Multiple Quantum Coherence NMR Research Articles

Preservation of heteronuclear multiple-quantum coherences in NMR by double-resonance irradiation

A heteronuclear double-resonance (HDR) method based on MLEV-32 or WALTZ-32 pulse sequences has been designed for the investigation of relaxation of heteronuclear multiple-quantum (MQ) coherences. The theoretical analysis of this technique uses average Hamiltonian theory (AHT) to treat the effects of coherent evolution associated with scalar couplings, offsets, and inhomogeneous radiofrequency (rf) fields during the pulse sequence. Under most conditions, the dynamics of the MQ coherences during the HDR sequence is not affected by rf inhomogeneities and scalar couplings for offsets as large as the nutation frequency. The predictions drawn from AHT are supported by numerical simulations and experiments.

Evolution of spin entanglement and an entanglement witness in multiple-quantum NMR experiments

We investigate the evolution of entanglement in multiple-quantum (MQ) NMR experiments in crystals with pairs of close nuclear 1/2-spins. The initial thermodynamic equilibrium state of the system in a strong external magnetic field evolves under the nonsecular part of the dipolar Hamiltonian. As a result, MQ coherences of the zeroth and plus/minus second orders appear. A simple condition for the emergence of entanglement is obtained. We show that the measure of the spin-pair entanglement—concurrence—coincides qualitatively with the intensity of MQ coherences of the plus/minus second order and, hence, the entanglement can be studied with MQ NMR methods. We introduce an entanglement witness using MQ NMR coherences of the plus/minus second order.

Intermolecular multiple-quantum coherence NMR signals modulated by double distant dipolar fields

A modified CRAZED pulse sequence was applied to obtain the intermolecular multiple-quantum coherence NMR signals from double distant dipolar fields in highly polarized spin systems. Complete theoretical analyses were explicitly derived from the dipolar field treatment combined with product operator formalism. Two typical samples containing several different components were chosen for the experimental verifications. The computer simulations and experimental observations are consistent with the theoretical predictions. The results presented herein provide a convenient way to understand the combined effects of multiple distant dipolar fields from the different components in complicated chemical or biological solutions. When experimental conditions such as selective radio-frequency pulses are not optimal, it may be helpful to identify possible unexpected signals or artefacts of high-resolution NMR spectroscopy in inhomogeneous fields.

Selection of intra- or inter-molecular multiple-quantum coherences in NMR of highly polarized solution

In highly polarized scalar-coupled liquid systems, nuclear magnetic resonance signals from multiple-quantum coherences (MQCs) may be formed by inter-molecular dipolar and/or intra-molecular scalar couplings. Selection of specific signals can simplify the spectra, which may help us understand the underlying physical mechanisms in complex coupled spin systems. In this paper, a pulse sequence with three selective radio-frequency (RF) pulses and phase cycling was designed for this purpose. For an I pS q ( p, q = 1 , 2 , 3 , … ) spin system, there are three kinds of MQC signals, which originate from intra-molecular I– S, inter-molecular I– S, and inter-molecular S– S (or I– I) coherences. These three kinds of signals can be detected separately by proper phase cycling of RF pulses, which is independent of coupling constants. The intra- and inter-molecular MQC signals can also be detected separately with specific preparation periods, but this method is sensitive to coupling constants. Our theoretical predictions are in good agreement with experimental observations. The method proposed herein can be extended to heteronuclear cases.

Line shapes of multiple quantum NMR coherences in one-dimensional quantum spin chains in solids

General formulae for intensities of multiple quantum (MQ) NMR coherences in systems of nuclear spins coupled by the dipole–dipole interactions are derived. The second moments of the MQ coherences of zero- and second orders are calculated for infinite linear chains in the approximation of the nearest neighbor interactions. Supercomputer simulations of intensities of MQ coherences of linear chains are performed at different times of preparation and evolution periods of MQ NMR experiments. The second moments obtained from the developed theory are compared with the results of the supercomputer analysis of MQ NMR dynamics. The linewidth information in MQ NMR experiments is discussed.

Evidence for slow motion in proteins by multiple refocusing of heteronuclear nitrogen/proton multiple quantum coherences in NMR.

