulin sample in isotropic and liquid crystalline phases.     

Applications of Variable-angle Sample Spinning Experiments to the Measurement of Scaled Residual Dipolar Couplings and <Superscript>15</Superscript>N CSA in Soluble Proteins

NMR spectra of ubiquitin in the presence of bicelles at a concentration of 32% w/v have been recorded at 700 MHz under sample spinning conditions at the magic angle (54.7°) and at an angle of 45.5°. At the magic angle, the ¹H–¹⁵N HSQC spectrum of ubiquitin in bicelles is virtually indistinguishable from the one recorded on the protein in solution. Spinning the sample at the magic angle creates an isotropic environment with no preferred bicelle orientations, thus allowing the determination of scalar coupling constants. For an angle of rotation of 45.5°, the bicelles orient with their normal perpendicular to the spinning axis leading to the observation of strong residual dipolar couplings and chemical shift variations of the ¹⁵N resonances. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Conformational studies of Lewis X and Lewis A trisaccharides using NMR residual dipolar couplings.

The conformations of the histo-blood group carbohydrate antigens Lewis X (Le(x)) and Lewis A (Le(a)) were studied by NMR measurements of one-bond C-H residual dipolar couplings in partially oriented liquid crystal solutions. A strategy for rapid calculation of the difference between theoretical and experimental dipolar couplings of a large number of model structures generated by computer simulations was developed, resulting in an accurate model structure for the compounds. Monte Carlo simulations were used to generate models for the trisaccharides, and orientations of each model were sought that could reproduce the experimental residual dipolar coupling values. For both, Le(a) and Le(x), single low energy models giving excellent agreement with experiment were found, implying a compact rigidly folded conformation for both trisaccharides. The new approach was also applied to the pentasaccharides lacto-N-fucopentaose 2 (LNF-2) and lacto-N-fucopentaose 3 (LNF-3) proving its consistency and robustness. For describing the conformation of tightly folded oligosaccharides, a definition for characterization of ring planes in pyranoside chairs is proposed and applied to the analysis of the relation between the fucose and galactose residues in the epitopes, revealing the structural similarity between them.     

Line narrowing in spectra of proteins dissolved in a dilute liquid crystalline phase by band-selective adiabatic decoupling: Application to 1HN-15N residual dipolar coupling measurements

Residual heteronuclear dipolar couplings obtained from partially oriented protein samples can provide unique NMR constraints for protein structure determination. However, partial orientation of protein samples also causes severe ¹ H line broadening resulting from residual ¹ H-¹H dipolar couplings. In this communication we show that band-selective ¹H homonuclear decoupling during data acquisition is an efficient way to suppress residual ¹H-¹H dipolar couplings, resulting in spectra that are still amenable to solution NMR analysis, even with high degrees of alignment. As an example, we present a novel experiment with improved sensitivity for the measurement of one-bond ¹ H^N-¹⁵N residual dipolar couplings in a protein sample dissolved in magnetically aligned liquid crystalline bicelles.     

Bicelle-based liquid crystals for NMR-measurement of dipolar couplings at acidic and basic pH values

It is demonstrated that mixtures of ditetradecyl- phosphatidylcholine or didodecyl-phoshatidylcholine and dihexyl- phosphatidylcholine in water form lyotropic liquid crystalline phases under similar conditions as previously reported for bicelles consisting of dimyristoyl-phosphatidylcholine (DMPC) and dihexanoyl- phosphatidylcholine (DHPC). The carboxy-ester bonds present in DMPC and DHPC are replaced by ether linkages in their alkyl analogs, which prevents acid- or base-catalyzed hydrolysis of these compounds. 15N-1H dipolar couplings measured for ubiquitin over the 2.3–10.4pH range indicate that this protein retains a backbone conformation which is very similar to its structure at pH 6.5 over this entire range.     

Molecular orientation and conformation of phosphatidylinositides in membrane mimetics using variable angle sample spinning (VASS) NMR

For many biological molecules, determining their geometry as they exist in a membrane environment is a crucial step in understanding their function. Variable angle sample spinning (VASS) NMR provides a new route to obtaining geometry information on membrane-associating molecules; it has been used here to scale and separate anisotropic contributions to phosphorus chemical shifts in NMR spectra of phosphatidylinositol phosphates. The procedure allows spectral assignment via correlation with isotropic chemical shifts and determination of a family of probable headgroup orientations via interpretation of anisotropic shift contributions. The molecules studied include phosphtidylinositol-4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). A membrane-like environment is provided by a dispersion of alkyl-poly(ethylene) glycols and n-alcohols that forms a field-orienting liquid crystal with a director that can be manipulated by varying the sample spinning axis. The experiments presented indicate that the variable angle sample spinning method will provide a direct approach for assignment and extraction of structural information from membrane-associating biomolecules labeled with a wider variety of NMR active isotopes.     

