Experimental Nuclear Overhauser Enhancements Research Articles

Structural Organization and Transport Properties of Novel Pyrrolidinium-Based Ionic Liquids with Perfluoroalkyl Sulfonylimide Anions

Room-temperature ionic liquids (RTILs) based on the N-butyl-N-methyl pyrrolidinium cation (PYR(14)(+)) combined with three different fluorinated anions have been prepared and characterized by NMR, conductivity, and rheological measurements. The anions are (trifluoromethanesulfonyl)(nonafluorobutanesulfonyl)imide (IM(14)(-)), bis(pentafluoroethanesulfonyl)imide (BETI(-)), and bis(trifluoromethanesulfonyl)imide (TFSI(-)). Intermolecular anion-cation nuclear Overhauser enhancements (NOEs) have been experimentally observed in all titled compounds. These findings indicate the formation of long-lived aggregates in the bulk liquids. The NOE patterns show marked selectivity and can be rationalized assuming that the perfluorinated moieties of the anions tend to adopt a preferential orientation with respect to the cations, with possible formation of mesoscopic fluorous domains. Self-diffusion coefficients D for the anion and the cation have been measured by DOSY NMR. Diffusion data show similar but not identical values for cation and anion, consistent with local ordering at the molecular level. The observed trend in diffusion coefficients, D(cation) > D(anion) for all compounds, is compatible with a higher degree of intermolecular organization of the anions. This nanoscale organization is connected to rather strong deviations of the experimental conductivities from those estimated from the ion diffusion coefficients through the Nernst-Einstein relationship. The measured viscosities and ion diffusion coefficients in PYR(14)IM(14) and in PYR(14)TFSI have similar temperature dependencies, leading to very close values of the activation energies for these processes. Ab initio density functional calculations on models of a PYR(14)TFSI ion pair lead to the identification of several local minima, whose structure and energy can be qualitatively related to the experimental NOE signals and activation energies.

Comparing atomistic simulation data with the NMR experiment: How much can NOEs actually tell us?

Simulated molecular dynamics trajectories of proteins and nucleic acids are often compared with nuclear magnetic resonance (NMR) data for the purposes of assessing the quality of the force field used or, equally important, trying to interpret ambiguous experimental data. In particular, nuclear Overhauser enhancement (NOE) intensities or atom-atom distances derived from them are frequently calculated from the simulated ensembles because the distance restraints derived from NOEs are the key ingredient in NMR-based protein structure determination. In this study, we ask how diverse and nonnative-like an ensemble of structures can be and still match the experimental NOE distance upper bounds well. We present two examples in which simulated ensembles of highly nonnative polypeptide structures (an unfolded state ensemble of the villin headpiece and a high-temperature denatured ensemble of lysozyme) are shown to match fairly well the experimental NOE distance upper bounds from which the corresponding native structures were derived. For example, the unfolded ensemble of villin headpiece, which is on average 0.90 +/- 0.13 nm root-mean-square deviation away from the native NMR structure, deviates from the experimental restraints by only 0.027 nm on average. However, this artificially good agreement is largely a consequence of 1) the highly nonlinear effects of r(-6) (or r(-3)) averaging and 2) focusing only on the experimentally observed set of NOE bounds. Namely, in addition to the experimentally observed NOEs, both simulated ensembles (especially the villin ensemble) also predict a large number of NOEs, which are not seen in the experiment. If these are taken into account, the agreement between simulation and experiment gets markedly worse, as it should, given the nonnative nature of the underlying simulated ensembles. In light of the examples given, we conclude that comparing experimental NOE distance restraints with large simulated ensembles provides just by itself only limited information about the quality of simulation.

Completely automated, highly error-tolerant macromolecular structure determination from multidimensional nuclear overhauser enhancement spectra and chemical shift assignments.

The major rate-limiting step in high-throughput NMR protein structure determination involves the calculation of a reliable initial fold, the elimination of incorrect nuclear Overhauser enhancement (NOE) assignments, and the resolution of NOE assignment ambiguities. We present a robust approach to automatically calculate structures with a backbone coordinate accuracy of 1.0-1.5 A from datasets in which as much as 80% of the long-range NOE information (i.e., between residues separated by more than five positions in the sequence) is incorrect. The current algorithm differs from previously published methods in that it has been expressly designed to ensure that the results from successive cycles are not biased by the global fold of structures generated in preceding cycles. Consequently, the method is highly error tolerant and is not easily funnelled down an incorrect path in either three-dimensional structure or NOE assignment space. The algorithm incorporates three main features: a linear energy function representation of the NOE restraints to allow maximization of the number of simultaneously satisfied restraints during the course of simulated annealing; a method for handling the presence of multiple possible assignments for each NOE cross-peak which avoids local minima by treating each possible assignment as if it were an independent restraint; and a probabilistic method to permit both inactivation and reactivation of all NOE restraints on the fly during the course of simulated annealing. NOE restraints are never removed permanently, thereby significantly reducing the likelihood of becoming trapped in a false minimum of NOE assignment space. The effectiveness of the algorithm is demonstrated using completely automatically peak-picked experimental NOE data from two proteins: interleukin-4 (136 residues) and cyanovirin-N (101 residues). The limits of the method are explored using simulated data on the 56-residue B1 domain of Streptococcal protein G.