A novel NMR method characterizes slow motions in proteins by multiple refocusing of double- and zero-quantum coherences of amide protons and nitrogen-15 nuclei. If both nuclei experience changes in their isotropic chemical shifts because of internal motions on slow time scales (mus - ms), this leads to a difference in the relaxation rates of double- and zero-quantum coherences. This is due to CSM/CSM (chemical shift modulation) cross-correlation effects that are related to the well-known chemical exchange contribution Rex to the decay rate R2 = 1/T2 of nitrogen-15 nuclei. The CSM/CSM contributions can be distinguished from other mechanisms through their dependence on the repetition rate of a Carr-Purcell-Meiboom-Gill (CPMG) multiple refocusing sequence. In ubiquitin, motional processes can be identified that could hitherto not be observed by conventional CPMG nitrogen-15 NMR.

The catalytic mechanism of glucose 6-phosphate dehydrogenases: assignment and 1H NMR spectroscopy pH titration of the catalytic histidine residue in the 109 kDa Leuconostoc mesenteroides enzyme.

The chemical shifts of the C(epsilon1) and C(delta2) protons of His-240 from the 109 kDa Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase (G6PD) were assigned by comparing 1H and 13C spectra of the wild-type and mutant G6PDs containing the His-240 to asparagine mutation (H240N). Unambiguous assignment of the His-240 1H(epsilon1) resonance was obtained from comparing 13C-1H heteronuclear multiple quantum coherence NMR spectra of wild-type and H240N G6PDs that were selectively labeled with 13C(epsilon1) histidine. The results from NOESY experiments with wild-type and H240N variants were consistent with these assignments and the three-dimensional structure of G6PD. pH titrations show that His-240 has a pK(a) of 6.4. This value is, within experimental error, identical to the value of 6.3 derived from the pH dependence of kcat [Viola, R. E. (1984) Arch. Biochem. Biophys. 228, 415-424], suggesting that the pK(a) of His-240 is unperturbed in the apoenzyme despite being part of a His-Asp catalytic dyad. The results obtained for this 109 kDa enzyme indicate that 1H NMR spectroscopy in combination with heteronuclear methods can be a useful tool for functional analysis of large proteins.

Enzymatic Synthesis of Inulin-Containing Hydrogels

The Bacillus subtilis protease Proleather FG-F catalyzed the transesterification of inulin with vinyl acrylate (VA) in dimethylformamide (DMF). The reaction conversion for different VA concentrations was greater than 57% after 96 h at 50 degrees C. The degree of substitution (DS, defined as the amount of acrylate groups per 100 inulin fructofuranoside residues) with acrylate moieties can be controlled by varying the molar ratio of VA to inulin. Reasonable yields were obtained (44-51%, 2 days) using a two-step purification methodology. Inulin derivatized with VA (Inul-VA) was characterized by gel permeation chromatography, and its structure was established by (1)H, (13)C, and (1)H-(1)H correlation spectroscopy and (1)H-(13)C heteronuclear multiple quantum coherence NMR. The main positional isomer was at the 6 position of the fructofuranoside residue and two other minor isomers were observed at the 3 and 4 positions. Thus, the enzymatic reaction was largely regioselective. Furthermore, the inulin fructose residues were monosubstituted. Gels with swelling ratios at equilibrium of up to ca. 20 were prepared by free radical polymerization of aqueous solutions of Inul-VA with different DS and monomer concentrations. Gel pore sizes were calculated from swelling experiments and range from 19 to 57 A. To our knowledge, this work reports the first successful enzymatic modification of a polysaccharide solubilized in 100% DMF solution.

Direct measurement of transverse relaxation time of intermolecular multiple quantum coherences in NMR

A one-dimensional NMR method is presented for measuring the transverse relaxation time, T2,n, of intermolecular multiple quantum coherences (IMQCs) of coherence order n in highly polarized spin systems. The pulse sequence proposed in this paper effectively suppresses the effects of radiation damping, molecular diffusion, inhomogeneity of magnetic field and variations of dipolar correlation distance, all of which may affect quantitation of T2,n. This pulse sequence can be used to measure not only IMQC transverse relaxation time T2,n(n>1) quickly and directly, but also the conventional transverse relaxation time. Experimental results demonstrate that the quantitative relationship between T2,n(n?1) and T2 is T2,n?T2/n. These results will be helpful for understanding the fundamental properties and mechanisms of IMQCs.

