NMR experiments for the measurement of proton-proton and carbon-carbon residual dipolar couplings in uniformly labelled oligosaccharides

A 2D-HSQC-carbon selective/proton selective-constant time COSY, 2D-HSQC-(sel C, sel H)-CT COSY experiment, which is applicable to uniformly ¹³C isotopically enriched samples (U-¹³C) of oligosaccharides or oligonucleotides is proposed for the measurement of proton–proton RDC in crowded regions of 2D-spectra. In addition, a heteronuclear constant time-COSY experiment, ¹³C-¹³C CT-COSY, is proposed for the measurement of one bond carbon–carbon RDC in these molecules. These two methods provide an extension, to U-¹³C molecules, of the original homonuclear constant time-COSY experiment proposed by Tian et al. (1999) for saccharides. The combination of a number of these RDC with NOE data may provide the method of choice to study oligosaccharide conformation in the free and receptor-bound state.     

Conformational studies of blood group A and blood group B oligosaccharides using NMR residual dipolar couplings

The conformations of two synthetic trisaccharides of blood group A and B (alpha-L-Fucp-(1-->2)-[alpha-D-GalpNAc-(1-->3)]-alpha-D-Galp and alpha-L-Fucp-(1-->2)-[alpha-D-Galp-(1-->3)]-alpha-D-Galp, respectively) and of a type A tetrasaccharide alditol, Fucp-(1-->2)-[alpha-D-GalpNAc-(1-->3)]-beta-D-Galp-(1-->3)-GalNAc-ol, were studied by NMR measurements of one-bond C-H residual dipolar couplings in partially oriented liquid crystal solutions. The conformations of the three oligosaccharides were analyzed by generating thousands of structures using a Monte-Carlo method. Two different strategies were applied to calculate theoretical dipolar couplings for these structures. In the first method, the orientation of the molecule was calculated from the optimal fit of the molecular model to the experimental data, while in the second method the orientation tensor was calculated directly from the moment of inertia of the molecular model. Both methods of analysis give similar results but with slightly better agreement with experiment for the former one. The analysis of the results implies a single unique conformation for both blood group epitopes in solution in disagreement with theoretical models suggesting the existence of two conformers in solution.     

NMR investigations of allosteric processes in a two-domain Thermus thermophilus Hsp70 molecular chaperone

Hsp70 chaperones are two-domain proteins that assist in intra-cellular protein (re) folding processes in all species. The protein folding activity of the substrate binding domain of the Hsp70s is regulated by nucleotide binding at the nucleotide-binding domain through an as yet undefined heterotropic allosteric mechanism. The available structures of the isolated domains of Hsp70s have given very limited indications of nucleotide-induced conformational changes that could modulate the affinity for substrate proteins. Here, we present a multi-dimensional NMR study of a prokaryotic Hsp70 homolog, Thermus thermophilus DnaK, using a 54kDa construct containing both nucleotide binding domain and most of the substrate binding domain. It is determined that the nucleotide binding domain and substrate binding domain are closely associated in all ligand states studied. Comparison of the assigned NMR spectra of the two-domain construct with those of the previously studied isolated nucleotide binding domain, allowed the identification of the nucleotide binding domain-substrate binding domain interface. A global three-dimensional structure was obtained for the two-domain construct on the basis of this information and of NMR residual dipolar couplings measurements. This is the first experimental elucidation of the relative positioning of the nucleotide binding domain and substrate binding domain for any Hsp70 chaperone. Comparisons of NMR data between various ligand states including nucleotide-free, ATP, ADP.Pi and ADP.Pi+ peptide bound, identified residues involved in the allosteric inter-domain communication. In particular, peptide binding to the substrate binding domain was found to cause conformational changes in the NBD extending to the nucleotide binding pocket. Detailed analysis suggests that the inter-domain interface becomes tighter in the (nucleotide binding domain ligation/substrate binding domain ligation) order ATP/apo, ADP.Pi/apo ADP.Pi/peptide.     

Accurate determination of leucine and valine side-chain conformations using U-[15N/13C/2H]/[1H-(methine/methyl)-Leu/Val] isotope labeling, NOE pattern recognition, and methine Cgamma-Hgamma/Cbeta-Hbeta residual dipolar couplings: application to the 34-kDa enzyme IIA(chitobiose)

An isotope labeling scheme is described in which specific protonation of methine and methyl protons of leucine and valine is obtained on a ¹⁵N/¹³C labeled background with uniform deuteration of all other non-exchangeable protons. The presence of a protonated methine group has little effect on the favorable relaxation properties of the methyl protons of Leu and Val. This labeling scheme permits the rotameric state of leucine side-chains to be readily determined by simple inspection of the pattern of Hγ(i)–H_N(i) and Hγ(i)–H_N(i+1) NOEs in a 3D ¹⁵N-separated NOE spectrum free of complications arising from spectral overlap and spin-diffusion. In addition, one-bond residual dipolar couplings for the methine ¹³C–¹H bond vectors of Leu and Val can be accurately determined from an intensity J-modulated constant-time HCCH-COSY experiment and used to accurately orient the side-chains of Leu and Val. Incorporation of these data into structure refinement improves the accuracy with which the conformations of Leu and Val side-chains can be established. This is important to ensure optimal packing both within the protein core and at intermolecular interfaces. The impact of the method on protein structure determination is illustrated by application to enzyme IIA^Chitobiose, a 34 kDa homotrimeric phosphotransferase protein.     

