Application Of Residual Dipolar Couplings Research Articles

Stereochemical investigation of flexible macrocyclic cembranes depending on residual dipolar couplings method

It is a great challenge to unambiguously validate stereochemistry for flexible macrocyclic compounds, such as cembrane diterpenoids. In the present study, the feasibility of residual dipolar couplings (RDCs) method is verified using ten cembranes with the configurations confirmed by X-ray diffraction. The result showed that the application of RDCs is reliable for the stereochemical elucidation of most compounds. But it is less determination for the stereochemistry of some other compounds. Nevertheless, a new idea was provided that the reconstructed correlation data which was processed by tick out the flexible fragments in superimposed conformers, which relied on the occasioned problem that the correct configuration should have the best agreement with the experimental result for RDCs.

Advances in NMR Spectroscopy of Weakly Aligned Biomolecular Systems.

The measurement and application of residual dipolar couplings (RDCs) in solution NMR studies of biological macromolecules has become well established over the past quarter of a century. Numerous methods for generating the requisite anisotropic orientational molecular distribution have been demonstrated, each with its specific strengths and weaknesses. In parallel, an enormous number of pulse schemes have been introduced to measure the many different types of RDCs, ranging from the most widely measured backbone amide 15N-1H RDCs, to 1H-1H RDCs and couplings between low-γ nuclei. Applications of RDCs range from structure validation and refinement to the determination of relative domain orientations, the measurement of backbone and domain motions, and de novo structure determination. Nevertheless, it appears that the power of the RDC methodology remains underutilized. This review aims to highlight the practical aspects of sample preparation and RDC measurement while describing some of the most straightforward applications that take advantage of the exceptionally precise information contained in such data. Some emphasis will be placed on more recent developments that enable the accurate measurement of RDCs in larger systems, which is key to the ongoing shift in focus of biological NMR spectroscopy from structure determination toward gaining improved understanding of how molecular flexibility drives protein function.

Stereochemical Elucidation of Natural Products from Residual Chemical Shift Anisotropies in a Liquid Crystalline Phase.

Determination of the stereochemistry of organic molecules still represents one of the major obstacles in the structure elucidation procedure in drug discovery. Although the application of residual dipolar couplings (RDCs) has revolutionized this field, residual chemical shift anisotropies (RCSAs) which contain valuable structural information for nonprotonated carbons have only been scarcely employed so far. In this study, we present a simple but highly effective solution to extract RCSAs of the analytes in a liquid crystalline phase formed by AAKLVFF oligopeptides. This method does not require any special instruments, devices, or correction during postacquisition data analysis and thus can be easily applied in any chemistry laboratory. To illustrate the potential of this method, the relative configurations of four known natural products (1-4) belonging to different structural classes were confirmed. Moreover, we unambiguously elucidated the stereochemistry of spiroepicoccin A (5), a rare thiodiketopiperazine marine natural product whose configuration could not be assigned based on conventional NMR methods.

Application of Residual Dipolar Couplings and Selective Quantitative NOE to Establish the Structures of Tetranortriterpenoids from Xylocarpus rumphii.

Nine triterpenoid derivatives were isolated from the heartwood of Xylocarpus rumphii and were identified as xylorumphiins E (1), C (2), L (3), and M-R (4-9). Compounds 4-9 have a hemiacetal group in the triterpenoid side chain, making them impossible to purify. Purification was achieved after acetylation and subsequent separation of the epimeric mixtures of acetates; however differentiaition of the R and S epimers was not possible using standard NMR techniques. In one case, the relative configuration of a remotely located stereocenter with respect to the stereocenters in the main skeleton was unambiguously determined using residual dipolar couplings. Dipolar couplings were collected from the sample oriented in compressed poly(methyl methacrylate) gels swollen in CDCl3. In another case, the relative configuration was determined using 1D selective quantitative NOE experiments. Xylorumphiin K (10), xyloccensin E, taraxer-14-en-3β-ol, (22S)-hydroxytirucalla-7,24-diene-3,23-dione, and 25-hydroxy-(20S,24S)-epoxydammaran-3-one were isolated from the bark of the same plant. Compounds 3-10 are new compounds. Compounds 1-6 and xyloccensin E were tested at one concentration, 1 × 10-5 M, in the NCI59 cell one-dose screen but did not show significant activity.

Perspectives in the application of residual dipolar couplings in the structure elucidation of weakly aligned small molecules.

This perspective article aims to review the general methodology in the application of residual dipolar couplings (RDCs) in the structure elucidation of small molecules and give the author's view on challenges for future applications. Recent improvements in the availability of alignment media, new pulse sequences for the measurement of couplings and improvements in the analysis software have garnered widespread interest in the technique. However, further generalization is needed in order to make RDC analysis into a truly "routine" method. Copyright © 2016 John Wiley & Sons, Ltd.