Hydrogen Bonds in RNA Base Pairs Investigated by Cross-Correlated Relaxation of Multiple-Quantum Coherence in NMR

Cross-correlated relaxation of multiple-quantum coherences in NMR was used to investigate hydrogen bonds in Watson-Crick base pairs, based on the interference between various relaxation mechanisms. [on SciFinder (R)]

Amidation of Salicyluric Acid and Gentisuric Acid: A Possible Role for Peptidylglycine α-Amidating Monooxygenase in the Metabolism of Aspirin

Bifunctional peptidylglycine α-amidating monooxygenase (PAM) catalyzes the copper-, ascorbate-, and O2-dependent cleavage of C-terminal glycine-extended peptides, N-acylglycines, and the bile acid glycine conjugates to the corresponding amides and glyoxylate. Two known metabolites of aspirin, salicyluric acid and gentisuric acid, are also substrates for PAM, leading to the formation of salicylamide and gentisamide. The time course for O2 consumption and glyoxylate production indicates that salicylurate amidation is a two-step reaction. Salicylurate is first converted to N-salicyl-α-hydroxyglycine, which is ultimately dealkylated to salicylamide and glyoxylate. The enzymatically generated salicylamide and N-salicyl-α-hydroxyglycine were characterized by mass spectrometry and two-dimensional 1H–13C heteronuclear multiple quantum coherence NMR.

Heteronuclear NMR studies of the specificity of the post-translational modification of biotinyl domains by biotinyl protein ligase

The lipoyl domains of 2-oxo acid dehydrogenase multienzyme complexes and the biotinyl domains of biotin-dependent enzymes have homologous structures, but the target lysine residue in each domain is correctly selected for post-translational modification by lipoyl protein ligase and biotinyl protein ligase, respectively. We have applied two-dimensional heteronuclear NMR spectroscopy to investigate the interaction between the apo form of the biotinyl domain of the biotin carboxyl carrier protein of acetyl-CoA carboxylase and the biotinyl protein ligase (BPL) from Escherichia coli. Heteronuclear multiple quantum coherence NMR spectra of the 15N-labelled biotinyl domain were recorded in the presence and absence of the ligase and backbone amide 1H and 15N chemical shifts were evaluated. Small, but significant, changes in chemical shift were found in two regions, including the tight β-turn that houses the lysine residue targetted for biotinylation, and the β-strand 2 and the loop that precedes it in the domain. When compared with the three-dimensional structure, sequence alignments of other biotinyl and lipoyl domains, and mutagenesis data, these results give a clear indication of how the biotinyl domain is both recognised by BPL and distinguished from the structurally related lipoyl domain to ensure correct post-translational modification.

Determination of NADH-dependent glutamate synthase (GOGAT) in Spodoptera frugiperda (Sf9) insect cells by a selective 1H/ 15N NMR in vitro assay

This is the second of two papers [Drews, M., Doverskog, M., Öhman, L., Chapman, B.E., Jacobsson, U., Kuchel, P.W., Häggström, L., 2000. Pathways of glutamine metabolism in Spodoptera frugiperda (Sf9) insect cells: evidence for the presence of the nitrogen assimilation system, and a metabolic switch by 1H/ 15N NMR. J. Biotechnol. 78, 23–37]. where the general goal has been to determine and characterise the glutamine metabolism in Sf9 cells. The presence of glutamate synthase (GOGAT) activity was investigated in cell-free extracts of S. frugiperda (Sf9) insect cells by modified 1H/ 15N spin-echo and gradient enhanced multiple quantum coherence NMR spectroscopy techniques. Cell-free extracts were prepared from cells cultured in a serum-free medium. The assay conditions were based on conventional spectrophotometric and chromatographic methods. NMR data showed that nitrogen from [5- 15N] glutamine was selectively incorporated into 2-oxoglutarate forming [2- 15N] glutamate with a specific activity of 4.15±0.21 nmol [2- 15N] glutamate min −1 (mg total protein) −1 in the cell-free extracts. The enzyme activity was exclusively dependent on NADH as coenzyme and was completely inhibited by 1 mM azaserine. From the results obtained, we conclude that Sf9 cells possess NADH-GOGAT activity. Furthermore, the high specificity of the NMR method enables distinction of competing reactions from glutaminase and glutamate dehydrogenase.

