ABSTRACT
In this study, 18 animals were fed two forage-based diets: red clover (RC) and grass silage (GS), in a crossover-design experiment in which methane (CH4) emissions were recorded in respiration chambers. Rumen samples obtained through naso-gastric sampling tubes were analysed by NMR. Methane yield (g/kg DM) was significantly lower from animals fed RC (17.8 ± 3.17) compared to GS (21.2 ± 4.61) p = 0.008. In total 42 metabolites were identified, 6 showing significant differences between diets (acetate, propionate, butyrate, valerate, 3-phenylopropionate, and 2-hydroxyvalerate). Partial least squares discriminant analysis (PLS-DA) was used to assess which metabolites were more important to distinguish between diets and partial least squares (PLS) regressions were used to assess which metabolites were more strongly associated with the variation in CH4 emissions. Acetate, butyrate and propionate along with dimethylamine were important for the distinction between diets according to the PLS-DA results. PLS regression revealed that diet and dry matter intake are key factors to explain CH4 variation when included in the model. Additionally, PLS was conducted within diet, revealing that the association between metabolites and CH4 emissions can be conditioned by diet. These results provide new insights into the methylotrophic methanogenic pathway, confirming that metabolite profiles change according to diet composition, with consequences for CH4 emissions.
Subject(s)
Rumen , Silage , Animals , Cattle , Diet/veterinary , Methane/metabolism , Poaceae/metabolism , Rumen/metabolism , Silage/analysisABSTRACT
This study presents the application of metabolomics to evaluate changes in the rumen metabolites of beef cattle fed with three different diet types: forage-rich, mixed and concentrate-rich. Rumen fluid samples were analysed by 1H-NMR spectroscopy and the resulting spectra were used to characterise and compare metabolomic profiles between diet types and assess the potential for NMR metabolite signals to be used as proxies of methane emissions (CH4 in g/kg DMI). The dataset available consisted of 128 measurements taken from 4 experiments with CH4 measurements taken in respiration chambers. Predictive modelling of CH4 was conducted by partial least squares (PLS) regression, fitting calibration models either using metabolite signals only as predictors or using metabolite signals as well as other diet and animal covariates (DMI, ME, weight, BW0.75, DMI/BW0.75). Cross-validated R2 were 0.57 and 0.70 for the two models respectively. The cattle offered the concentrate-rich diet showed increases in alanine, valerate, propionate, glucose, tyrosine, proline and isoleucine. Lower methane yield was associated with the concentrate-rich diet (p < 0.001). The results provided new insight into the relationship between rumen metabolites, CH4 production and diets, as well as showing that metabolites alone have an acceptable association with the variation in CH4 production from beef cattle.
Subject(s)
Cattle/metabolism , Methane/analysis , Rumen/metabolism , Animals , Diet/veterinary , Female , Magnetic Resonance Spectroscopy/methods , Male , Metabolomics , Rumen/chemistryABSTRACT
Unravelling structures of molecules contained in complex, chromatographically inseparable mixtures is a challenging task. Due to the number of overlapping resonances in NMR spectra of these mixtures, unambiguous chemical shift correlations attributable to individual molecules cannot be achieved and thus their structure determination is elusive by this technique. Placing a tag carrying an NMR active nucleus onto a subset of molecules enables (i) to eliminate signals from the non-tagged molecules, and (ii) to obtain a set of correlated chemical shifts and coupling constants belonging to a single molecular type. This approach provides an opportunity for structure determination without the need for compound separation. Focusing on the most abundant functional groups of natural organic matter molecules, the carboxyl and hydroxyl groups were converted into esters and ethers, respectively by introducing (13)CH3O groups. A set of (13)C-filtered nD NMR experiments was designed yielding structures/structural motives of tagged molecules. The relative sensitivity of these experiments was compared and a step-by-step guide how to use these experiments to analyse the structures of methylated phenolics is provided. The methods are illustrated using an operational fraction of soil organic matter, fulvic acid isolated from a Scottish peat bog. Analysis of 33 structures identified in this sample revealed a correlation between the position of the methoxy cross-peaks in the (1)H, (13)C HSQC spectra and the compound type. This information enables profiling of phenolic compounds in natural organic matter without the need to acquire a full set of experiments described here or access to high field cryoprobe NMR spectrometers.