Variable helix elongation as a tool to modulate RNA alignment and motional couplings

The application of residual dipolar couplings (RDCs) in studies of RNA structure and dynamics can be complicated by the presence of couplings between collective helix motions and overall alignment and by the inability to modulate overall alignment of the molecule by changing the ordering medium. Here, we show for a 27-nt TAR RNA construct that variable levels of helix elongation can be used to alter both overall alignment and couplings to collective helix motions in a semi-predictable manner. In the absence of elongation, a four base-pair helix II capped by a UUCG apical loop exhibits a higher degree of order compared to a six base-pair helix I ( ϑ I / ϑ II = 0.56 ± 0.1 ). The principal S zz direction is nearly parallel to the axis of helix II but deviates by ∼40° relative to the axis of helix I. Elongating helix I by three base-pairs equalizes the alignment of the two helices and pushes the RNA into the motional coupling limit such that the two helices have comparable degrees of order ( ϑ I / ϑ II = 0.92 ± 0.04 ) and orientations relative to S zz (∼17°). Increasing the length of elongation further to 22 base-pairs pushes the RNA into the motional decoupling limit in which helix I dominates alignment ( ϑ II / ϑ I = 0.45 ± 0.05 ), with S zz orientated nearly parallel to its helix axis. Many of these trends can be rationalized using PALES simulations that employ a previously proposed three-state dynamic ensemble of TAR. Our results provide new insights into motional couplings, offer guidelines for assessing their extent, and suggest that variable degrees of helix elongation can allow access to independent sets of RDCs for characterizing RNA structural dynamics.

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.

Application of Residual Dipolar Couplings in Organic Compounds

AbstractFor Abstract see ChemInform Abstract in Full Text.

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.

Short-range, Long-range and Transition State Interactions in the Denatured State of ACBP from Residual Dipolar Couplings

Residual dipolar couplings in the denatured state of bovine acyl-coenzyme A binding protein (ACBP) oriented in strained polyacrylamide gels have been shown to be a sensitive, sequence-specific probe for residual secondary structure. Results supporting this were obtained by comparing residual dipolar couplings under different denaturing conditions. The data were analyzed using the program molecular fragment replacement (MFR), which demonstrated α-helix propensity in four isolated stretches along the protein backbone, and these coincide with the location of native helices. This is in full agreement with earlier findings based on secondary chemical shift values. Furthermore, N–H residual dipolar couplings provided direct evidence for the existence of native-like hydrophobic interactions in the acid-denatured state of ACBP at pH 2.3. It was shown that replacement of the hydrophobic side-chain of residue Ile27 with alanine in helix A2 leads to large decreases of residual dipolar couplings in residues that form helix A4 in the native state. It is suggested that the Ile to Ala mutation changes the probability for the formation of long-range interactions, which are present in the acid-denatured state of the wild-type protein. These long-range interactions are similar to those proposed to form in the transition state of folding of ACBP. Therefore, the application of residual dipolar couplings in combination with a comparative mutation study has demonstrated the presence of precursors to the folding transition state under acid-unfolding conditions.

High-resolution X-ray and NMR Structures of the SMN Tudor Domain: Conformational Variation in the Binding Site for Symmetrically Dimethylated Arginine Residues

The SMN protein, which is linked to spinal muscular atrophy (SMA), plays an important role in the assembly of the spliceosomal small nuclear ribonucleoprotein complexes. This function requires binding of SMN to the arginine-glycine (RG) rich C-terminal tails of the Sm proteins, which contain symmetrically dimethylated arginine residues (sDMA) in vivo. Using NMR titrations, we show that the SMN Tudor domain recognizes these sDMAs in the methylated RG repeats. Upon complex formation a cluster of conserved aromatic residues in the SMN Tudor domain interacts with the sDMA methyl groups. We present two high resolution structures of the uncomplexed SMN Tudor domain, a 1.8 Å crystal structure and an NMR structure that has been refined against a large number of backbone and side-chain residual dipolar couplings. The backbone conformation of both structures is very similar, however, differences are observed for the cluster of conserved aromatic side-chains in the sDMA binding pocket. In order to validate these variations we introduce a novel application of residual dipolar couplings for aromatic rings. We show that structural information can be derived from aromatic ring residual dipolar couplings, even in the presence of internal motions such as ring flipping. These residual dipolar couplings and ring current shifts independently confirm that the SMN Tudor domain adopts two different conformations in the sDMA binding pocket. The observed structural variations may play a role for the recognition of sDMAs.

Incorporating residual dipolar couplings into the NMR solution structure determination of nucleic acids.

NMR solution structures of nucleic acids are generally less well defined than similar-sized proteins. Most NMR structures of nucleic acids are defined only by short-range interactions, such as intrabase-pair or sequential nuclear Overhauser effects (NOEs), and J-coupling constants, and there are no long-range structural data on the tertiary structure. Residual dipolar couplings represent an extremely valuable source of distance and angle information for macromolecules but they average to zero in isotropic solutions. With the recent advent of general methods for partial alignment of macromolecules in solution, residual dipolar couplings are rapidly becoming indispensable constraints for solution NMR structural studies. These residual dipolar couplings give long-range global structural information and thus complement the strictly local structural data obtained from standard NOE and torsion angle constraints. Such global structural data are especially important in nucleic acids due to the more elongated, less-globular structure of many DNAs and RNAs. Here we review recent progress in application of residual dipolar couplings to structural studies of nucleic acids. We also present results showing how refinement procedures affect the final solution structures of nucleic acids.

Editorial: Structure-function of nucleic acids?following the Tinoco path

Editorial: Structure-function of nucleic acids?following the Tinoco path