Assignment Of NMR Spectra Research Articles

Sequential assignment of NMR spectra of peptides at natural isotopic abundance with zero- and ultra-low-field total correlation spectroscopy (ZULF-TOCSY).

A novel method dubbed ZULF-TOCSY results from the combination of Zero and Ultra-Low Field (ZULF) with high-field, high-resolution NMR, leading to a generalization of the concept of total correlation spectroscopy (TOCSY). ZULF-TOCSY is a new building block for NMR methods, which has the unique property that the polarization is evenly distributed among all NMR-active nuclei such as 1H, 13C, 15N, 31P, etc., provided that they belong to the same coupling network, and provided that their relaxation is not too fast at low fields, as may occur in macromolecules. Here, we show that ZULF-TOCSY correlations can be observed for peptides at natural isotopic abundance, such as the protected hexapeptide Boc-Met-enkephalin. The analysis of ZULF-TOCSY spectra readily allows one to make sequential assignments, thus offering an alternative to established heteronuclear 2D experiments like HMBC. For Boc-Met-enkephalin, we show that ZULF-TOCSY allows one to observe all expected cross-peaks between carbonyl carbons and α-CH protons, while the popular HMBC method provides insufficient information.

Sequence-specific assignments in NMR spectra of paramagnetic systems: A non-systematic approach

The complete assignment of 1H, 13C and 15N protein signals, which is a straightforward task for diamagnetic proteins provided they are folded, soluble and with a molecular mass below 30,000 Da, often becomes an intractable problem in the presence of a paramagnetic center. Indeed, the hyperfine interaction quenches signal intensity; this prevents the detection of scalar and dipolar connectivities and the sequential assignment of protein regions close to the metal ion(s). However, many experiments can be optimized and novel experiments can be designed to circumvent the problem and to revive coherences invisible in standard experiments. The small HiPIP protein PioC provides an interesting case to address this issue: the prosthetic group is a [Fe4S4]2+ cluster that is bound to the 54 amino acids protein via four cysteine residues. The four cluster-bound cysteine residues adopt different binding conformations and therefore each cysteine is affected by paramagnetic relaxation to different extent. A network of tailored experiments succeeded to obtain the complete resonance assignment of cluster bound residues.

Evaluation of the ID number method for structure generation and complete assignment of NMR spectra of erythromycin A

The present paper describes the evaluation of the ID number method for the structure generation and complete assignments of proton and carbon NMR spectra of erythromycin A. This study revised the errors found in previous assignments of two carbon signals in the spectra of this compound.

Ensemble-Based Modeling of the NMR Spectra of Solid Solutions: Cation Disorder in Y2(Sn,Ti)2O7.

The sensitivity of NMR to the local environment, without the need for any long-range order, makes it an ideal tool for the characterization of disordered materials. Computational prediction of NMR parameters can be of considerable help in the interpretation and assignment of NMR spectra of solids, but the statistical representation of all possible chemical environments for a solid solution is challenging. Here, we illustrate the use of a symmetry-adapted configurational ensemble in the simulation of NMR spectra, in combination with solid-state NMR experiments. We show that for interpretation of the complex and overlapped lineshapes that are typically observed, it is important to go beyond a single-configuration representation or a simple enumeration of local environments. The ensemble method leads to excellent agreement between simulated and experimental spectra for Y2(Sn,Ti)2O7 pyrochlore ceramics, where the overlap of signals from different local environments prevents a simple decomposition of the experimental spectral lineshapes. The inclusion of a Boltzmann weighting confirms that the best agreement with experiment is obtained at higher temperatures, in the limit of full disorder. We also show that to improve agreement with experiment, in particular at low dopant concentrations, larger supercells are needed, which might require alternative simulation approaches as the complexity of the system increases. It is clear that ensemble-based modeling approaches in conjunction with NMR spectroscopy offer great potential for understanding configurational disorder, ultimately aiding the future design of functional materials.

