UltraSOFAST HMQC NMR and the repetitive acquisition of 2D protein spectra at Hz rates

Following unidirectional biophysical events such as the folding of proteins or the equilibration of binding interactions, requires experimental methods that yield information at both atomic-level resolution and at high repetition rates. Toward this end a number of different approaches enabling the rapid acquisition of 2D NMR spectra have been recently introduced, including spatially encoded "ultrafast" 2D NMR spectroscopy and SOFAST HMQC NMR. Whereas the former accelerates acquisitions by reducing the number of scans that are necessary for completing arbitrary 2D NMR experiments, the latter operates by reducing the delay between consecutive scans while preserving sensitivity. Given the complementarities between these two approaches it seems natural to combine them into a single tool, enabling the acquisition of full 2D protein NMR spectra at high repetition rates. We demonstrate here this capability with the introduction of "ultraSOFAST" HMQC NMR, a spatially encoded and relaxation-optimized approach that can provide 2D protein correlation spectra at approximately 1 s repetition rates for samples in the approximately 2 mM concentration range. The principles, relative advantages, and current limitations of this new approach are discussed, and its application is exemplified with a study of the fast hydrogen-deuterium exchange characterizing amide sites in Ubiquitin.     

Real-time monitoring of chemical transformations by ultrafast 2D NMR spectroscopy

An approach enabling the acquisition of 2D nuclear magnetic resonance (NMR) spectra within a single scan has been recently proposed. A promising application opened up by this "ultrafast" data acquisition format concerns the monitoring of chemical transformations as they happen, in real time. The present paper illustrates some of this potential with two examples: (i) following an H/D exchange process that occurs upon dissolving a protonated protein in D2O, and (ii) real-time in situ tracking of a transient Meisenheimer complex that forms upon rapidly mixing two organic reactants inside the NMR observation tube. The first of these measurements involved acquiring a train of 2D 1H-15N HSQC NMR spectra separated by ca. 4 s; following an initial dead time, this allowed us to monitor the kinetics of hydrogen exchange in ubiquitin at a site-resolved level. The second approach enabled us to observe, within ca. 2 s after the triggering of the reaction, a competition between thermodynamic and kinetic controls via changes in a series of 2D TOCSY patterns. The real-time dynamic experiments hereby introduced thus add to an increasing family of fast characterization techniques based on 2D NMR; their potential and limitations are briefly discussed.     

Spec2D: a structure elucidation system based on 1H NMR and H-H COSY spectra in organic chemistry

A system for structure elucidation based on proton NMR spectra has been developed. The system, named Spec2D (system for spectra from 2D-NMR), incorporates 1H NMR and H-H correlation spectroscopy (COSY) spectral information obtained from 2D-NMR experiments. 2D-NMR is important for the structure elucidation because it provides information about the relationships among differently situated protons in the structures of unknown compounds. The system uses the concepts of molecular graphs. The improved representation of substructures as well as several novel algorithms for structure generation have been devised to solve the combinatorial problem and to reduce the processing time. Spec2D consists of a knowledge base, an analysis module, and a candidate structure generator module. Spec2D proposes candidate structures from only 1H NMR and H-H COSY spectral information of an unknown compound without any 13C NMR spectral or structural information, such as molecular formulas. Spec2D has the capability to propose the "new" structure of an unknown compound, if the corresponding substructures are included in the knowledge base.     

Fuzzy structure generation: a new efficient tool for Computer-Aided Structure Elucidation (CASE)

Contemporary Computer-Aided Structure Elucidation (CASE) systems are heavily based on the utilization of 2D NMR spectra. The utilization of HMBC/GHMBC and COSY/GCOSY correlations generally assumes that these correlations result from (2-3)JCH and (2-3)JHH spin-spin couplings, respectively, and consequently these values are used as the default setting in these systems. Our previous studies1,2 have shown that about half of the problems studied actually contain some correlations of 4-6 bonds, so-called "nonstandard" correlations. In such cases the initial 2D NMR data are contradictory, and the correct solution is therefore not directly attainable. Unfortunately nonstandard correlations and the number of intervening bonds usually cannot be identified experimentally. In this work we suggest a new approach that we term Fuzzy Structure Generation. This allows the solution of structural problems whose 2D NMR data contain an unknown number of nonstandard correlations having different and unknown lengths. Suggested methods for the application of Fuzzy Structure Generation are described, and their application is illustrated by a series of real-world examples. We conclude that Fuzzy Structure Generation is efficient, and there is no real alternative at present in terms of a universal practical method for the structure elucidation of organic molecules from 2D NMR data.     

