13C-31P couplings in the study of conformational properties of some diastereomeric nucleoside-3'-thiophosphate derivatives

Synthesis and stereochemical characterization of enantiomerically pure nucleoside-3'-phosphorothioate esters and salts are reported. Vicinal carbon phosphorus couplings reflect different predominance of the epsilon conformation in the isomeric (Rp and Sp) esters, while for the salts the epsilont conformation prevails in both stereoisomers. The influence of solvent and temperature on the conformational preferences is also described.     

Solution NMR conformation of glycosaminoglycans

Nuclear magnetic resonance (NMR) spectroscopy has been giving a pivotal contribution to the progress of glycomics, mostly by elucidating the structural, dynamical, conformational and intermolecular binding aspects of carbohydrates. Particularly in the field of conformation, NOE resonances, scalar couplings, residual dipolar couplings, and chemical shift anisotropy offsets have been the principal NMR parameters utilized. Molecular dynamics calculations restrained by NMR-data input are usually employed in conjunction to generate glycosidic bond dihedral angles. Glycosaminoglycans (GAGs) are a special class of sulfated polysaccharides extensively studied worldwide. Besides regulating innumerous physiological processes, these glycans are also widely explored in the global market as either clinical or nutraceutical agents. The conformational aspects of GAGs are key regulators to the quality of interactions with the functional proteins involved in biological events. This report discusses the solution conformation of each GAG type analyzed by one or more of the above-mentioned methods.     

Conformational properties of diastereomeric 5'-O-DMT-2'deoxythymidine 3'-O-(S-methyl-methanephosphonothiolate)s and -(se-methyl-methanephosphonoselenolate)s in solution as studied by NMR methods

Stereochemical characterization of diastereomerically pure 5'-O-DMT-2'deoxythymidine 3'-O-(S-methyl-methanephosphonothiolate)s and -(Se-methyl-methane-phosphono-selenolate)s by NMR methods are reported. (1)H-(1)H, (1)H-(31)P, and (13)C-(31)P coupling constants and nuclear overhauser enhancement (NOE) connectivities from transverse cross-relaxation experiments in rotating frame (T-ROESY) were measured to correlate the conformational properties of the isomers with the absolute configurations at the phosphorus obtained from X-ray studies of the relatives of S(p) configured isomers. Conformational differences between the stereoisomers were found to be restricted to the different orientation of the C3'-O3'-P bond. The NMR data reflected the preferred epsilon(-) conformation for the S(p) isomers, while in the R(p) isomers the conformational equilibrium was shifted toward the epsilon(t) domain. These results also indicated that for 5'-protected mononucleotides the absolute configuration at the phosphorus atom can be inferred from the NOE experiments and the trends observed in vicinal carbon-phosphorus coupling constants. Copyright 2000 Wiley-Liss, Inc.     

Orienting domains in proteins using dipolar couplings measured by liquid-state NMR: differences in solution and crystal forms of maltodextrin binding protein loaded with beta-cyclodextrin

Protein function is often regulated by conformational changes that occur in response to ligand binding or covalent modification such as phosphorylation. In many multidomain proteins these conformational changes involve reorientation of domains within the protein. Although X-ray crystallography can be used to determine the relative orientation of domains, the crystal-state conformation can reflect the effect of crystal packing forces and therefore may differ from the physiologically relevant form existing in solution. Here we demonstrate that the solution-state conformation of a multidomain protein can be obtained from its X-ray structure using an extensive set of dipolar couplings measured by triple-resonance multidimensional NMR spectroscopy in weakly aligning solvent. The solution-state conformation of the 370-residue maltodextrin-binding protein (MBP) loaded with beta-cyclodextrin has been determined on the basis of one-bond (15)N-H(N), (15)N-(13)C', (13)C(alpha)-(13)C', two-bond (13)C'-H(N), and three-bond (13)C(alpha)-H(N) dipolar couplings measured for 280, 262, 276, 262, and 276 residues, respectively. This conformation was generated by applying hinge rotations to various X-ray structures of MBP seeking to minimize the difference between the experimentally measured and calculated dipolar couplings. Consistent structures have been derived in this manner starting from four different crystal forms of MBP. The analysis has revealed substantial differences between the resulting solution-state conformation and its crystal-state counterpart (Protein Data Bank accession code 1DMB) with the solution structure characterized by an 11(+/-1) degrees domain closure. We have demonstrated that the precision achieved in these analyses is most likely limited by small uncertainties in the intradomain structure of the protein (ca 5 degrees uncertainty in orientation of internuclear vectors within domains). In addition, potential effects of interdomain motion have been considered using a number of different models and it was found that the structures derived on the basis of dipolar couplings accurately represent the effective average conformation of the protein.     

