Force field dependence of NMR-Based,restrained molecular dynamics DNA structure calculations including an analysis of the influence of residual dipolar coupling restraints

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.     

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.     

Partial alignment and measurement of residual dipolar couplings of proteins under high hydrostatic pressure

High-pressure NMR spectroscopy has emerged as a complementary approach for investigating various structural and thermodynamic properties of macromolecules. Noticeably absent from the array of experimental restraints that have been employed to characterize protein structures at high hydrostatic pressure is the residual dipolar coupling, which requires the partial alignment of the macromolecule of interest. Here we examine five alignment media that are commonly used at ambient pressure for this purpose. We find that the spontaneous alignment of Pf1 phage, d(GpG) and a C12E5/n-hexnanol mixture in a magnetic field is preserved under high hydrostatic pressure. However, DMPC/DHPC bicelles and collagen gel are found to be unsuitable. Evidence is presented to demonstrate that pressure-induced structural changes can be identified using the residual dipolar coupling.     

NMR-Based Simulation Studies of Pf1 Coat Protein in Explicit Membranes

As time- and ensemble-averaged measures, NMR observables contain information about both protein structure and dynamics. This work represents a computational study to extract such information for membrane proteins from orientation-dependent NMR observables: solid-state NMR chemical shift anisotropy and dipolar coupling, and solution NMR residual dipolar coupling. We have performed NMR-restrained molecular dynamics simulations to refine the structure of the membrane-bound form of Pf1 coat protein in explicit lipid bilayers using the recently measured chemical shift anisotropy, dipolar coupling, and residual dipolar coupling data. From the simulations, we have characterized detailed protein-lipid interactions and explored the dynamics. All simulations are stable and the NMR restraints are well satisfied. The C-terminal transmembrane (TM) domain of Pf1 finds its optimal position in the membrane quickly (within 6 ns), illustrating efficient solvation of TM domains in explicit bilayer environments. Such rapid convergence also leads to well-converged interaction patterns between the TM helix and the membrane, which clearly show the interactions of interfacial membrane-anchoring residues with the lipids. For the N-terminal periplasmic helix of Pf1, we identify a stable, albeit dynamic, helix orientation parallel to the membrane surface that satisfies the amphiphatic nature of the helix in an explicit lipid bilayer. Such detailed information cannot be obtained solely from NMR observables. Therefore, the present simulations illustrate the usefulness of NMR-restrained MD refinement of membrane protein structure in explicit membranes.     

NMR-Based Simulation Studies of Pf1 Coat Protein in Explicit Membranes

As time- and ensemble-averaged measures, NMR observables contain information about both protein structure and dynamics. This work represents a computational study to extract such information for membrane proteins from orientation-dependent NMR observables: solid-state NMR chemical shift anisotropy and dipolar coupling, and solution NMR residual dipolar coupling. We have performed NMR-restrained molecular dynamics simulations to refine the structure of the membrane-bound form of Pf1 coat protein in explicit lipid bilayers using the recently measured chemical shift anisotropy, dipolar coupling, and residual dipolar coupling data. From the simulations, we have characterized detailed protein-lipid interactions and explored the dynamics. All simulations are stable and the NMR restraints are well satisfied. The C-terminal transmembrane (TM) domain of Pf1 finds its optimal position in the membrane quickly (within 6 ns), illustrating efficient solvation of TM domains in explicit bilayer environments. Such rapid convergence also leads to well-converged interaction patterns between the TM helix and the membrane, which clearly show the interactions of interfacial membrane-anchoring residues with the lipids. For the N-terminal periplasmic helix of Pf1, we identify a stable, albeit dynamic, helix orientation parallel to the membrane surface that satisfies the amphiphatic nature of the helix in an explicit lipid bilayer. Such detailed information cannot be obtained solely from NMR observables. Therefore, the present simulations illustrate the usefulness of NMR-restrained MD refinement of membrane protein structure in explicit membranes.     

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.     

Quaternary structure built from subunits combining NMR and small-angle x-ray scattering data

A new principle in constructing molecular complexes from the known high-resolution domain structures joining data from NMR and small-angle x-ray scattering (SAXS) measurements is described. Structure of calmodulin in complex with trifluoperazine was built from N- and C-terminal domains oriented based on residual dipolar couplings measured by NMR in a dilute liquid crystal, and the overall shape of the complex was derived from SAXS data. The residual dipolar coupling data serves to reduce angular degrees of freedom, and the small-angle scattering data serves to confine the translational degrees of freedom. The complex built by this method was found to be consistent with the known crystal structure. The study demonstrates how approximate tertiary structures of modular proteins or quaternary structures composed of subunits can be assembled from high-resolution structures of domains or subunits using mutually complementary NMR and SAXS data.     