Influence of the distal his in imparting imidazolate character to the proximal his in heme peroxidase: (1)h NMR spectroscopic study of cyanide-inhibited his42-->ala horseradish peroxidase

The functional higher oxidation states of heme peroxidases have been proposed to be stabilized by the significant imidazolate character of the proximal His. This is induced by a "push-pull" combination effect produced by the proximal Asp that abstracts ("pulls") the axial His ring N(delta)H, along with the distal protonated His that contributes ("pushes") a strong hydrogen bond to the distal ligand. The molecular and electronic structure of the distal His mutant of cyanide-inhibited horseradish peroxidase, H42A-HRPCN, has been investigated by NMR. This complex is a valid model for the active site hydrogen-bonding network of HRP compound II. The (1)H and (15)N NMR spectral parameters characterize the relative roles of the distal His42 and proximal Asp247 in imparting imidazolate character to the axial His. 1D/2D spectra reveal a heme pocket molecular structure that is highly conserved in the mutant, except for residues in the immediate proximity of the mutation. This conserved structure, together with the observed dipolar shifts of numerous active site residue protons, allowed a quantitative determination of the orientation and anisotropies of the paramagnetic susceptibility tensor, both of which are only minimally perturbed relative to wild-type HRPCN. The quantitated dipolar shifts allowed the factoring of the hyperfine shifts to reveal that the significant changes in hyperfine shifts for the axial His and ligated (15)N-cyanide result primarily from changes in contact shifts that reflect an approximately one-third reduction in the axial His imidazolate character upon abolishing the distal hydrogen-bond to the ligated cyanide. Significant changes in side chain orientation were found for the distal Arg38, whose terminus reorients to partially fill the void left by the substituted His42 side chain. It is concluded that 1D/2D NMR can quantitate both molecular and electronic structural changes in cyanide-inhibited heme peroxidase and that, while both residues contribute, the proximal Asp247 is more important than the distal His42 in imparting imidazole character to the axial His 170.     

Ultrafast-based projection-reconstruction three-dimensional nuclear magnetic resonance spectroscopy

Recent years have witnessed increased efforts toward the accelerated acquisition of multidimensional nuclear magnetic resonance (nD NMR) spectra. Among the methods proposed to speed up these NMR experiments is "projection reconstruction," a scheme based on the acquisition of a reduced number of two-dimensional (2D) NMR data sets constituting cross sections of the nD time domain being sought. Another proposition involves "ultrafast" spectroscopy, capable of completing nD NMR acquisitions within a single scan. Potential limitations of these approaches include the need for a relatively slow 2D-type serial data collection procedure in the former case, and a need for at least n high-performance, linearly independent gradients and a sufficiently high sensitivity in the latter. The present study introduces a new scheme that comes to address these limitations, by combining the basic features of the projection reconstruction and the ultrafast approaches into a single, unified nD NMR experiment. In the resulting method each member within the series of 2D cross sections required by projection reconstruction to deliver the nD NMR spectrum being sought, is acquired within a single scan with the aid of the 2D ultrafast protocol. Full nD NMR spectra can thus become available by backprojecting a small number of 2D sets, collected using a minimum number of scans. Principles, opportunities, and limitations of the resulting approach, together with demonstrations of its practical advantages, are here discussed and illustrated with a series of three-dimensional homo- and heteronuclear NMR correlation experiments.     

Hydrogen ion titration of alkyldimethylamine oxides by 13C and 1H NMR and conventional methods

In the hydrogen ion titration of micelles, the degree of ionization of the micelle at a given pH has to be evaluated to obtain a pKa value of micelles (Ka being the proton dissociation constant) at the pH. We compared the degree of ionization obtained from 13C and 1H NMR spectra with that obtained from the stoichiometric method. We used dodecyldimethylamine oxide (C12DMAO) and hexyldimethylamine oxide (C6DMAO) to examine the titration behavior of micelles and monomers, respectively. We determined pKa values of amine oxides both in H2O and D2O. As to the monomer (C6DMAO), the degree of ionization from NMR, alpha(NMR), coincided with that from the conventional stoichiometric method alpha. The difference of pK1 of amine oxide monomer between D2O and H2O was about 0.5: pK1(D) approximately pK1(H) + 0.5. The difference was about the same as that for carboxylic acids. As to the C12DMAO micelle, alphaNMR did not coincide with alpha over a considerable range of alpha. The NMR chemical shift might be influenced by micellar structure changes induced by the ionization, such as the sphere-to-rod transition. The intrinsic logarithmic dissociation constants of the micelle were 5.9+/-0.1 for H2O, and 6.5+/-0.1 for D2O.     