Carbon-13 NMR in conformational analysis of nucleic acid fragments. 4. The torsion angle distribution about the C3''-O3'' bond in DNA constituents.

Carbon-13 and proton NMR spectra of a series of oligodeoxynucleotides (d(CT), d(CC), d(TA), d(AT), d(CG), d(GC), d(AG), d(AAA), d(TATA) and d(GGTAAT] were measured at various temperatures. The three coupling constants that are related to the magnitude of backbone angle epsilon (J(C4'-P), J(C2'-P) and J(H3'-P] are analyzed in terms of a three-state equilibrium about this bond. Two epsilon (trans) angles occur, which differ in magnitude depending on the conformation (N or S) of the adjoining deoxyribose ring. The S-type deoxyribose ring is associated with a smaller epsilon (trans) angle: epsilon (t,S) = 192 degrees. The N-type deoxyribose ring is associated with a larger epsilon (trans) angle epsilon (t,N) = 212 degrees. The third rotamer participating in the conformational equilibrium, is a gauche(-) (epsilon (-] conformer and occurs exclusively in combination with the S-type sugar ring (epsilon (-,S) = 266 degrees). Within the limits of experimental error, the magnitude of these three angles appears to be independent of the particular base sequence, except in the case of d(CG) where a slightly larger epsilon (t,S) angle (197 degrees) is indicated. A simple equation is proposed which may be used to calculate the population of epsilon (t,S) conformer in cases where only J(H3'-P) is known.     

Sucrose in aqueous solution revisited, Part 1: molecular dynamics simulations and direct and indirect dipolar coupling analysis

Although the crystal structure of the disaccharide sucrose was solved more than 30 years ago, its conformational distribution in aqueous solution is still a matter of debate. We report here a variety of molecular dynamics simulations (mostly of 100 ns) using the GLYCAM06 force field and various water models, paying particular attention to comparisons to NMR measurements of residual dipolar couplings and electron-mediated spin-spin couplings. We focus on the glycosidic linkage conformation, the puckering phase angle of the fructose ring, and intramolecular hydrogen bonds between the two sugars. Our results show that sucrose is indeed a dynamic molecule, but the crystal conformation is qualitatively the dominant one in dilute solution. A second conformational basin, populated in many force fields, is probably overstabilized in the calculations.     

Concerted motions in HIV-1 TAR RNA may allow access to bound state conformations: RNA dynamics from NMR residual dipolar couplings.

Ground-state dynamics in RNA is a critical precursor for structural adaptation observed ubiquitously in protein-RNA recognition. A tertiary conformational analysis of the stem-loop structural element in the transactivation response element (TAR) from human immunodeficiency virus type 1 (HIV-I) RNA is presented using recently introduced NMR methods that rely on the measurement of residual dipolar couplings (RDC) in partially oriented systems. Order matrix analysis of RDC data provides evidence for inter-helical motions that are of amplitude 46(+/-4) degrees, of random directional character, and that are executed about an average conformation with an inter-helical angle between 44 degrees and 54 degrees. The generated ensemble of TAR conformations have different organizations of functional groups responsible for interaction with the trans-activator protein Tat, including conformations similar to the previously characterized bound-state conformation. These results demonstrate the utility of RDC-NMR for simultaneously characterizing RNA tertiary dynamics and average conformation, and indicate an avenue for TAR complex formation involving tertiary structure capture.     