ABSTRACT
The 155-kDa glycoprotein, complement factor H (CFH), is a regulator of complement activation that is abundant in human plasma. Three-dimensional structures of over half the 20 complement control protein (CCP) modules in CFH have been solved in the context of single-, double- and triple-module segments. Proven binding sites for C3b occupy the N and C termini of this elongated molecule and may be brought together by a bend in CFH mediated by its central CCP modules. The C-terminal CCP 20 is key to the ability of the molecule to adhere to polyanionic markers on self-surfaces where CFH acts to regulate amplification of the alternative pathway of complement. The surface patch on CCP 20 that binds to model glycosaminoglycans has been mapped using nuclear magnetic resonance (NMR), as has a second glycosaminoglycan-binding patch on CCP 7. These patches include many of the residue positions at which sequence variations have been linked to three complement-mediated disorders: dense deposit disease, age-related macular degeneration and atypical haemolytic uraemic syndrome. In one plausible model, CCP 20 anchors CFH to self-surfaces via a C3b/polyanion composite binding site, CCP 7 acts as a 'proof-reader' to help discriminate self- from non-self patterns of sulphation, and CCPs 1-4 disrupt C3/C5 convertase formation and stability.
Subject(s)
Complement Factor H/genetics , Amino Acid Sequence , Binding Sites , Complement C3/immunology , Complement Factor H/chemistry , Complement Factor H/immunology , Humans , Models, Molecular , Molecular Sequence Data , Mutation , Protein Conformation , Sequence Alignment , Structure-Activity RelationshipABSTRACT
The solution-state NMR spectra of a per-6-substituted gamma-cyclodextrin show some interesting dynamic properties. At high temperature (353 K), the (1)H NMR spectrum shows dynamic averaging of the different conformations. This averaging is no longer observed on cooling of the cyclodextrin solution to 278 K, resulting in NMR spectra with a large (1)H and (13)C chemical shift dispersion. The complete assignment of the eight unique glucosyl residues was achieved using COSY, HSQC and exchange spectroscopy. A ROESY spectrum, with a short mixing time to reduce the effects of exchange, gives correlations that lead to the determination of the connectivity of all eight glucosyl residues. On the NMR time-scale, the cyclodextrin is highly dynamic; the lower temperature minimum energy conformation has one of the aromatic rings self-complexed and a distorted cyclodextrin torus.
Subject(s)
Magnetic Resonance Spectroscopy/methods , Sodium/chemistry , gamma-Cyclodextrins/chemistry , Magnetic Resonance Spectroscopy/standards , Molecular Conformation , TemperatureABSTRACT
The relative configuration of the two xanthene units of neosartorin, a new ergochrome biosynthesised by the soil mould Neosartorya fischeri, was determined using a 1D double-pulsed field gradient spin-echo NOESY experiment. It was found that both units have the same relative stereochemistry. Long-range nonbonding interactions between the substituents of different xanthene units stabilise the nonplanar configuration of the two aromatic rings A and A' connecting both monomer units of neosartorin.
ABSTRACT
The complement control protein (CCP) module (also known as SCR, CCP or sushi domain) is prevalent amongst proteins that regulate complement activation. Functional and mutagenesis studies have shown that in most cases two or more neighbouring CCP modules form specific binding sites for other molecules. Hence the orientation in space of a CCP module with respect to its neighbours and the flexibility of the intermodular junction are likely to be critical for function. Vaccinia virus complement control protein (VCP) is a complement regulatory protein composed of four tandemly arranged CCP modules. The solution structure of the carboxy-terminal half of this protein (CCP modules 3 and 4) has been solved previously. The structure of the central portion (modules 2 and 3, VCP approximately 2,3) has now also been solved using NMR spectroscopy at 37 degrees C. In addition, the backbone dynamics of VCP approximately 2,3 have been characterised by analysis of its (15)N relaxation parameters. Module 2 has a typical CCP module structure while module 3 in the context of VCP approximately 2,3 has some modest but significant differences in structure and dynamics to module 3 within the 3,4 pair. Modules 2 and 3 do not share an extensive interface, unlike modules 3 and 4. Only two possible NOEs were identified between the bodies of the modules, but a total of 40 NOEs between the short intermodular linker of VCP approximately 2,3 and the bodies of the two modules determines a preferred, elongated, orientation of the two modules in the calculated structures. The anisotropy of rotational diffusion has been characterised from (15)N relaxation data, and this indicates that the time-averaged structure is more compact than suggested by (1)H-(1)H NOEs. The data are consistent with the presence of many intermodular orientations, some of which are kinked, undergoing interconversion on a 10(-8)-10(-6) second time-scale. A reconstructed representation of modules 2-4 allows visualisation of the spatial arrangement of the 11 substitutions that occur in the more potent complement inhibitor from Variola (small pox) virus.