The Enzymatic Formation of Novel Bile Acid Primary Amides

Bifunctional peptidylglycine α-amidating monooxygenase (PAM) catalyzes the copper-, ascorbate-, and O2-dependent cleavage of C-terminal glycine-extended peptides and N-acylglycines to the corresponding amides and glyoxylate. The α-amidated peptides and the long-chain acylamides are hormones in humans and other mammals. Bile acid glycine conjugates are also substrates for PAM leading to the formation of bile acid amides. The (VMAX/Km)app values for the bile acid glycine conjugates are comparable to other known PAM substrates. The highest (VMAX/Km)app value, 3.1 ± 0.12 × 105 M−1 s−1 for 3-sulfolithocholylglycine, is 6.7-fold higher than that for d-Tyr–Val–Gly, a representative peptide substrate. The time course for O2 consumption and glyoxylate production indicates that bile acid glycine conjugate amidation is a two-step reaction. The bile acid glycine conjugate is first converted to an N-bile acyl-α-hydroxyglycine intermediate which is ultimately dealkylated to the bile acid amide and glyoxylate. The enzymatically produced bile acid amides and the carbinolamide intermediates were characterized by mass spectrometry and two-dimensional 1H–13C heteronuclear multiple quantum coherence NMR.

Calmodulin binding properties of peptide analogues and fragments of the calmodulin-binding domain of simian immunodeficiency virus transmembrane glycoprotein 41

The calcium-regulatory protein calmodulin (CaM) can bind with high affinity to a region in the cytoplasmic C-terminal tail of glycoprotein 41 of simian immunodeficiency virus (SIV). The amino acid sequence of this region is (1)DLWETLRRGGRW(13)ILAIPRRIRQGLELT(28)L. In this work, we have used near- and far-uv CD, and fluorescence spectroscopy, to study the orientation of this peptide with respect to CaM. We have also studied biosynthetically carbon-13 methyl-Met calmodulin by (1)H, (13)C heteronuclear multiple quantum coherence NMR spectroscopy. Two Trp-substituted peptides, SIV-W3F and SIV-W12F, were utilized in addition to the intact SIV peptide. Two half-peptides, SIV-N (residues 1-13) and SIV-C (residues 13-28) were also synthesized and studied. The spectroscopic results obtained with the SIV-W3F and SIV-W12F peptides were generally consistent with those obtained for the native SIV peptide. Like the native peptide, these two analogues bind with an alpha-helical structure as shown by CD spectroscopy. Fluorescence intermolecular quenching studies suggested binding of Trp3 to the C-lobe of CaM. Our NMR results show that SIV-N can bind to both lobes of calcium-CaM, and that it strongly favors binding to the C-terminal hydrophobic region of CaM. The SIV-C peptide binds with relatively low affinity to both halves of the protein. These data reveal that the intact SIV peptide binds with its N-terminal region to the carboxy-terminal region of CaM, and this interaction initiates the binding of the peptide. This orientation is similar to that of most other CaM-binding domains.

Recognition of a Surface Loop of the Lipoyl Domain Underlies Substrate Channelling in the Pyruvate Dehydrogenase Multienzyme Complex

In the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus, the interaction between the pyruvate decarboxylase (E1p) component and the lipoyl domain of the dihydrolipoyl acetyltransferase (E2) component was investigated using a combination of site-directed mutagenesis and NMR spectroscopy. Residues 11 to 15 (EGIHE) of the lipoyl domain, part of a surface loop close in space to the β-turn containing the lipoyl-lysine residue (position 42), were deleted or replaced. The mutant domains all retained their three-dimensional structures and ability to become lipoylated, but in the absence of the loop the lipoyl-lysine residue could no longer be reductively acetylated by E1p. A mutation (N40A) in the N- terminal part of the lipoyl-lysine hairpin showed that it is involved in recognition of the domain by E1p but other mutations in the loop (E15A) and close to the lipoyl-lysine hairpin (V44S, V45S and E46A) were without effect. The heteronuclear multiple quantum coherence NMR spectra of 15N-labelled lipoyl domain in the presence and absence of B. stearothermophilusE1p were recorded. Of the 85 amino acid residues in the lipoyl domain, 13 exhibited significant differences in chemical shift. These differences, most of which were associated with residues in the surface loop between positions 8 and 15 and in, or close to, the lipoyl-lysine hairpin, indicate that E1p makes contact with the lipoyl domain in these areas. The combined results of directed mutagenesis and NMR spectroscopy point to the surface loop as a major determinant of the interaction of lipoyl domain with E1p. The specificity of this essential interaction provides the molecular basis of substrate channelling in this, the first committed, step of the enzyme reaction mechanism.