13C-31P Couplings in the Study of Conformational Properties of Some Diastereomeric Nucleoside-3′-Thiophosphate Derivatives

Synthesis and stereochemical characterization of enantiomerically pure nucleoside-3′-phosphorothioate esters and salts are reported. Vicinal carbon–phosphorus couplings reflect different predominance of the ? conformation in the isomeric (Rp and Sp) esters, while for the salts the ?^t conformation prevails in both stereoisomers. The influence of solvent and temperature on the conformational preferences is also described.     

Tetrahydrobiopterin analogues: solution conformations of 6-methyltetrahydropterin, 7-methyltetrahydropterin, and cis- and trans-6,7-dimethyltetrahydropterins as determined by proton nuclear magnetic resonance

The conformation of tetrahydrobiopterin analogues in aqueous solution at 23 degrees C has been determined by analyzing the 200-MHz 1H NMR spectral parameters of the enzymatically active 6-methyltetrahydropterin, 7-methyltetrahydropterin, and cis- and trans-6,7-dimethyl-5,6,7,8-tetrahydropterins. Each of these cofactors, with the exception of the cis-6,7-dimethyl isomer, exhibited an unusually small trans 6H-7H spin-spin coupling (8.5-9.1 Hz). An empirical equation that accounts for the effects of substituent electronegativity and orientation on vicinal couplings [Haasnoot, C. A. G., deLeeuw, F. A. A. M., & Altona, C. (1980) Tetrahedron 36, 2783-2792] predicted this coupling to be 11.3-11.6 Hz. We attribute the discrepancy between the calculated and experimentally observed values of this coupling to hyperconjugation of the axially oriented C7-H bond with the pi orbital of the vinylogous amide protein of the pterin ring (N8-C8a = C4a-C4 = O) rather than conformational averaging. The trans 6H-7H interproton distance in the 6-methyl analogue is calculated to be 3.0 A from the measured decrease in the spin-lattice relaxation rate of the axially oriented C7 proton after specific deuteration at C6. This is consistent with the single-conformer interpretation. Chemical shift comparisons of the methyl resonances of these analogues, NOE measurements from selectively deuterated analogues, and the differential sensitivities of axially vs. equatorially disposed ring protons to protonation at N5 all indicate that (i) the methyl substituents at both the C6 and C7 positions markedly prefer equatorial-like orientations and (ii) the tetrahydropterin ring is, with the exception of a pronounced pucker at C6, nearly planar.(ABSTRACT TRUNCATED AT 250 WORDS)     

All-atom molecular dynamics simulations using orientational constraints from anisotropic NMR samples

Orientational constraints obtained from solid state NMR experiments on anisotropic samples are used here in molecular dynamics (MD) simulations for determining the structure and dynamics of several different membrane-bound molecules. The new MD technique is based on the inclusion of orientation dependent pseudo-forces in the COSMOS-NMR force field. These forces drive molecular rotations and re-orientations in the simulation, such that the motional time-averages of the tensorial NMR properties approach the experimentally measured parameters. The orientational-constraint-driven MD simulations are universally applicable to all NMR interaction tensors, such as chemical shifts, dipolar couplings and quadrupolar interactions. The strategy does not depend on the initial choice of coordinates, and is in principle suitable for any flexible molecule. To test the method on three systems of increasing complexity, we used as constraints some deuterium quadrupolar couplings from the literature on pyrene, cholesterol and an antimicrobial peptide embedded in oriented lipid bilayers. The MD simulations were able to reproduce the NMR parameters within experimental error. The alignment of the three membrane-bound molecules and some aspects of their conformation were thus derived from the NMR data, in good agreement with previous analyses. Furthermore, the new approach yielded for the first time the distribution of segmental orientations with respect to the membrane and the order parameter tensors of all three systems.     