"Multi-scan single shot" quantitative 2D NMR: a valuable alternative to fast conventional quantitative 2D NMR

Quantitative Ultrafast (UF) 2D NMR is a very promising methodology enabling the acquisition of 2D spectra in a single scan. The analytical performances of UF 2D NMR have been highly increased in the last few years, however little is known about the sensitivity of ultrafast experiments versus conventional 2D NMR. A fair and relevant comparison has to consider the Signal-to-Noise Ratio (SNR) per unit of time, in order to answer the following question: for a given experiment time, should we run a conventional 2D experiment or is it preferable to accumulate ultrafast acquisitions? To answer this question, we perform here a systematic comparison between accumulated ultrafast experiments and conventional ones, for different experiment durations. Sensitivity issues and other analytical aspects are discussed for the COSY experiment in the context of quantitative analysis. The comparison is first carried out on a model sample, and then extended to model metabolic mixtures. The results highlight the high analytical performance of the "multi-scan single shot" approach versus conventional 2D NMR acquisitions. This result is attributed to the absence of t(1) noise in spatially encoded experiments. The multi-scan single shot approach is particularly interesting for quantitative applications of 2D NMR, whose occurrence in the literature has been greatly increasing in the last few years.     

13C‐15N Connectivity networks via unsymmetrical indirect covariance processing of 1H‐13C HSQC and 1H‐15N IMPEACH spectra

Long‐range ¹H‐¹⁵N heteronuclear shift correlation methods at natural abundance to facilitate the elucidation of small molecule structures have assumed a role of growing importance over the past decade. Recently, there has also been a high level of interest in the exploration of indirect covariance NMR methods. From two coherence transfer experiments, A→B and A→C, it is possible to indirectly determine B?C. We have shown that unsymmetrical indirect covariance methods can be employed to indirectly determine several types of hyphenated 2D NMR data from higher sensitivity experiments. Examples include the calculation of hyphenated 2D NMR spectra such as 2D GHSQC‐COSY and GHSQC‐NOESY from the discrete component 2D NMR experiments. We now wish to report the further extension of unsymmetrical indirect covariance NMR methods for the combination of ¹H‐¹³C GHSQC and ¹H‐¹⁵N longrange (GHMBC, IMPEACH‐MBC, CIGAR‐HMBC, etc.) heteronuclear chemical shift correlation spectra to establish ¹³C‐¹⁵N correlation pathways.     

Fourier transform ion cyclotron resonance mass spectrometric detection of small Ca2+-induced conformational changes in the regulatory domain of human cardiac troponin C

Troponin C (TnC), a calcium-binding protein of the thin filament of muscle, plays a regulatory role in skeletal and cardiac muscle contraction. NMR reveals a small conformational change in the cardiac regulatory N-terminal domain of TnC (cNTnC) on binding of Ca2+ such that the total exposed hydrophobic surface area increases very slightly from 3090 +/- 86 A2 for apo-cNTnC to 3108 +/- 71 A2 for Ca(2+)-cNTnC. Here, we show that measurement of solvent accessibility for backbone amide protons by means of solution-phase hydrogen/deuterium (H/D) exchange followed by pepsin digestion, high-performance liquid chromatography, and electrospray ionization high-field (9.4 T) Fourier transform Ion cyclotron resonance mass spectrometry is sufficiently sensitive to detect such small ligand binding-induced conformational changes of that protein. The extent of deuterium incorporation increases significantly on binding of Ca2+ for each of four proteolytic segments derived from pepsin digestion of the apo- and Ca(2+)-saturated forms of cNTnC. The present results demonstrate that H/D exchange monitored by mass spectrometry can be sufficiently sensitive to detect and identify even very small conformational changes in proteins, and should therefore be especially informative for proteins too large (or too insoluble or otherwise intractable) for NMR analysis.     

CEESY: characterizing the conformation of unobservable protein states

Protein conformations that are only marginally populated often play important roles as intermediate states in many processes such as ligand binding, enzyme catalysis, allostery, and protein folding. An NMR method is presented that can give valuable information about the structure of these "excited states" by measuring the relative position of exchanging excited- and ground-state resonances using a single 2D spectrum. This new approach can be applied to any nucleus, which will facilitate a complete structural characterization of these states.     