The global conformation of the hammerhead ribozyme determined using residual dipolar couplings

The global structure of the hammerhead ribozyme was determined in the absence of Mg(2+) by solution NMR experiments. The hammerhead ribozyme motif forms a branched structure consisting of three helical stems connected to a catalytic core. The (1)H-(15)N and (1)H-(13)C residual dipolar couplings were measured in a set of differentially (15)N/(13)C-labeled ribozymes complexed with an unlabeled noncleavable substrate. The residual dipolar couplings provide orientation information on both the local and the global structure of the molecule. Analysis of the residual dipolar couplings demonstrated that the local structure of the three helical stems in solution is well modeled by an A-form conformation. However, the global structure of the hammerhead in solution in the absence of Mg(2+) is not consistent with the Y-shaped conformation observed in crystal structures of the hammerhead. The residual dipolar couplings for the helical stems were combined with standard NOE and J coupling constant NMR data from the catalytic core. The NOE data show formation of sheared G-A base pairs in domain 2. These NMR data were used to determine the global orientation of the three helical stems in the hammerhead. The hammerhead forms a rather extended structure under these conditions with a large angle between stems I and II ( approximately 153 degrees ), a smaller angle between stems II and III ( approximately 100 degrees ), and the smallest angle between stems I and III ( approximately 77 degrees ). The residual dipolar coupling data also contain information on the dynamics of the molecule and were used here to provide qualitative information on the flexibility of the helical domains in the hammerhead ribozyme-substrate complex.     

Nuclear magnetic resonance in the era of structural genomics

Current interests in structural genomics, and the associated need for high through-put structure determination methods, offer an opportunity to examine new nuclear magnetic resonance (NMR) methodology and the impact this methodology can have on structure determination of proteins. The time required for structure determination by traditional NMR methods is currently long, but improved hardware, automation of analysis, and new sources of data such as residual dipolar couplings promise to change this. Greatly improved efficiency, coupled with an ability to characterize proteins that may not produce crystals suitable for investigation by X-ray diffraction, suggests that NMR will play an important role in structural genomics programs.     

The use of NMR residual dipolar couplings in aqueous dilute liquid crystalline medium for conformational studies of complex oligosaccharides

C-H dipolar coupling values were measured for a natural-abundance sample of the pentasaccharide beta-D-Galp-(1-->3)-[alpha-L-Fucp-(1-->4)]-beta-D-GlcNAcp-(1 -->3)-beta-D- Galp-(1-->4)-beta-D-Glcp ('lacto-N-fucopentaose 2') (LNF-2), in a 7.5% solution of dimyristoyl phosphatidylcholine-dihexanoyl phosphatidylcholine bicelle liquid crystals oriented in the NMR magnetic field. Interpretation of the dipolar coupling data and NOE confirms the conformational model for the Lewis(a) trisaccharide epitope based on NOE, molecular dynamics simulations, and scalar coupling data and provided new structural information for the remaining residues of the pentasaccharide. Since residual dipolar coupling provides information on long-range order, it is a valuable complement to other types of NMR data such as NOE and scalar coupling for exploring conformations of complex oligosaccharides.     

Weak alignment offers new NMR opportunities to study protein structure and dynamics

Protein solution nuclear magnetic resonance (NMR) can be conducted in a slightly anisotropic environment, where the orientational distribution of the proteins is no longer random. In such an environment, the large one-bond internuclear dipolar interactions no longer average to zero and report on the average orientation of the corresponding vectors relative to the magnetic field. The desired very weak ordering, on the order of 10(-3), can be induced conveniently by the use of aqueous nematic liquid crystalline suspensions or by anisotropically compressed hydrogels. The resulting residual dipolar interactions are scaled down by three orders of magnitude relative to their static values, but nevertheless can be measured at high accuracy. They are very precise reporters on the average orientation of bonds relative to the molecular alignment frame, and they can be used in a variety of ways to enrich our understanding of protein structure and function. Applications to date have focused primarily on validation of structures, determined by NMR, X-ray crystallography, or homology modeling, and on refinement of structures determined by conventional NMR approaches. Although de novo structure determination on the basis of dipolar couplings suffers from a severe multiple minimum problem, related to the degeneracy of dipolar coupling relative to inversion of the internuclear vector, a number of approaches can address this problem and potentially can accelerate the NMR structure determination process considerably. In favorable cases, where large numbers of dipolar couplings can be measured, inconsistency between measured values can report on internal motions.     

Localization of the binding site for the oligosaccharide moiety of Gb3 on verotoxin 1 using NMR residual dipolar coupling measurements

By use of NMR residual dipolar coupling measurements in a dilute liquid-crystalline solvent, the solution structure has been determined of the complex between the oligosaccharide moiety of globotriaosylceramide (Gb(3)-OS) and the B-subunit homopentamer of verotoxin 1 (VTB). The dipolar coupling data indicate that Gb(3)-OS binds in a single binding site per monomer, which is identical to one of three sites inferred from the X-ray structure of the same complex. We find no evidence within experimental error for occupancy at either of the two additional binding sites observed per monomer in the crystal structure.     