The determination of the conformational properties of nucleic acids in solution from NMR data

A program, NUCFIT, has been written for simulating the effects of conformational averaging on nuclear Overhauser enhancement (NOE) intensities for the spin systems found in nucleic acids. Arbitrary structures can be generated, and the NOE time courses can be calculated for truncated one-dimensional NOEs, two-dimensional NOE and rotating frame NOE spectroscopy (NOESY and ROESY) experiments. Both isotropic and anisotropic molecular rotation can be treated, using Woessner's formalism (J. Chem. Phys. (1962) 37, 647-654). The effects of slow conformational averaging are simulated by taking population-weighted means of the conformations present. Rapid motions are allowed for by using order parameters which can be supplied by the user, or calculated for specific motional models using the formalism of Tropp (J. Chem. Phys. (1980) 72, 6035-6043). NOE time courses have been simulated for a wide variety of conformations and used to determine the quality of structure determinations using NMR data for nucleic acids. The program also allows grid-searching with least-squares fitting of structures to experimental data, including the effects of spin-diffusion, conformational averaging and rapid internal motions. The effects of variation of intra and internucleotide conformational parameters on NOE intensities has been systematically explored. It is found that (i) the conformation of nucleotides is well determined by realistic NOE data sets, (ii) some of the helical parameters, particularly the base pair roll, are poorly determined even for extensive, noise-free data sets, (iii) conformational averaging of the sugars by pseudorotation has at most second-order influence on the determination of other parameters and (iv) averaging about the glycosidic torsion bond also has, in most cases, an insignificant effect on the determination of the conformation of nucleotides.     

Regulation of the F0F1-ATP synthase: the conformation of subunit epsilon might be determined by directionality of subunit gamma rotation

F(0)F(1)-ATP synthase couples ATP synthesis/hydrolysis with transmembrane proton transport. The catalytic mechanism involves rotation of the gamma epsilon c(approximately 10)-subunits complex relative to the rest of the enzyme. In the absence of protonmotive force the enzyme is inactivated by the tight binding of MgADP. Subunit epsilon also modulates the activity: its conformation can change from a contracted to extended form with C-terminus stretched towards F(1). The latter form inhibits ATP hydrolysis (but not synthesis). We propose that the directionality of the coiled-coil subunit gamma rotation determines whether subunit epsilon is in contracted or extended form. Block of rotation by MgADP presumably induces the extended conformation of subunit epsilon. This conformation might serve as a safety lock, stabilizing the ADP-inhibited state upon de-energization and preventing spontaneous re-activation and wasteful ATP hydrolysis. The hypothesis merges the known regulatory effects of ADP, protonmotive force and conformational changes of subunit epsilon into a consistent picture.     

Conformational dynamics of sialyl Lewisx in aqueous solution and its interaction with selectinE. A study by molecular dynamics

Three dimensional structures of sialyl Lewis(x) (SLe(x)) in aqueous solution and bound to selectinE are described based on an exhaustive conformational analysis and several long molecular dynamics simulations using different glycosidic regions as starting conformations. It appears from this study that when the oligosaccharide is free in solution the NeuNAcalpha(2-3)Gal segment favors glycosidic conformation in three different regions in the (Phi,Psi) plane with propensity of populations in the ratio 1:8:1. Each one of these structures is characteristically stabilized by specific hydrogen bonding interaction between NeuNAc and Gal. On the other hand, the Gal-GlcNAc-Fuc segment can exist in four different conformational states. Based on the topology of SLe(x) we are able to predict that out of all the allowed conformations in solution only two of these structures possess a geometry that would fit without steric clashes into the binding location of selectinE. In both of these binding modes, segment Gal-GlcNAc-Fuc adopts a unique conformation. The only difference between the two SLe(x) conformers that can successfully bind to selectinE is given by two possible regions in glycosidic space in the fragment NeuNAcalpha(2-3)Gal. A large conformational departure from the crystallographic data is observed for two lysine residues at the binding site of selectinE. These two residues play an important role when SLe(x) binds selectinE in aqueous solution. These findings help reconcile the X-ray data, in which these residues appear to be 1 nm away from SLe(x), with recent liquid NMR data reporting couplings between these protein residues and the sugar.     