Control of disaccharide conformation by π-stacking

The conformations of a series of derivatives of the disaccharide α-L-fucopyranosyl-(1[Formula: see text]3)-2-acetamido-2-deoxy-D-glucopyranoside, part of the Lex determinant, were studied by molecular modelling using the MM3* forcefield and by 1H NMR spectroscopy. Unusually shielded O-benzyl protons were observed in the 1H NMR spectrum of phenyl 2,3,4-tri-O-benzyl-α-L-fucopyranosyl-(1[Formula: see text]3)-2-deoxy-2-phthalimido-1-thio-α-D-glucopyranoside and assigned to the 2-O-benzyl group. This observation was explained by a shift in the population of the conformational mixture present about the glycosidic linkage from the positive Ψ region in the unsubstituted disaccharide to the negative Ψ region induced by π-stacking between the phthalimide and the 2-O-benzyl phenyl ring. The experimental nuclear Overhauser enhancements confirm the accuracy of the calculations.Key words: disaccharide, conformation, π-stacking, Lex determinant, NOE measurements, MM3 calculations.

Transferred nuclear Overhauser enhancement (NOE) and rotating-frame NOE experiments reflect the size of the bound segment of the Forssman pentasaccharide in the binding site of Dolichos biflorus lectin.

A complex between the Forssman pentasaccharide alpha-D-GalNAc-(1-->3)-beta-D-GalNAc-(1-->3)-alpha-D-Gal-(1-->4)-beta-D- Gal-(1-->4)-D-Glc and the seed lectin from Dolichos biflorus was studied using transfer-NOESY and transfer rotating frame NOE spectroscopy (ROESY) experiments. The evolution of transferred NOEs and ROEs as a function of the pentasaccharide/lectin ratio was different for the non-reducing disaccharide moiety alpha-D-GalNAc-(1-->3)-beta-D-GalNac compared to the rest of the molecule, which reflects distinct relaxation properties and effects of exchange broadening of the corresponding ligand resonances. Significantly, several intermolecular transferred NOEs were observed between protons of the nonreducing disaccharide moiety alpha-D-GalNAc-(1-->3)-beta-D-GalNAc and aliphatic as well as aromatic amino acid side chain protons in the binding pocket of the lectin. It is concluded that the non-reducing disaccharide fragment is buried in the lectin-binding pocket, whereas the reducing trisaccharide portion alpha-D-Gal-(1-->4)-beta-D-Gal-(1-->4)-D-Glc has no immediate contacts with the protein. The experimental transfer NOE data were qualitatively compared to theoretical proton-proton distances from a model that was based on a previous homology modeling study of a complex between the disaccharide fragment alpha-D-GalNAc-(1-->3)-beta-D-GalNAc and D. biflorus lectin. It was found that all intermolecular transferred NOEs matched short interatomic distances between ligand protons and aliphatic or aromatic amino acid side chain protons predicted by the theoretical model.

Determination of the stereochemistry of natural products from nuclear magnetic resonance data by constrained molecular dynamics

We have developed a molecular modeling procedure to determine the relative configuration of a chiral molecule from nuclear magnetic resonance (NMR) data. Our procedure uses constrained molecular mechanics, and the constraints are interproton distances derived from the experimental nuclear Overhauser enhancement (NOE) data. The main feature is a period of high-temperature dynamics in which frequent inversions occur at most chiral sites. This allows the distance constraints to guide the molecule into configurations consistent with the NOE data. For molecules with complex ring systems, high-temperature dynamics alone may fail to invert certain chiral centers with sufficient frequency. We have countered this by allowing as an option additional inversions of selected chiral centers. The procedure tested successfully on organic molecules of known stereochemistry, with 5 to 17 chiral centers, provided that the number of available constraints was at least twice the number of chiral centers. The procedure is tolerant of large errors in the estimated interproton distances and is reasonably rapid. For a series of sugars, the time required increases less than quadratically with the number of atoms. © 1996 by John Wiley & Sons, Inc.

Conformation of Deoxy Oligosaccharides: Solution Conformation and Flexibility of the Antibiotic Kijanimicin Studied by NMR Spectroscopy and MMC Calculations