Breaking symmetry in the structure determination of (large) symmetric protein dimers

We demonstrate a novel methodology to disrupt the symmetry in the NMR spectra of homodimers. A paramagnetic probe is introduced sub-stoichiometrically to create an asymmetric system with the paramagnetic probe residing on only one monomer within the dimer. This creates sufficient magnetic anisotropy for resolution of symmetry-related overlapped resonances and, consequently, detection of pseudocontact shifts and residual dipolar couplings specific to each monomeric component. These pseudocontact shifts can be readily incorporated into existing structure refinement calculations and enable determination of monomer orientation within the dimeric protein. This methodology can be widely used for solution structure determination of symmetric dimers.     

NMR methodology for the study of nucleic acids.

During the past few years, NMR methodology for the study of nucleic acids has benefited from new developments that greatly improved state-of-the-art technology for the precise determination of three-dimensional structures. Substantial progress has been made in designing experimental protocols for the measurement of residual dipolar couplings, in sensitivity optimization of triple-resonance experiments and in detection of hydrogen bonds and in developing computational methods for structure refinement using NMR restraints.     

The utility of residual dipolar couplings in detecting motion in carbohydrates: application to sucrose

The solution structure and dynamics of sucrose are examined using a combination of NMR residual dipolar coupling and molecular mechanics force fields. It is found that the alignment tensors of the individual rings are different, and that fitting 35 measured residual dipolar couplings to structures with specific phi, psi values indicates the presence of three major conformations: phi, psi=(120 degrees ,270 degrees), (45 degrees, 300 degrees) and (90 degrees ,180 degrees). Furthermore, fitting two structures simultaneously to the 35 residual dipolar couplings results in a substantial improvement in the fits. The existence of multiple conformations having similar stabilities is a strong indication of motion, due to the interconversion among these states. Results from four molecular mechanics force fields are in general agreement with the experimental results. However, there are major disagreements between force fields. Because fits of residual dipolar couplings to structures are dependent on the force field used to calculate the structures, multiple force fields were used to interpret NMR data. It is demonstrated that the pucker of the fructofuranosyl ring affects the calculated potential energy surface, and the fit to the residual dipolar couplings data. Previously published 13C nuclear relaxation results suggesting that sucrose is rigid are not inconsistent with the present results when motional timescales are considered.     

Conformational studies of human milk oligosaccharides using (1)H-(13)C one-bond NMR residual dipolar couplings

1H-(13)C one-bond dipolar coupling values were measured for natural abundance samples of the human milk oligosaccharides "lacto-N-fucopentaose" (LNF-1 LNF-2, and LNF-3), "lacto-N-difucohexaose" (LND-1), "lacto-N-tetraose" (LNT), and "lacto-N-neo-tetraose" (LNnT), four of which have Lewis blood group epitopes. Each oligosaccharide was dissolved in a 7.5% solution of 1, 2-dimyristoyl-sn-glycero-3-phosphocholine/1, 2-dihexanoyl-sn-glycero-3-phosphocholine (DMPC/DHPC) bicelle liquid crystals oriented in the NMR magnetic field. The dipolar coupling data and NOE were fitted to conformational models with calculations of an optimum orientation tensor which best represents the dipolar coupling values for a fragment hypothesized to adopt a single conformation. In the case of LNF-1, LNF-2, LNF-3, and LND-1, the models confirm previous conformational models for the Lewis epitopes based on NOE and molecular dynamics simulations. Extensions of the model provided new structural data for the remaining residues. In all cases, upper limits for the errors in the glycosidic angles of the models were estimated. Since residual dipolar coupling provides information on long-range order, it is a valuable complement to other types of NMR data such as NOE and scalar coupling for exploring conformations of complex oligosaccharides.     

NMR solution structure of calerythrin, an EF-hand calcium-binding protein from Saccharopolyspora erythraea.

The structure of calerythrin, a prokaryotic 20 kDa calcium-binding protein has been determined by solution NMR spectroscopy. Distance, dihedral angle, J coupling, secondary chemical shift, residual dipolar coupling and radius of gyration restraints reveal four EF-hand motifs arranged in a compact globular structure. A tight turn in the middle of the amino acid sequence brings the two halves, each comprising a pair of EF-hands, close together. The structural similarity between calerythrin and the eukaryotic sarcoplasmic calcium-binding proteins is notable.     

High resolution MAS-NMR in combinatorial chemistry

High-resolution magic angle spinning (hr-MAS) NMR is a powerful tool for characterizing organic reactions on solid support. Because magic angle spinning reduces the line-broadening due to dipolar coupling and variations in bulk magnetic susceptibility, line widths approaching those obtained in solution-phase NMR can be obtained. The magic angle spinning method is amenable for use in conjunction with a variety of NMR-pulse sequences, making it possible to perform full-structure determinations and conformational analysis on compounds attached to a polymer support. Diffusion-weighted MAS-NMR methods such as SPEEDY (Spin-Echo-Enhanced Diffusion-Filtered Spectroscopy) can be used to remove unwanted signals from the solvent, residual reactants, and the polymer support from the MAS-NMR spectrum, leaving only those signals arising from the resin-bound product. This review will present the applications of high-resolution magic angle spinning NMR for use in combinatorial chemistry research.