NMR studies of carbohydrates and carbohydrate-mimetic peptides recognized by an anti-group B Streptococcus antibody

As part of a program to investigate the origins of peptide-carbohydrate mimicry, the conformational preferences of peptides that mimic the group B streptococcal type III capsular polysaccharide have been investigated by NMR spectroscopy. Detailed studies of a dodecapeptide, FDTGAFDPDWPA, a molecular mimic of the polysaccharide antigen, and two new analogs, indicated a propensity for beta-turn formation. Different beta-turn types were found to be present in the trans and cis (Trp-10-Pro-11) isomers of the peptide: the trans isomer favored a type I beta-turn from residues Asp-7-Trp-10, whereas the cis isomer exhibited a type VI beta-turn from residues Asp-9-Ala-12. The interaction of the dodecapeptide FDTGAFDPDWPA with a protective anti-group B Streptococcus monoclonal antibody has also been investigated, by transferred nuclear Overhauser effect NMR spectroscopy and saturation-transfer difference NMR spectroscopy (STD-NMR). The peptide was found to adopt a type I beta-turn conformation on binding to the antibody; the peptide residues (Asp-7-Trp-10) forming this turn are recognized by the antibody, as demonstrated by STD-NMR experiments. STD-NMR studies of the interactions of oligosaccharide fragments of the capsular polysaccharide have also been performed and provide evidence for the existence of a conformational epitope.     

Characterization of a novel type VII beta-turn conformation for a bio-active tetrapeptide rigin A synergy between theoretical and experimental results.

The conformational analysis of an immunomodulating tetrapeptide rigin (H-Gly-Gln-Pro-Arg-OH), shown to possess diverse immunological activity, has been investigated both theoretically and experimentally for its conformational preferences. Unrestrained molecular dynamics simulation studies in implicit dimethylsulfoxide provide strong support for the existence of a significant population of ordered reverse turn structures for the major trans isomer. Of the three different energy minimized families, generated from computer molecular modelling, only one could be complemented by most of the 1D and 2D 1H NMR parameters obtained in dimethylsulfoxide-d6. A variable temperature NMR experiment in dimethylsulfoxide-d6 revealed that the preferred conformation is not stabilized by an intramolecular hydrogen bonding interaction. An analysis of the 2D ROESY experiment provides evidence in favour of an uncommonly observed, rather ill-defined type VII beta-turn structure. A survey of the observed specific inter-and intra-residue NOE connectivities and their comparison with one of the predicted low-energy conformations, demonstrates synergy between the theoretical molecular modelling and experimentally determined NMR spectral data. The primary structure, rather than long-range interactions, appears to be critical in determining the folding behaviour of the bio-active rigin. The present structural attributes may be valuable in peptide drug design and development of the rigin analogs having more effective stimulating activity.     

Contribution of NMR spectroscopy to the study of structure-function relations of proteins and peptides

NMR spectroscopy provides a unique means to study molecular conformation, mechanisms of action and structure-function relationships for peptides and proteins in solution under conditions approaching those of their physiological environment. Development of NMR techniques, especially directed to the peptide and protein conformational analysis, is considered under the topics of two-level signal assignment and structural significance of homo- and heteronuclear spin-spin couplings. The results of NMR conformational analysis are presented for solution spatial structure of valinomy cin and gramicidin A antibiotics, honey-bee neurotoxin apamin, scorpion insectotoxin I5A and snake venom neurotoxins of "short" and "long" types. The structure-function relationships are discussed for these biologically active molecules.     

Steroid structure and function:I. Conformational transmission in 17alpha-acetoxy progesterone

The molecular conformation of 17alpha-acetoxy progesterone has been determined crystallographically and is compared with that of progesterone. The 17alpha-acetate substituent restricts the flexibility of the progesterone side chain, strains bond lengths in the C- and D-rings, and has long range effects on the A-ring conformation. The A-ring adopts a perfect sofa conformation similar to that observed in one conformational isomer of progesterone. Consequently this progesterone isomer is proposed to be that best suited to binding in the rabbit and human uterus.     

