Dipolar couplings in multiple alignments suggest alpha helical motion in ubiquitin

Recently, residual dipolar couplings (RDCs) of backbone N-HN vectors measured in 11 different alignment media were analyzed with respect to structure and dynamics in a model-free way in terms of generalized order parameters and motional anisotropies. The anisotropies in the central alpha-helix were found to be strikingly uniformly distributed. In this communication, these parameters are further interpreted in terms of physically feasible cooperative reorientational motion of the helix with respect to the core of the protein. The RDCs are compatible with a model in which all N-HN vectors of the alpha-helix of ubiquitin exhibit correlated anisotropic excursions with amplitudes of 21 degrees and 12 degrees along two orthogonal directions x' ' and y' ' of a coordinate system C' ', if z' ' represents the helix axis. Such motion contradicts neither NOE data nor molecular force-field calculations.     

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.     

Spatial structure of peptides determined by residual dipolar couplings analysis

The gated decoupled (13)C NMR spectra of a dipeptide (Glu-Trp) and a tetrapeptide (NAc-Ser-Phe-Val-Gly-OMe) were recorded in D(2)O and in a lyotropic alignment medium (pentaethylene glycol monododecyl ether/n-hexanol). The residual dipolar couplings were extracted as the differences between the observed couplings for the magnetic nuclei dissolved in the latter and former media. Using a computational optimization, the spatial structures of the compounds were calculated starting from their respective low energy conformations obtained on a semiempirical basis. The uniformity of each conformation was confirmed by the solid-state (13)C NMR spectra of powder samples. Differences between the starting structures and final ones, optimized when employing residual dipolar couplings, are discussed.     

Model-free analysis of protein backbone motion from residual dipolar couplings

On the basis of the measurement of NH residual dipolar couplings (RDCs) in 11 different alignment media, an RDC-based order parameter is derived for each residue in the protein ubiquitin. Dipolar couplings are motionally averaged in the picosecond to millisecond time range and, therefore, reflect motion slower than the inverse overall tumbling correlation time of the protein. It is found that there is considerable motion that is slower than the correlation time and could not be detected with previous NMR methodology. Amplitudes and anisotropies of the motion can be derived from the model-free analysis. The method can be applied provided that at least five sufficiently different alignment media can be found for the biomolecule under investigation.     

Conformational analysis of a flexible oligosaccharide using residual dipolar couplings.

We present a new approach to the analysis of the conformational and the motional properties of an oligosaccharide, methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside. The approach relies on an order matrix analysis of residual dipolar couplings in the solution state. By combining a number of different types of couplings, (1)D(CH), (2)D(CH), and D(HH), an order matrix is solved for each ring of the trimannoside. The resulting order parameters indicate the internal motion at the alpha (1,3) linkage to be limited, while significant motion is suggested at the alpha (1,6) linkage. Two structures for the trimannoside were determined by aligning the order tensor principal axes obtained from two different orienting media, bicelles and phage. The very similar conformations at the alpha (1,3) linkage of these two structures confirm that the internal motion at the alpha (1,3) linkage is small and the conformation is a good representation of a single preferred structure. The different conformations at the alpha (1,6) linkage suggest that the motional amplitudes are large and the conformations must be viewed as virtual conformers. Compared with traditional NMR methods, data acquisition is easy and data analysis is straightforward.     

Application of residual dipolar couplings in organic compounds

Residual dipolar couplings (RDCs) induced by anisotropic media are a powerful tool for the structure determination of biomolecules through NMR spectroscopy. Recent advances have proven it to be a valuable tool for determination of the stereochemistry of organic molecules. By simple inspection or order matrix calculations, RDCs provide unambiguous information about the relative configurations or complete stereochemistry of organic compounds.     

Lanthanide-binding peptides for NMR measurements of residual dipolar couplings and paramagnetic effects from multiple angles

Lanthanide-binding peptide tags (LBTs) containing a single cysteine residue can be attached to proteins via a disulfide bond, presenting a flexible means of tagging proteins site-specifically with a lanthanide ion. Here we show that cysteine residues placed in different positions of the LBT can be used to expose the protein to different orientations of the magnetic susceptibility anisotropy (delta chi) tensor and to generate different molecular alignments in a magnetic field. Delta chi tensors determined by nuclear magnetic resonance (NMR) spectroscopy for LBT complexes with Yb3+, Tm3+, and Er3+ suggest a rational way of producing alignment tensors with different orientations. In addition, knowledge of the delta chi tensor of LBT allows modeling of the protein-LBT structures. Despite evidence for residual mobility of the LBTs with respect to the protein, the pseudocontact shifts and residual dipolar couplings displayed by proteins disulfide-bonded to LBTs are greater than those achievable with most other lanthanide binding tags.     

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.     

Physical interpretation of residual dipolar couplings in neutral aligned media

A novel method is described for rapidly calculating alignment tensors from hydrodynamic shape, required for the prediction of residual dipolar couplings in neutral aligned media. Simulations of alignment were used to show that for steric restriction at a planar surface, the alignment process is dependent on linear hydrodynamic length. However, as discussed, previous methods are not in agreement with this observation. Therefore, the method presented here is the first to provide simple, accurate predictions of the alignment tensor for neutral and dilute media, while being consistent with simulations of alignment. It provides predictions in a fraction of the time of a simulation approach, while aiding physical intuition by providing a direct link between shape and alignment. Not only is this physically gratifying, but it also permits residual dipolar couplings to be applied in demanding situations where simulations of alignment are not desirable, such as in studies of molecular dynamics.     