On-line NMR detection of microgram quantities of heparin-derived oligosaccharides and their structure elucidation by microcoil NMR

The isolation and purification of sufficient quantities of heparin-derived oligosaccharides for characterization by NMR is a tedious and time-consuming process. In addition, the structural complexity and microheterogeneity of heparin makes its characterization a challenging task. The improved mass-sensitivity of microcoil NMR probe technology makes this technique well suited for characterization of mass-limited heparin-derived oligosaccharides. Although microcoil probes have poorer concentration sensitivity than conventional NMR probes, this limitation can be overcome by coupling capillary isotachophoresis (cITP) with on-line microcoil NMR detection (cITP-NMR). Strategies to improve the sensitivity of on-line NMR detection through changes in probe design and in the cITP-NMR experimental protocol are discussed. These improvements in sensitivity allow acquisition of cITP-NMR survey spectra facilitating tentative identification of unknown oligosaccharides. Complete structure elucidation for microgram quantities of the purified material can be carried out through acquisition of 2D NMR spectra using a CapNMR microcoil probe. Survey NMR spectrum obtained by cITP-NMR using a second-generation probe (the microcoil of which is shown) facilitates tentative identification of unknown oligosaccharides (e.g., the heparin-derived tetrasaccharide illustrated)     

Metal ion binding to parvalbumin. A proton NMR study

The 1H NMR spectra of carp parvalbumin saturated with Ca2+, Cd2+, La3+ and Lu3+ were compared, using 2D 1H NMR techniques as well as conventional 1H NMR spectra. The Ca2+ and Cd2+ saturated parvalbumin (with both high affinity Ca2+-binding sites occupied) gave rise to very similar spectra. This shows that these two species have almost identical protein conformations. The 1H NMR spectrum from the Ln3+ saturated parvalbumins deviated from the other two and it was therefore concluded that Cd2+ is a better probe for Ca2+ than Ln3+ in parvalbumin and probably also for related calcium binding proteins. The addition of excess of divalent metal ions, such as Mg2+ or Ca2+, causes small changes in the chemical shift of some methyl resonances. This is presumably caused by binding of these metal ions to a third site close to the CD site which is made up of the carboxylic groups from Glu 60 and Asp 61.     

You cannot fight the pressure: Structural rearrangements of active pharmaceutical ingredients under magic angle spinning

Although solid-state nuclear magnetic resonance (NMR) is a versatile analytical tool to study polymorphs and phase transitions of pharmaceutical molecules and products, this work summarizes examples of spontaneous and unexpected (and unwanted) structural rearrangements and phase transitions (amorphous-to-crystalline and crystalline-to-crystalline) under magic angle spinning (MAS) conditions, some of them clearly being due to the pressure experienced by the samples. It is widely known that such changes can often be detected by X-ray powder diffraction (XRPD); here, the capability of solid-state NMR experiments with a special focus on ¹H-¹³C frequency-switched Lee–Goldburg heteronuclear correlation (FSLG HETCOR)/MAS NMR experiments to detect even subtle changes on a molecular level not observable by conventional 1D NMR experiments or XRPD is presented. Furthermore, it is shown that a polymorphic impurity combined with MAS can induce a crystalline-to-crystalline phase transition. This showcases that solid-state NMR is not always noninvasive and such changes upon MAS should be considered in particular when compounds are studied over longer time spans.     

Real-time 2D NMR identification of analytes undergoing continuous chromatographic separation

We have recently proposed and demonstrated an approach that enables the acquisition of multidimensional nuclear magnetic resonance (NMR) spectra within a single scan. A promising application opened up by this new accelerated form of data acquisition concerns the possibility of monitoring in real time the chemical nature of analytes subject to a continuous flow. The present paper illustrates such potential, with the real-time acquisition of a series of 2D 1H NMR spectra arising from a mixture of compounds subject to a continuous liquid chromatography (LC) separation. This real-time 2D NMR identification of chemicals eluted minutes apart under usual LC-NMR conditions differs from the way in which LC-2D NMR has hitherto been carried out, which relies on stopped-flow modes of operations whereby fractions are first collected and then subject to individual, aliquot-by-aliquot analyses. The real-time LC-2D NMR experiment hereby introduced can be implemented in a straightforward manner using modern commercial LC-NMR hardware, thus opening up immediate possibilities in high-throughput characterizations of complex molecules.     