Rapid and accurate structure determination of coiled-coil domains using NMR dipolar couplings: application to cGMP-dependent protein kinase Ialpha

Coiled-coil motifs play essential roles in protein assembly and molecular recognition, and are therefore the targets of many ongoing structural and functional studies. However, owing to the dynamic nature of many of the smaller coiled-coil domains, crystallization for X-ray studies is very challenging. Determination of elongated structures using standard NMR approaches is inefficient and usually yields low-resolution structures due to accumulation of small errors over long distances. Here we describe a solution NMR approach based on residual dipolar couplings (RDCs) for rapid and accurate structure determination of coiled-coil dimers. Using this approach, we were able to determine the high-resolution structure of the coiled-coil domain of cGMP-dependent protein kinase Ialpha, a protein of previously unknown structure that is critical for physiological relaxation of vascular smooth muscle. This approach can be extended to solve coiled-coil structures with higher order assemblies.     

Evaluation of uncertainty in alignment tensors obtained from dipolar couplings

Residual dipolar couplings and their corresponding alignment tensors are useful for structural analysis of macromolecules. The error in an alignment tensor, derived from residual dipolar couplings on the basis of a known structure, is determined not only by the accuracy of the measured couplings but also by the uncertainty in the structure (structural noise). This dependence is evaluated quantitatively on the basis of simulated structures using Monte-Carlo type analyses. When large numbers of dipolar couplings are available, structural noise is found to result in a systematic underestimate of the magnitude of the alignment tensor. Particularly in cases where only few dipolar couplings are available, structural noise can cause significant errors in best-fitted alignment tensor values, making determination of the relative orientation of small fragments and evaluation of local backbone mobility from dipolar couplings difficult. An example for the protein ubiquitin demonstrates the inherent limitations in characterizing motions on the basis of local alignment tensor magnitudes.     

Efficient and precise measurement of H(alpha)-C(alpha), C(alpha)-C', C(alpha)-C(beta) and H(N)-N residual dipolar couplings from 2D H(N)-N correlation spectra

A suite of experiments are presented for the measurement of H–C, C–C, C–C and H^N–N couplings from uniformly ¹⁵N, ¹³C labeled proteins. Couplings are obtained from a series of intensity modulated two-dimensional H^N–N spectra equivalent to the common ¹H–¹⁵N–HSQC spectra, alleviating many overlap and assignment issues associated with other techniques. To illustrate the efficiency of this method, H–C, C–C, and H^N–N isotropic scalar couplings were determined for ubiquitin from data collected in less than 4.5 h, C–C data collection required 10 h. The resulting couplings were measured with an average error of ±0.06, ±0.05, ±0.04 and ±0.10 Hz, respectively. This study also shows H–C and C–C couplings, valuable because they provide orientation of bond vectors outside the peptide plane, can be measured in a uniform and precise way. Superior accuracy and precision to existing 3D measurements for C–C couplings and increased precision compared to IPAP measurements for H^N–N couplings are demonstrated. Minor modifications allow for acquisition of modulated H^N–C 2D spectra, which can yield additional well resolved peaks and significantly increase the number of measured RDCs for proteins with crowded ¹H–¹⁵N resonances.     

Use of residual dipolar couplings as restraints in ab initio protein structure prediction

NMR residual dipolar couplings (RDCs), in the form of the projection angles between the respective internuclear bond vectors, are used as structural restraints in the ab initio structure prediction of a test set of six proteins. The restraints are applied using a recently developed SICHO (SIde-CHain-Only) lattice protein model that employs a replica exchange Monte Carlo (MC) algorithm to search conformational space. Using a small number of RDC restraints, the quality of the predicted structures is improved as reflected by lower RMSD/dRMSD (root mean square deviation/distance root mean square deviation) values from the corresponding native structures and by the higher correlation of the most cooperative mode of motion of each predicted structure with that of the native structure. The latter, in particular, has possible implications for the structure-based functional analysis of predicted structures.     

Refinement of d(GCGAAGC) hairpin structure using one- and two-bond residual dipolar couplings

The structure of the ¹³C,¹⁵N-labeled d(GCGAAGC) hairpin, as determined by NMR spectroscopy and refined using molecular dynamics with NOE-derived distances, torsion angles, and residual dipolar couplings (RDCs), is presented. Although the studied molecule is of small size, it is demonstrated that the incorporation of diminutive RDCs can significantly improve local structure determination of regions undefined by the conventional restraints. Very good correlation between the experimental and back-calculated small one- and two-bond ¹H-¹³C, ¹H-¹⁵N, ¹³C-¹³C and ¹³C-¹⁵N coupling constants has been attained. The final structures clearly show typical features of the miniloop architecture. The structure is discussed in context of the extraordinary stability of the d(GCGAAGC) hairpin, which originates from a complex interplay between the aromatic base stacking and hydrogen bonding interactions.     

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.     

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.     

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.