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.     

Structural characterization of a mannose-binding protein-trimannoside complex using residual dipolar couplings

The ligand-binding properties of a 53 kDa homomultimeric trimer from mannose-binding protein (MBP) have been investigated using residual dipolar couplings (RDCs) that are easily measured from NMR spectra of the ligand and isotopically labeled protein. Using a limited set of 1H-15N backbone amide NMR assignments for MBP and orientational information derived from the RDC measurements in aligned media, an order tensor for MBP has been determined that is consistent with symmetry-based predictions of an axially symmetric system. 13C-1H couplings for a bound trisaccharide ligand, methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside (trimannoside) have been determined at natural abundance and used as orientational constraints. The bound ligand geometry and orientational constraints allowed docking of the trimannoside ligand in the binding site of MBP to produce a structural model for MBP-oligosaccharide interactions.     

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.     

Kinetics of drug release from a hyaluronan-steroid conjugate investigated by NMR spectroscopy

The methylprednisolone steroid ester of hyaluronan was hydrolyzed under physiological conditions in vitro, and the kinetics of drug release was investigated by NMR spectroscopy. Transverse relaxation times are correlated with the molecular rotational freedom, which undergoes large changes for methylprednisolone when released. Multi-exponential decays were observed, which together with the corresponding population gave valuable insights into the conformational changes that occur in the biopolymer during hydrolysis. The biomaterial exists in aqueous solution in two conformations, ‘collapsed’ and ‘water-exposed’, in equilibrium. Under physiological conditions, the methylprednisolone is completely released within 48 h. Transverse relaxation times proved to be an appropriate tool for monitoring the drug release in vitro.     

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.     

Bioactive conformation of a potent stromelysin inhibitor determined by X-nucleus filtered and multidimensional NMR spectroscopy

The biologically active conformation of a novel, very potent, nonpeptidic stromelysin inhibitor was determined by X-nucleus filtered and multidimensional NMR spectroscopy. This bound conformer was subsequently docked into the stromelysin catalytic domain (SCD) using intermolecular distance constraints derived from NOE data. The complex showed the S1′ pocket of stromelysin to be the major site of enzyme-inhibitor interaction with other portions of the inhibitor spanning the S2′ and S1 binding sites. Theoretical predictions of SCD-inhibitor binding from molecular modeling studies were consistent with the NMR data. Comparison of modeled enzyme-inhibitor complexes for stromelysin and collagenase revealed an alternate binding mode for the inhibitor in collagenase, suggesting a similar binding interaction might also be possible for stromelysin. The NMR results, however, revealed a single SCD-inhibitor binding mode and provided a structural template for the design of more potent stromelysin inhibitors.     

Determination of the residue-specific 15N CSA tensor principal components using multiple alignment media

The individual components of the backbone ¹⁵N CSA tensor, σ₁₁, σ₂₂, σ₃₃, and the orientation of σ₁₁ relative to the NH bond described by the angle β have been determined for uniformly labeled ¹⁵N, ¹³C ubiquitin from partial alignment in phospholipid bicelles, Pf1 phage, and poly(ethylene glycol) by measuring the residue-specific residual dipolar couplings and chemical shift deviations. No strong correlation between any of the CSA tensor components is observed with any single structural feature. However, the experimentally determined tensor components agree with the previously determined average CSA principal components [Cornilescu and Bax (2000) J. Am. Chem. Soc. 122, 10143–10154]. Significant deviations from the averages coincide with residues in β-strand or extended regions, while α-helical residue tensor components cluster close to the average values.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

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.     

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.     

Evidence of molecular alignment fluctuations in aqueous dilute liquid crystalline media

Protein dynamics can be studied by NMR measurements of aqueous dilute liquid crystalline samples. However, the measured residual dipolar couplings are sensitive not only to internal fluctuations but to all changes in internuclear vectors relative to the laboratory frame. We show that side-chain fluctuations and bond librations in the ps–ns time scale perturb the molecular shape and charge distribution of a small globular protein sufficiently to cause a noticeable variation in the molecular alignment. The alignment variation disperses the bond vectors of a conformational ensemble even further from the dispersion already caused by internal fluctuations of a protein. Consequently RDC-probed order parameters are lower than those obtained by laboratory frame relaxation measurements.     

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