Structure elucidation from 2D NMR spectra using the StrucEluc expert system: detection and removal of contradictions in the data

The elucidation of chemical structures from 2D NMR data commonly utilizes a combination of COSY, HMQC/HSQC, and HMBC data. Generally COSY connectivities are assumed to mostly describe the separation of protons that are separated by 1 skeletal bond (3JHH), while HMBC connectivities represent protons separated from carbon atoms by 1 to 2 skeletal bonds (2JCH and 3JCH). Obviously COSY and HMBC connectivities of lengths greater than those described have been detected. Though experimental techniques have recently been described to aid in the identification of the nature of the couplings the detection of whether a coupling is 2-bond or greater still remains a challenge in most laboratories. In the StrucEluc software system the common lengths of the connectivities, 1-bond for COSY and 1- or 2-bond for HMBC, derived from 2D NMR data are set as the default. Therefore, in the presence of any extended connectivities contradictions can appear in the 2D NMR data. In this article, algorithmic methods for the detection and removal of contradictions in 2D NMR data that have been developed in support of StrucEluc are described. The methods are based on the analysis of molecular connectivity diagrams, MCDs. These methods have been implemented in the StrucEluc system and tested by solving 50 structural problems with 2D NMR spectral data containing contradictions. The presence of contradictions was detected by the algorithm in 90% of the cases, and the contradictions were automatically removed in approximately 50% of the problems. A method of "fuzzy" structure generation in the presence of contradictions has been suggested and successfully tested in this work. This work will demonstrate examples of the application of developed methods to a number of structural problems.     

Separate-local-field NMR spectroscopy on half-integer quadrupolar nuclei

New approaches to the characterization of resonances in the solid-state NMR spectroscopy of half-integer quadrupolar nuclei are explored, on the basis of the acquisition of heteronuclear separate-local-field spectra on rotating solids. In their two-dimensional version, these experiments correlate for each chemical site a second-order quadrupolar MAS powder pattern with the dipolar MAS sideband pattern to nearby heteronuclei. As 3D NMR sequences, such 2D anisotropic correlation spectra become separated for inequivalent chemical sites along a third, isotropic dimension. Extending in such manner separate-local-field NMR approaches to quadrupoles facilitates the assignment of inequivalent resonances to specific structural environments, and provides new tools for the investigation of dynamics in solids. Details about these 2D and 3D NMR experiments are given, and their application is illustrated with 1H-23Na recoupling experiments on mononucleotides possessing multiple bound cations.     

Structure of a missing-caged metallofullerene: La2@C72

The La2@C72 metallofullerene having the so-called "missing" C72 fullerene cage was structurally elucidated by using 13C NMR and 139La NMR spectroscopy. The obtained structure of La2@C72 does not satisfy fullerene's structural golden rule, that is, the isolated-pentagon rule. The structure is consistent with a non-IPR D2-C72 (#10611) cage structure where each La atom is situated close to one of the two-fused pentagons.     

Ultrafast Single-Scan 2D NMR Spectroscopic Detection of a PHIP-Hyperpolarized Protease Inhibitor

Two-dimensional NMR spectroscopy is one of the most important spectroscopic tools for the investigation of biological macromolecules. However, due to the low sensitivity of NMR spectroscopy, it takes usually from several minutes to many hours to record such spectra. Here, the possibility of detecting a bioactive derivative of the sunflower trypsin inhibitor-1 (SFTI-1), a tetradecapeptide, by combining parahydrogen-induced polarization (PHIP) and ultrafast 2D NMR spectroscopy is shown. The PHIP activity of the inhibitor was achieved by labeling with O-propargyl-l -tyrosine. In 1D PHIP experiments a signal enhancement of a factor of approximately 1200 compared to standard NMR was found. This enhancement permits measurement of 2D NMR correlation spectra of low-concentrated SFTI-1 in less than 10 seconds, employing ultrafast single-scan 2D NMR detection. As experimental examples PHIP-assisted ultrafast single-scan TOCSY spectra of SFTI-1 are shown.     

In situ NMR spectroelectrochemistry for the structure elucidation of unstable intermediate metabolites

In situ NMR spectroelectrochemistry is presented in this study as a useful hybrid technique for the chemical structure elucidation of unstable intermediate species. An experimental setting was designed to follow the reaction in real time during the experimental electrochemical process. The analysis of ¹H NMR spectra recorded in situ permitted us (1) to elucidate the reaction pathway of the electrochemical oxidation of phenacetin and (2) to reveal the quinone imine as a reactive intermediate species without using any trapping reaction. Phenacetin has been considered as hepatotoxic at high therapeutic amounts, which is why it was chosen as a model to prove the applicability of the analytical method. The use of 1D and 2D NMR experiments led to the elucidation of the major species produced from the oxidation process. We demonstrated that in situ NMR spectroelectrochemistry constitutes a fast way for monitoring unstable quinone imines and elucidating their chemical structures.