Global structure of a DNA three-way junction by solution NMR: towards prediction of 3H fold

Three-way junctions (3H) are the simplest and most commonly occurring branched nucleic acids. They consist of three double helical arms (A to C), connected at the junction point, with or without a number of unpaired bases in one or more of the three different strands. Three-way junctions with two unpaired bases in one strand (3HS2) have a high tendency to adopt either of two alternative stacked conformations in which two of the three arms A, B and C are coaxially stacked, i.e. A/B-stacked or A/C-stacked. Empirical stacking rules, which successfully predict for DNA 3HS2 A/B-stacking preference from sequence, have been extended to A/C-stacked conformations. Three novel DNA 3HS2 sequences were designed to test the validity of these extended stacking rules and their conformational behavior was studied by solution NMR. All three show the predicted A/C-stacking preference even in the absence of multivalent cations. The stacking preference for both classes of DNA 3HS2 can thus be predicted from sequence. The high-resolution NMR solution structure for one of the stacked 3HS2 is also reported. It shows a well-defined local and global structure defined by an extensive set of classical NMR restraints and residual dipolar couplings. Analysis of its global conformation and that of other representatives of the 3H family, shows that the relative orientations of the stacked and non-stacked arms, are restricted to narrow regions of conformational space, which can be understood from geometric considerations. Together, these findings open up the possibility of full prediction of 3HS2 conformation (stacking and global fold) directly from sequence.     

Structural Dynamics and Conformational Equilibria of SERCA Regulatory Proteins in Membranes by Solid-State NMR Restrained Simulations

Solid-state NMR spectroscopy is emerging as a powerful approach to determine structure, topology, and conformational dynamics of membrane proteins at the atomic level. Conformational dynamics are often inferred and quantified from the motional averaging of the NMR parameters. However, the nature of these motions is difficult to envision based only on spectroscopic data. Here, we utilized restrained molecular dynamics simulations to probe the structural dynamics, topology and conformational transitions of regulatory membrane proteins of the calcium ATPase SERCA, namely sarcolipin and phospholamban, in explicit lipid bilayers. Specifically, we employed oriented solid-state NMR data, such as dipolar couplings and chemical shift anisotropy measured in lipid bicelles, to refine the conformational ensemble of these proteins in lipid membranes. The samplings accurately reproduced the orientations of transmembrane helices and showed a significant degree of convergence with all of the NMR parameters. Unlike the unrestrained simulations, the resulting sarcolipin structures are in agreement with distances and angles for hydrogen bonds in ideal helices. In the case of phospholamban, the restrained ensemble sampled the conformational interconversion between T (helical) and R (unfolded) states for the cytoplasmic region that could not be observed using standard structural refinements with the same experimental data set. This study underscores the importance of implementing NMR data in molecular dynamics protocols to better describe the conformational landscapes of membrane proteins embedded in realistic lipid membranes.     

Structural Dynamics and Conformational Equilibria of SERCA Regulatory Proteins in Membranes by Solid-State NMR Restrained Simulations

Solid-state NMR spectroscopy is emerging as a powerful approach to determine structure, topology, and conformational dynamics of membrane proteins at the atomic level. Conformational dynamics are often inferred and quantified from the motional averaging of the NMR parameters. However, the nature of these motions is difficult to envision based only on spectroscopic data. Here, we utilized restrained molecular dynamics simulations to probe the structural dynamics, topology and conformational transitions of regulatory membrane proteins of the calcium ATPase SERCA, namely sarcolipin and phospholamban, in explicit lipid bilayers. Specifically, we employed oriented solid-state NMR data, such as dipolar couplings and chemical shift anisotropy measured in lipid bicelles, to refine the conformational ensemble of these proteins in lipid membranes. The samplings accurately reproduced the orientations of transmembrane helices and showed a significant degree of convergence with all of the NMR parameters. Unlike the unrestrained simulations, the resulting sarcolipin structures are in agreement with distances and angles for hydrogen bonds in ideal helices. In the case of phospholamban, the restrained ensemble sampled the conformational interconversion between T (helical) and R (unfolded) states for the cytoplasmic region that could not be observed using standard structural refinements with the same experimental data set. This study underscores the importance of implementing NMR data in molecular dynamics protocols to better describe the conformational landscapes of membrane proteins embedded in realistic lipid membranes.