Automated assignment of NMR spectra of macroscopically oriented proteins using simulated annealing

An automated technique for the sequential assignment of NMR backbone resonances of oriented protein samples has been developed and tested based on 15N-15N homonuclear exchange and spin-exchanged separated local-field spectra. By treating the experimental spectral intensity as a pseudopotential, the Monte-Carlo Simulated Annealing algorithm has been employed to seek lowest-energy assignment solutions over a large sampling space where direct enumeration would be unfeasible. The determined sequential assignments have been scored based on the positions of the crosspeaks resulting from the possible orders for the main peaks. This approach is versatile in terms of the parameters that can be specified to achieve the best-fit result. At a minimum the algorithm requires a continuous segment of the main-peak chemical shifts obtained from a uniformly labeled sample and a spin-exchanged experimental spectrum represented as a 2D matrix array. With selective labeling experiments, groups of chemical shifts corresponding to specific locations in the protein backbone can be fixed, thereby decreasing the sampling space. The output from the program consists of a list of top-score main peak assignments, which can be subjected to further scoring criteria until a consensus solution is found. The algorithm has first been tested on a synthetic spectrum with randomly generated chemical shifts and dipolar couplings for the main peaks. The original assignments have been successfully recovered for as many as 100 main peaks when residue-type information was used even in the presence of substantial spectral peak overlap. The algorithm was then applied to assigning two sets of experimental spectra to recover and confirm the previously established assignments in an automated fashion. For the 20-residue transmembrane domain of Pf1 coat protein reconstituted in magnetically aligned bicelles, the original assignment by Park et al. (2010) was recovered by the automated algorithm with additional input from 5 selectively labeled amino acid spectra. The second case considered was the 46 residue Pf1 bacteriophage from Thiriot et al. (2005) and Knox et al. (2010), of which 38 residues were fit. Automated fitting resulted in several possible assignments but not exactly the original assignment. By using a post-fitting filtering procedure based on the number of missed cross peaks and Pf1 helical structure, a consensus spectroscopic assignment is proposed covering 84% of the original assignment. While the automated assignment works best in spectra with well-resolved crosspeaks, it also tolerates substantial spectral crowding to yield reasonable assignments in the cases where ambiguity and degeneracy of possible assignment solutions are inevitable.

Data handling in NMR facilities and assignment of NMR spectra in synthetic chemistry labs: Why electronic structure validation should become part of the routine.

Data handling in NMR facilities and assignment of NMR spectra in synthetic chemistry labs: Why electronic structure validation should become part of the routine.

Selective excitation for spectral editing and assignment in separated local field experiments of oriented membrane proteins

Spectroscopic assignment of NMR spectra for oriented uniformly labeled membrane proteins embedded in their native-like bilayer environment is essential for their structure determination. However, sequence-specific assignment in oriented-sample (OS) NMR is often complicated by insufficient resolution and spectral crowding. Therefore, the assignment process is usually done by a laborious and expensive “shotgun” method involving multiple selective labeling of amino acid residues. Presented here is a strategy to overcome poor spectral resolution in crowded regions of 2D spectra by selecting resolved “seed” residues via soft Gaussian pulses inserted into spin-exchange separated local-field experiments. The Gaussian pulse places the selected polarization along the z-axis while dephasing the other signals before the evolution of the 1H-15N dipolar couplings. The transfer of magnetization is accomplished via mismatched Hartmann-Hahn conditions to the nearest-neighbor peaks via the proton bath. By optimizing the length and amplitude of the Gaussian pulse, one can also achieve a phase inversion of the closest peaks, thus providing an additional phase contrast. From the superposition of the selective spin-exchanged SAMPI4 onto the fully excited SAMPI4 spectrum, the 15N sites that are directly adjacent to the selectively excited residues can be easily identified, thereby providing a straightforward method for initiating the assignment process in oriented membrane proteins.

Molecular Structure of Phenytoin: NMR, UV-Vis and Quantum Chemical Calculations

Due to the presence of the carbonyl and imide groups in the structure of 5,5-diphenylhydantoin (DPH), the possibility for this com- pound to be involved in hydrogen bonding intermolecular interactions is obvious. Even though such interactions are presumably responsible for the mechanism of action of this drug, however, to the best of our knowledge, the self-hydrogen bonding interactions between the DPH monomers have not been addressed till now. Furthermore, studies reporting on the spectroscopic characteristics of this molecule are scarcely reported in the literature. Here we report on the possible dimers of DPH, investigated by quantum chemical calculations at B3LYP/6-31+G(2d,2p) level of theory. Twelve unique DPH dimers were structurally optimized in gas-phase, as well as in ethanol and DMSO and then were used to compute the population-averaged UV-Vis and NMR spectra using Boltzmann statistics. UV-Vis and NMR techniques were employed to assess experimentally the spectroscopical response of this compound. DFT calculations are also used to investigate the structural transformations between the solid and liquid phase, as well as for describing the electronic transitions and for the assignment of NMR spectra of DPH.

Automated structure determination from NMR spectra.