Measurement of carbon-proton dipolar couplings in liquid crystals using DAPT

Dipolar couplings provide valuable information on order and dynamics in liquid crystals. For measuring heteronuclear dipolar couplings in oriented systems, a new separated local field experiment is presented here. The method is based on the dipolar assisted polarization transfer (DAPT) pulse sequence proposed recently (Chem. Phys. Lett. 2007, 439, 407) for transfer of polarization between two spins I and S. DAPT utilizes the evolution of magnetization of the I and S spins under two blocks of phase shifted BLEW-12 pulses on the I spin separated by a 90 degree pulse on the S spin. Compared to the rotating frame techniques based on Hartmann-Hahn match, this approach is easy to implement and is independent of any matching conditions. DAPT can be utilized either as a proton encoded local field (PELF) technique or as a separated local field (SLF) technique, which means that the heteronuclear dipolar coupling can be obtained by following either the evolution of the abundant spin like proton (PELF) or that of the rare spin such as carbon (SLF). We have demonstrated the use of DAPT both as a PELF and as a SLF technique on an oriented liquid crystalline sample at room temperature and also have compared its performance with PISEMA. We have also incorporated modifications to the original DAPT pulse sequence for (i) improving its sensitivity and (ii) removing carrier offset dependence.     

Determination of long range dipolar couplings in ferroelectric liquid crystals

Long range dipolar coupling constants have been determined in three ferroelectric liquid crystals in their racemic forms using ¹³C NMR. Two of these liquid crystals are esters of α-chloroacids and 4-octyloxy-4'-hydroxybiphenyl, and have a very large spontaneous polarization in the smectic C* phase. The strategy used in the present study is the observation and measurement of ²H-¹³C splittings in the ¹³C spectra of monodeuterated compounds. The order parameters were calculated from the 1D spectra, and some of the coupling constants are compared with the ¹H-¹³C coupling constants previously obtained from 2D experiments. In addition, the deuterium quadrupole splitting of these compounds was determined from their ²H NMR spectra. The experiments were carried out over the whole mesomorphic ranges of the liquid crystals, covering the smectic A and smectic C phases.     

Configuration assignment in small organic molecules via residual dipolar couplings

Here we propose a new method to assign relative configurations of stereocenters in small organic molecules by using residual dipolar couplings; the main advantage of this method is that spatial proximity of the stereocenters is not required.     

Technique for generating three-dimensional alignments of multiple ligands from one-dimensional alignments

We describe and demonstrate a method for the simultaneous, fully flexible alignment of multiple molecules with a common biological activity. The key aspect of the algorithm is that the alignment problem is first solved in a lower dimensional space, in this case using the one-dimensional representations of the molecules. The three-dimensional alignment is then guided by constraints derived from the one-dimensional alignment. We demonstrate using 10 hERG channel blockers, with a total of 72 rotatable bonds, that the one-dimensional alignment is able to effectively isolate key conserved pharmacophoric features and that these conserved features can effectively guide the three-dimensional alignment. Further using 10 estrogen receptor agonists and 5 estrogen receptor antagonists with publicly available cocrystal structures we show that the method is able to produce superpositions comparable to those derived from crystal structures. Finally, we demonstrate, using examples from peptidic CXCR3 agonists, that the method is able to generate reasonable binding hypotheses.     

Vibrational corrections of dipolar couplings: methylfluoride and 1,1-difluoroethene

Previously calculated vibrational corrections for methylfluoride are discussed and harmonic corrections are applied to 1,1-difluoroethane. For both molecules, vibrational motions do not account for inconsistencies between the measured anisotropic couplings and the geometry.     

On the origin of residual dipolar couplings from denatured proteins

Effects of steric obstruction on random flight chains are examined. Spatial probability distributions are elaborated to calculate residual dipolar couplings and residual chemical shift anisotropy, parameters that are acquired by NMR spectroscopy from solutes dissolved in dilute liquid crystals. Calculations yield chain length and residue position-dependent values in good agreement with simulations to provide understanding of recently acquired data from denatured proteins.     

Glycosidic torsional motions in a bicelle-associated disaccharide from residual dipolar couplings

An analysis of torsional motions about glycosidic bonds in a disaccharide is undertaken using residual dipolar coupling measurements and selective immobilization of the reducing end sugar to provide a suitable motional reference. The immobilization is accomplished by using the short chain of an alkyl glycoside to anchor the disaccharide to a bilayer medium aligned in magnetic field. Motions about the beta-(1-4) linkage of the n-butyl-4-O-beta-d-galactopyranosyl-alpha-d-mannopyranoside are shown to be substantial (+/-40 degrees ) and in good agreement with predictions of a fully solvated molecular dynamics simulation.     

Chi1 torsion angle dynamics in proteins from dipolar couplings.

Experiments are presented for the measurement of one-bond carbon-proton dipolar coupling values at CH and CH2 ositions in 13C-labeled, approximately 50% fractionally deuterated proteins. 13Cbeta-1Hbeta dipolar couplings have been measured for 38 of 49 possible residues in the 63-amino-acid B1 domain of peptostreptococcal protein L in two aligning media and interpreted in the context of side-chain chi1 torsion angle dynamics. The beta protons for 18 of the 25 beta-methylene-containing amino acids for which dipolar data are available can be unambiguously stereoassigned, and for those residues which are best fit to a single rotamer model the chi(1) angles obtained deviate from crystal structure values by only 5.2 degrees (rmsd). The results for 11 other residues are significantly better fit by a model that assumes jumps between the three canonical (chi1 approximately -60 degrees, 60 degrees, 180 degrees ) rotamers. Relative populations of the rotamers are determined to within +/-6% uncertainty on average and correlate with dihedral angles observed for the three molecules in the crystal asymmetric unit. Entropic penalties for quenching chi1 jumps are considered for six mobile residues thought to be involved in binding to human immunoglobulins. This study demonstrates that dipolar couplings may be used to characterize both the conformation of static residues and side-chain motion with high precision.     

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.