AbstractThe solution conformation of the macrolide antibiotic kijanimicin (1) was deduced by a combination of NMR spectroscopy and potential energy calculations of the carbohydrate parts using the HSEA force field. The study was based on temperature dependent NMR spectroscopic shift data, 1D nuclear Overhauser enhancements (NOE) of selected protons and 2D rotating frame NOE of the complete antibiotic. NOEs in the oligosaccharide part of the antibiotic indicated some unusual short distances between the α‐linked digitoxose residues A and B, as well as between not directly linked residues A and C. Calculation of preferred conformations revealed a global minimum with ΦA,B/ΨA,B ≈ 70°/50°, ΦB,C/ΨB,C ≈ 60°/30°, and ΦB,D/ΨB,D ≈ −60°/5° for the oligosaccharide moiety. MMC simulations of the conformational flexibility indicated a high rigidity of the carbohydrate. The range of the glycosidic angles covered during MMC simulations was 50°–70° for the α‐linkages of the oligosaccharide. The ß‐glycosidic linkages of residue D and E showed slightly more flexibility, covering a range of 120° – 270°. Minimum energy calculations of the oligosaccharide part revealed a second minimum with different orientation of sugar residues A‐B and an energy difference of 2 kcal/mol to the global minimum. This energy difference increased to 12 kcal/mol when the complete antibiotic was calculated and MMC simulations did not show any transitions between the two minima. The experimental NOE in the oligosaccharide moiety and the enhancements between carbohydrate and lactone part agree well with theoretical NOEs that were obtained by sampling the global minimum in MMC simulations. The conformation of the macrolide moiety was evaluated by comparing experimental NOEs with the crystal structure of the isolated aglycon. The conformation found in solution is slightly altered around the sites of attachment of the carbohydrate parts. Constancy of the proton chemical shifts over a temperature range of 70°C indicated the high degree of rigidity of the carbohydrate moiety and most of the macrolide structure. Flexibility was observed for the C‐13/C‐20 part of the lactone ring. The antibiotic has a fairly unpolar surface and adopts a well defined and quite rigid conformation. magnified image

Solution structure of [d(GCGTATACGC)] 2

The solution structure of the alternating pyrimidine-purine DNA duplex [d(GCGTATACGC)] 2 has been determined using two-dimensional nuclear magnetic resonance techniques and distance geometry methods. Backbone distance constraints derived from experimental nuclear Overhauser enhancement and J-coupling torsion angle constraints were required to adequately define the conformation of the inter-residue backbone linkages and to avoid underwinding of the duplex. The distance geometry structures were further refined by back-calculation of the two-dimensional nuclear Overhauser enhancement spectra to correct spin-diffusion distance errors. Fifteen final structures for [d(GCGTATACGC)] 2 were generated from the refined experimental distance bounds. These structures all exhibit fully wound B-form geometry with small penalty values (< 1.5 Å) against the distance bounds and small pair-wise root-mean-square deviation values (typically 0.6 Å to 1.5 Å). The final structures exhibit positive base-pair inclination with respect to the helix axis, a marked alternation in rise and twist, and are shorter and wider than classical fiber B-form DNA. The purines were found to adopt a sugar pucker close to the C-2′- endo conformation while pyrimidine sugars exhibited significantly lower pseudorotation phase angles in the C-1′- exo to C-2′- endo range. The minor groove crossstrand steric clashes at pyrimidine-purine steps that would exist in pure B-DNA are attenuated by an increased rise at these steps (and an increased roll angle at TpA steps). Concomitantly the backbone torsion angles of the pyrimidine moieties have larger γ values, larger ε values, and smaller ζ values than the purines. The structures generated by distance geometry methods were also compared with those obtained from restrained molecular dynamics with empirical force-field potentials. The results indicate that the nuclear magnetic resonance/distance geometry approach alone is capable of elucidating most of the salient structural features of double-stranded helical nucleic acids in solution without resorting to empirical energy potentials and without using any structural assumptions from crystallographic data.

Studies of individual carbon sites of proteins in solution by natural abundance carbon 13 nuclear magnetic resonance spectroscopy. Relaxation behavior

The aromatic regions in proton-decoupled natural abundance 13C Fourier transform nuclear magnetic resonance spectra (at 14.2 kG) of small native proteins contain broad methine carbon bands and narrow nonprotonated carbon resonances. Some factors that affect the use of natural abundance 13C Fourier transform NMR spectroscopy for monitoring individual nonprotonated aromatic carbon sites of native proteins in solution are discussed. The effect of protein size is evaluated by comparing the 13C NMR spectra of horse heart ferrocytochrome c, hen egg white lysozyme, horse carbon monoxide myoglobin, and human adult carbon monoxide hemoglobin. Numerous single carbon resonances are observed in the aromatic regions of 13C NMR spectra of cytochrome c, lysozyme, and myoglobin. The much larger hemoglobin yields few resolved individual carbon resonances. Theoretical and some experimental values are presented for the natural linewidths (W), spin-lattice relaxation times (T1), and nuclear Overhauser enhancements (NOE) of nonprotonated aromatic carbons and Czeta of arginine residues. In general, the 13C-1H dipolar mechanism dominates the relaxation of these carbons. 13C-14N dipolar relaxation contributes significantly to 1/T1 of C epsilon2 of tryptophan residues and Czeta of arginine residues of proteins in D2O. The NOE of each nonprotonated aromatic carbon is within experimental error of the calculated value of about 1.2. As a result, integrated intensities can be used for making a carbon count. Theoretical results are presented for the effect of internal rotation on W, T1, and the NOE. A comparison with the experimental T1 and NOE values indicates that if there is internal rotation of aromatic amino acid side chains, it is not fast relative to the over-all rotational motion of the protein.