Three-dimensional structures of proteins in solution can be calculated on the basis of conformational restraints derived from NMR measurements. This chapter gives an overview of the computational methods for NMR protein structure analysis highlighting recent automated methods for the assignment of NMR spectra, the collection of conformational restraints, and the structure calculation.

Resonance assignment of the NMR spectra of disordered proteins using a multi-objective non-dominated sorting genetic algorithm

A multi-objective genetic algorithm is introduced to predict the assignment of protein solid-state NMR (SSNMR) spectra with partial resonance overlap and missing peaks due to broad linewidths, molecular motion, and low sensitivity. This non-dominated sorting genetic algorithm II (NSGA-II) aims to identify all possible assignments that are consistent with the spectra and to compare the relative merit of these assignments. Our approach is modeled after the recently introduced Monte-Carlo simulated-annealing (MC/SA) protocol, with the key difference that NSGA-II simultaneously optimizes multiple assignment objectives instead of searching for possible assignments based on a single composite score. The multiple objectives include maximizing the number of consistently assigned peaks between multiple spectra ("good connections"), maximizing the number of used peaks, minimizing the number of inconsistently assigned peaks between spectra ("bad connections"), and minimizing the number of assigned peaks that have no matching peaks in the other spectra ("edges"). Using six SSNMR protein chemical shift datasets with varying levels of imperfection that was introduced by peak deletion, random chemical shift changes, and manual peak picking of spectra with moderately broad linewidths, we show that the NSGA-II algorithm produces a large number of valid and good assignments rapidly. For high-quality chemical shift peak lists, NSGA-II and MC/SA perform similarly well. However, when the peak lists contain many missing peaks that are uncorrelated between different spectra and have chemical shift deviations between spectra, the modified NSGA-II produces a larger number of valid solutions than MC/SA, and is more effective at distinguishing good from mediocre assignments by avoiding the hazard of suboptimal weighting factors for the various objectives. These two advantages, namely diversity and better evaluation, lead to a higher probability of predicting the correct assignment for a larger number of residues. On the other hand, when there are multiple equally good assignments that are significantly different from each other, the modified NSGA-II is less efficient than MC/SA in finding all the solutions. This problem is solved by a combined NSGA-II/MC algorithm, which appears to have the advantages of both NSGA-II and MC/SA. This combination algorithm is robust for the three most difficult chemical shift datasets examined here and is expected to give the highest-quality de novo assignment of challenging protein NMR spectra.

Systematic assignment of NMR spectra of 5‐substituted‐4‐thiopyrimidine nucleosides

Unambiguous characterization of 5-substituted-4-thiopyrimidine nucleosides (ribonucleosides and 2'-deoxynucleosides) was performed using NMR spectroscopy. Assignments of all proton and carbon signals of 5-bromo-4-thiouridine and related nucleosides were systematically carried out and firmly established by COSY and HMQC techniques. The NMR data of various 4-thiopyrimidine nucleosides are compared, and the key contributing factors discussed. The approach presented here is applicable to other modified nucleosides and nucleotides, as well as nucleobases.

High-resolution solid-state 13C NMR spectroscopy of the paramagnetic metal–organic frameworks, STAM-1 and HKUST-1

Solid-state (13)C magic-angle spinning (MAS) NMR spectroscopy is used to investigate the structure of the Cu(II)-based metal-organic frameworks (MOFs), HKUST-1 and STAM-1, and the structural changes occurring within these MOFs upon activation (dehydration). NMR spectroscopy is an attractive technique for the investigation of these materials, owing to its high sensitivity to local structure, without any requirement for longer-range order. However, interactions between nuclei and unpaired electrons in paramagnetic systems (e.g., Cu(II)-based MOFs) pose a considerable challenge, not only for spectral acquisition, but also in the assignment and interpretation of the spectral resonances. Here, we exploit the rapid T(1) relaxation of these materials to obtain (13)C NMR spectra using a spin-echo pulse sequence at natural abundance levels, and employ frequency-stepped acquisition to ensure uniform excitation of resonances over a wide frequency range. We then utilise selective (13)C isotopic labelling of the organic linker molecules to enable an unambiguous assignment of NMR spectra of both MOFs for the first time. We show that the monomethylated linker can be recovered from STAM-1 intact, demonstrating not only the interesting use of this MOF as a protecting group, but also the ability (for both STAM-1 and HKUST-1) to recover isotopically-enriched linkers, thereby reducing significantly the overall cost of the approach.