Subject(s)
Poxviridae/chemistry , Viral Proteins/chemistry , Amino Acid Sequence , Magnetic Resonance Spectroscopy , Models, Chemical , Models, Molecular , Molecular Sequence Data , Protein Conformation , Sequence Homology, Amino AcidABSTRACT
The structure and backbone dynamics of a double labelled (15N,13C) monomeric, 23.7 kD phosphoglycerate mutase (PGAM) from Schizosaccharomyces pombe have been investigated in solution using NMR spectroscopy. A set of 3125 NOE-derived distance restraints, 148 restraints representing inferred hydrogen bonds and 149 values of (3)J(HNHalpha) were used in the structure calculation. The mean rmsd from the average structure for all backbone atoms from residues 6-205 in the best 21 calculated structures was 0.59 A. The core of the enzyme includes an open, twisted, six-stranded beta-sheet flanked by four alpha-helices and a short 3(10)-helix. An additional smaller domain contains two short antiparallel beta-strands and a further pair of alpha-helices. The C(alpha) atoms of the S. pombe PGAM may be superimposed on their equivalents in one of the four identical subunits of Saccharomyces cerevisiae PGAM with an rmsd of 1.34 A (0.92 A if only the beta-sheet is considered). Small differences between the two structures are attributable partly to the deletion in the S. pombe sequence of a 25 residue loop involved in stabilising the S. cerevisiae tetramer. Analysis of 15N relaxation parameters indicates that PGAM tumbles isotropically with a rotational correlation time of 8.7 ns and displays a range of dynamic features. Of 178 residues analysed, only 77 could be fitted without invoking terms for fast internal motion or chemical exchange, and out of the remainder, 77 required a chemical exchange term. Significantly, 46 of the slowly exchanging (milli- to microsecond) residues lie in helices, and these account for two-thirds of all analysed helix residues. On the contrary, only one beta-sheet residue required an exchange term. In contrast to other analyses of backbone dynamics reported previously, residues in slow exchange appeared to correlate with architectural features of the enzyme rather than congregating close to ligand binding sites.
Subject(s)
Phosphoglycerate Mutase/chemistry , Phosphoglycerate Mutase/metabolism , Schizosaccharomyces/enzymology , Amino Acid Substitution/genetics , Crystallography, X-Ray , Models, Molecular , Molecular Weight , Nuclear Magnetic Resonance, Biomolecular , Phosphoglycerate Mutase/genetics , Protein Structure, Secondary , Saccharomyces cerevisiae/enzymology , Schizosaccharomyces/genetics , SolutionsABSTRACT
Fluorescence resonance energy transfer provides valuable long-range distance information about macromolecules in solution. Fluorescein and Cy3 are an important donor-acceptor pair of fluorophores; the characteristic Förster length for this pair on DNA is 56 A, so the pair can be used to study relatively long distances. Measurement of FRET efficiency for a series of DNA duplexes terminally labeled with fluorescein and Cy3 suggests that the Cy3 is close to the helical axis of the DNA. An NMR analysis of a self-complementary DNA duplex 5'-labeled with Cy3 shows that the fluorophore is stacked onto the end of the helix, in a manner similar to that of an additional base pair. This provides a known point from which distances calculated from FRET measurements are measured. Using the FRET efficiencies for the series of DNA duplexes as restraints, we have determined an effective position for the fluorescein, which is maximally extended laterally from the helix. The knowledge of the fluorophore positions can now be used for more precise interpretation of FRET data from nucleic acids.
Subject(s)
Carbocyanines , DNA/chemistry , Nucleic Acid Conformation , Oligodeoxyribonucleotides/chemistry , Base Sequence , Energy Transfer , Fluorescein , Fluorescent Dyes , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Oligodeoxyribonucleotides/chemical synthesis , Spectrometry, FluorescenceABSTRACT
We have used NMR spectroscopy to determine the three-dimensional (3D) structure, and to characterize the backbone dynamics, of a recombinant version of bovine beta-lactoglobulin (variant A) at pH 2. 6, where the protein is a monomer. The structure of this low-pH form of beta-lactoglobulin is very similar to that of a subunit within the dimer at pH 6.2. The root-mean-square deviation from the pH 6.2 (crystal) structure, calculated for backbone atoms of residues 6-160, is approximately 1.3 A. Differences arise from the orientation, with respect to the calyx, of the A-B and C-D loops, and of the flanking three-turn alpha-helix. The hydrophobic cavity within the calyx is retained at low pH. The E-F loop (residues 85-90), which moves to occlude the opening of the cavity over the pH range 7.2-6.2, is in the "closed" position at pH 2.6, and the side chain of Glu89 is buried. We also carried out measurements of (15)N T(1)s and T(2)s and (1)H-(15)N heteronuclear NOEs at pH 2.6 and 37 degrees C. Although the residues of the E-F loop (residues 86-89) have the highest crystallographic B-factors, the conformation of this loop is reasonably well defined by the NMR data, and its backbone is not especially mobile on the pico- to nanosecond time scale. Several residues (Ser21, Lys60, Ala67, Leu87, and Glu112) exhibit large ratios of T(1) to T(2), consistent with conformational exchange on a micro- to millisecond time scale. The positions of these residues in the 3D structure of beta-lactoglobulin are consistent with a role in modulating access to the hydrophobic cavity.