3-[(Phenylacetyl)amino]-2,6-piperidinedione Hydrolysis Studies with Improved Synthesis and Characterization of Hydrolysates

In an attempt to clarify ambiguities in earlier reports on the preclinical chemistry of Antineoplaston A10 (3-[(phenylacetyl)amino]-2,6-piperidinedione; PAP), we detail herein hydrolysis studies with improved synthesis and characterization of PAP hydrolysis products, (phenylacetyl)-glutamine (PAG), (phenylacetyl)isoglutamine (PAIG), and (phenylacetyl)-glutamic acid (PAGA). Flash chromatography proved superior to extraction in the isolation of synthetic standards and hydrolysates. Synthesis of PAIG directly from commercial isoglutamine showed consistently better yields than the previously reported method. The 1H and 13C NMR and HPLC–MS data from the synthesized standards matched the data from the isolated PAP hydrolysates formed under acid and alkaline degradation conditions. Multiple quantum coherence NMR methods (HMQC and HMBC) and HPLC–MS–MS methods were applied to provide unambiguous structural assignments for key isomers PAG and PAIG. Previous PAP hydrolysis studies are shown to be reproducible, and the structures of hydrolysis products are elucidated in detail.

17O and 1H- 15N heteronuclear multiple quantum coherence ( 1H- 15N HMQC) NMR of linear amides: evidence of an out-of-plane (torsion angle) deformation of the amide bond and pyramidicity at the amide nitrogen

17O and 1H- 15N heteronuclear multiple quantum coherence NMR studies of linear amides demonstrate the ready occurence of an out-of-plane (torsion angle) deformation of the amide bond and pyramidicity at the amide nitrogen for the sterically hindered trans-isomer of N-tert-butylformamide. Solvent accessibility, steric hindrance and, thus, torsion angle deformation is shown to be strongly solvent dependent.

Common metal ion coordination in LIM domain proteins.

The LIM motif is a cysteine- and histidine-rich sequence that was first identified in proteins involved in control of gene expression and cell differentiation. In order to characterize structural features of the LIM domain, we have carried out biophysical studies on two polypeptides that display LIM domains: the cysteine-rich intestinal protein (CRIP) and a fragment of the cysteine-rich protein (CRP). Bacterial expression vectors were constructed for the intact CRIP molecule and the C-terminal half of CRP, designated LIM2, such that each expressed protein contained a single LIM domain. Both proteins were recovered as soluble, Zn(II)-containing proteins. The metal coordination properties of these two distinct LIM domain proteins were highly similar, suggesting that a common structural architecture may exist in LIM domain proteins. Both proteins exhibit a maximum of two tetrahedrally bound Zn(II) ions per molecule. Electronic spectroscopy of Co(II) complexes and 113Cd NMR of Cd(II) complexes of CRIP and LIM2 revealed a similar ligand field pattern with one tetrathiolate (S4) site and one S3N1 site for divalent metal ions. The nitrogen ligand was shown to arise from a histidyl imidazole by heteronuclear multiple quantum coherence NMR. The eight conserved residues within the LIM domains of CRIP and LIM2 include seven cysteines and one histidine. It is likely that these conserved residues generate the S4 and S3N1 Zn(II)-binding sites. Metal binding to the two sites within a single LIM domain is sequential, with preferential occupancy of the S4 site. Slow metal ion exchange occurs between sites within an LIM domain, and metal exchange with exogenous metal ions is observed, with exchange at the S3N1 site being kinetically more facile. In the absence of metal binding both proteins appear to be substantially unfolded. Metal binding stabilizes a tertiary fold containing appreciable secondary structural elements. The common metal ion coordination in CRIP and LIM2 suggests that the LIM motif may constitute a structural module with conserved features.

Intermolecular multiple-quantum coherences and cross correlations in solution nuclear magnetic resonance

It was recently reported that multiple-quantum NMR coherences could apparently be observed in water and other concentrated samples, in direct violation of established theory. These results were previously explained in a dressed-state framework as manifestations of the coupling between the spins and the coil (quantized radiation damping). Here we provide details of previously communicated experimental explorations of these effects [J. Chem. Phys. 96, 1659 (1992)], and we extend these results to multicomponent samples. We observe cross peaks between independent molecules in solution in two-dimensional experiments, including spectra with multiple-quantum coherence transfer echoes; we also demonstrate coherence transfer between solvent and (dilute) solute molecules. However, we show that these intermolecular cross peaks are induced by a mechanism which is local in nature, and thus radiation damping (either classical or quantized) cannot provide the bulk of the explanation for their occurrence. Simulations and analytical results show that the dipolar demagnetizing field can account for many of these surprising effects, although a complete picture must be more complex.