Structure verification through computer-assisted spectral assignment of NMR spectra

The validation of a molecular organic structure on the basis of 1D and 2D HSQC, COSY and HMBC NMR spectra is proposed as an alternative to the methods that are mainly based on chemical shift prediction. The CCASA software was written for this purpose. It provides an updated and improved implementation of the preceding computer-assisted spectral assignment software. CCASA can be downloaded freely from http://www.univ-reims.fr/LSD/JmnSoft/CASA. Two bioactive natural products, a triterpene and a benzophenone, were selected from literature data as examples. The tentative matching between the structure and the NMR data interpretation of the triterpene unexpectedly leads to the hypothesis of an incorrect structure. The LSD software was used to find an alternative structure that improved the 2D NMR data interpretation and the carbon-13 chemical shift matching between experimental values and those produced by the nmrshiftdb2 prediction tool. The benzophenone example showed that signal assignment by means of chemical shift prediction can be replaced by elementary user-supplied chemical shift and multiplicity constraints.

Application of multivariate analysis of NMR spectra of poly(N-isopropylacrylamide) to assignment of stereostructures and prediction of tacticity distribution

In this paper, we report multivariate analyses, such as principal component analysis (PCA) and partial least-squares (PLS) regression, of NMR spectra of poly(N-isopropylacrylamide)s [poly(NIPAAm)s]. PCA successfully interpreted the assignments of NMR spectra of poly(NIPAAm)s in terms of stereostructures for the methine carbons at triad levels and for the methylene protons at tetrad levels. Furthermore, triad tacticity was successfully predicted by PLS regression of 1H NMR spectra of the methine and methylene groups, although the low resolution of the signals of the methine protons confines determination of tacticities by conventional methods to the diad levels. Consequently, it is assumed that chemometric approaches are useful for assigning NMR spectra in terms of stereostructures and for predicting tacticity distributions.

NMR Spectroscopic Assignment and Structure Determination of Membrane Proteins Reconstituted in Magnetically Aligned Bicelles

Magnetically aligned bicelles represent a nearly ideal mimetic for the structural studies of membrane proteins in their native lipid environment. Structures of several proteins reconstituted in bicelles have been obtained by solid-state NMR, including MerF, VPU, and Pf1.However, the assignment of NMR spectra of membrane proteins is usually performed by a laborious preparation of multiple selectively labeled samples, and a helical wheel-like arrangement of peaks is assumed. Here we have used a novel spectroscopic technique [1,2] to obtain spin correlations between the resonances and assigned the spectrum of uniformly 15N labeled Pf1 protein reconstituted in aligned bicelles. The peaks generally follow the (i, i+1) connectivity pattern, and yield the assignment identical to the previously established, albeit without using multiple labeled samples [3]. The method is generally applicable to membrane proteins of arbitrary topology. Long-range correlations (up to 6.7 A) can be observed that have a potential for establishing spectroscopic contacts between the neighboring transmembrane helices.Furthermore, we have developed an algorithm [4] that calculates three-dimensional structures of membrane proteins solely based on the dipolar couplings between the covalently bound spins. Structures of protein G and a helical hairpin of bacteriorhodopsin have been calculated from simulated solid-state NMR spectra as illustrative examples. Effect of experimental uncertainty has been assessed. Application of the algorithm to fully assigned three-dimensional NMR data of doubly (15N, 13C) labeled membrane proteins may allow one to obtain the entire backbone structure from a single spectrum.[1] A.A. Nevzorov, J. Am. Chem. Soc. 130 (2008) 11282-11283.[2] R.W. Knox, J. Lu, S.J. Opella, A.A. Nevzorov, J. Am. Chem. Soc. 132 (2010) 8255-8257.[3] S.J. Opella, A.C. Zeri, S.H. Park, Annu. Rev. Phys. Chem. 59 (2008) 635-57.[4] Y.Y. Yin, A.A. Nevzorov, J. Magn. Reson. 212 (2011) 64-73.

SAGA: rapid automatic mainchain NMR assignment for large proteins

Here we describe a new algorithm for automatically determining the mainchain sequential assignment of NMR spectra for proteins. Using only the customary triple resonance experiments, assignments can be quickly found for not only small proteins having rather complete data, but also for large proteins, even when only half the residues can be assigned. The result of the calculation is not the single best assignment according to some criterion, but rather a large number of satisfactory assignments that are summarized in such a way as to help the user identify portions of the sequence that are assigned with confidence, vs. other portions where the assignment has some correlated alternatives. Thus very imperfect initial data can be used to suggest future experiments.

Dinuclear fluoro-peroxovanadium(v) complexes with symmetric and asymmetric peroxo bridges: syntheses, structures and DFT studies

Two new dinuclear fluoro peroxovanadium(v) complexes, Cs3[V2O2(O2)4F] x H2O (1) and Cs3[V2O2(O2)3F3] x 2HF x H2O (2), were prepared and characterized by elemental analysis, IR spectroscopy, thermal analysis and X-ray crystallography. While the anion in possesses an asymmetric structure with a micro-eta1:eta2 bridging peroxo group, the [V2O2(O2)3F3]3- ion in exhibits a symmetrical structure with a unique mu-fluoro and micro-eta2:eta2 peroxo double bridge. The X-ray structure data were compared with equilibrium and vibrationally-averaged (effective) DFT calculated geometries. The decomposition reactions of and in aqueous solution were studied by 51V NMR spectroscopy. The calculations of vibrationally averaged NMR chemical shifts (DFT-GIAO) were used to support the empirical assignment of NMR signals and afforded excellent agreement with experimental values for the studied peroxovanadium species. The ESI mass spectra of the prepared compounds are in accordance with the assignment of NMR spectra and with DFT study.

13C, 15N CPMAS NMR and GIAO DFT calculations of stereoisomeric oxindole alkaloids from Cat's Claw ( Uncaria tomentosa)

Oxindole alkaloids, isolated from the bark of Uncaria tomentosa [Willd. ex Schult.] Rubiaceae, are considered to be responsible for the biological activity of this herb. Five pentacyclic and two tetracyclic alkaloids were studied by solid-state NMR and theoretical GIAO DFT methods. The 13C and 15N CPMAS NMR spectra were recorded for mitraphylline, isomitraphylline, pteropodine (uncarine C), isopteropodine (uncarine E), speciophylline (uncarine D), rhynchophylline and isorhynchophylline. Theoretical GIAO DFT calculations of shielding constants provide arguments for identification of asymmetric centers and proper assignment of NMR spectra. These alkaloids are 7 R/7 S and 20 R/20 S stereoisomeric pairs. Based on the 13C CP MAS chemical shifts the 7 S alkaloids ( δ C3 70–71 ppm) can be easily and conveniently distinguished from 7 R ( δC3 74.5–74.9 ppm), also 20 R ( δC20 41.3–41.7 ppm) from the 20 S ( δC20 36.3–38.3 ppm). The epiallo-type isomer (3 R, 20 S) of speciophylline is characterized by a larger 15N MAS chemical shift of N4 (64.6 ppm) than the allo-type (3 S, 20 S) of isopteropodine ( δN4 53.3 ppm). 15N MAS chemical shifts of N1–H in pentacyclic alkaloids are within 131.9–140.4 ppm.

The unambiguous assignment of NMR spectra of per-O-methylated 6-mono and 6,6-diamino-β-cyclodextrins

The step-by-step analysis of high-resolution proton and 13C NMR spectra of per-O-methylated-6-monoamino and 6,6-diamino-A,D-β-cyclodextrins is described. Selected 2-D experiments (COSY, NOESY, HSQC, DEPT, and HMBC) allowed us to challenge the current interpretation. The full assignment of the proton and carbon-13 NMR spectra of these two compounds was performed enabling us to complete the assignment of NMR spectra acquired at 500 and 800 MHz for these important synthetic intermediates.Key words: COSY, NOESY, HSQC, DEPT, HMBC, NMR, 6-amino-β-cyclodextrins, 6,6-diamino-β-cyclodextrins.

Silicon uptake and metabolism of the marine diatom Thalassiosira pseudonana: Solid-state 29Si NMR and fluorescence microscopic studies

Uptake and metabolism of silicon by diatoms are studied by the combined use of solid-state 29Si NMR spectroscopy and confocal laser fluorescence microscopy especially with respect to the presence and nature of an intracellular silicon-storage pool. Cells of the marine diatom Thalassiosira pseudonana were synchronized by silicon starvation and frozen without any freeze-drying or chemical treatment in order to analyze integer and unmodified diatoms. The frozen samples were investigated by solid-state 29Si NMR spectroscopy to identify potential silica precursors. The developmental state of the cell culture and the formation of new siliceous girdle bands and valves were monitored by laser fluorescence microscopic studies. A comparison of fluorescence microscopic and NMR data allows the assignment of NMR spectra to the various developmental stages of the dividing diatom cells. A detailed analysis of solid-state 29Si NMR spectra suggests that the silicon-storage pool—if present—consists of four-coordinated, condensed silicon; possibly a silica sol.