Subject(s)
Lactoglobulins/chemistry , Lactoglobulins/metabolism , Animals , Cattle , Crystallization , Crystallography, X-Ray , Dimerization , Hydrogen-Ion Concentration , Nuclear Magnetic Resonance, Biomolecular/methods , Peptide Fragments/chemistry , Protein Structure, Secondary , Solutions , Structure-Activity RelationshipABSTRACT
Two 3D experiments, (H)CCH(3)-TOCSY and H(C)CH(3)-TOCSY, are proposed for resonance assignment of methyl-containing amino acid side chains. After the initial proton-carbon INEPT step, during which either carbon or proton chemical shift labeling is achieved (t(1)), the magnetization is spread along the amino acid side chains by a carbon spin lock. The chemical shifts of methyl carbons are labeled (t(2)) during the following constant time interval. Finally the magnetization is transferred, in a reversed INEPT step, to methyl protons for detection (t(3)). The proposed experiments are characterized by high digital resolution in the methyl carbon dimension (t(2max) = 28.6 ms), optimum sensitivity due to the use of proton decoupling during the long constant time interval, and an optional removal of CH(2), or CH(2) and CH, resonances from the F(2)F(3) planes. The building blocks used in these experiments can be implemented in a range of heteronuclear experiments focusing on methyl resonances in proteins. The techniques are illustrated using a (15)N, (13)C-labeled E93D mutant of Schizosacharomyces pombe phosphoglycerate mutase (23.7 kDa).
Subject(s)
Magnetic Resonance Spectroscopy/methods , Proteins/chemistryABSTRACT
Based on the HSQC scheme, we have designed a 2D heterocorrelated experiment which combines constant time (CT) 13C and variable time (VT) 15N chemical shift labelling. Although applicable to all carbons, this mode is particularly suitable for simultaneous recording of methyl-carbon and nitrogen chemical shifts at high digital resolution. The methyl carbon magnetisation is in the transverse plane during the whole CT period (1/J(CC) = 28.6 ms). The magnetisation originating from NH protons is initially stored in the 2HzNz state, then prior to the VT chemical shift labelling period is converted into 2HzNy coherence. The VT -15N mode eliminates the effect of 1J(N,CO) and 1,2J(N,CA) coupling constants without the need for band-selective carbon pulses. An optional editing procedure is incorporated which eliminates signals from CH2 groups, thus removing any potential overlap with the CH3 signals. The CT-13CH3,VT-15N HSQC building block is used to construct two 3D experiments: 3D NOESY-CH3NH and 3D 13C,15N HSQC-NOESY-CH3NH. Combined use of these experiments yields proton and heteronuclear chemical shifts for moieties experiencing NOEs with CH3 and NH protons. These NOE interactions are resolved as a consequence of the high digital resolution in the carbon and nitrogen chemical shifts of CH3 and NH groups, respectively. The techniques are illustrated using a double labelled sample of the CH domain from calponin.
Subject(s)
Magnetics , Nuclear Magnetic Resonance, Biomolecular/methods , Algorithms , Animals , Calcium-Binding Proteins/chemistry , Carbon Isotopes , Electronic Data Processing , Humans , Microfilament Proteins , Nitrogen Isotopes , Protein Structure, Tertiary , Time Factors , CalponinsABSTRACT
MeCP2 is an abundant mammalian protein that binds methylated CpG (mCpG) sequences within double-stranded DNA, represses transcription by recruiting histone deacetylases, and is essential for embryonic development. It is one of a family of proteins which mediate the biological consequences of DNA methylation. These proteins each possess a sequence motif of about 70 residues which, in MeCP2, form a domain necessary and sufficient for binding to mCpG. The solution structure of the mCpG-binding domain (MBD) from MeCP2 has been solved and the DNA-binding surface of the domain mapped using NMR spectroscopy. Residues 95-162 of MeCP2 adopt a novel fold forming a wedge-shaped structure. An N-terminal four-stranded antiparallel beta-sheet forms one face of the wedge, while the other face is formed mainly by a C-terminal helical region. The thin end of the wedge is extended by a long loop between beta-strands B and C containing many basic residues. The B-C loop together with residues in strands B, C and D, and at the N terminus of the alpha-helix, appears to form an interface with methylated DNA. Unstructured residues at the NH2 terminus of the domain are also involved in formation of the complex. The presence of numerous arginine and lysine side-chains on the DNA-binding surface of MBD is consistent with the requirement for the mCpG site to be flanked by non-specific sequences of base-pairs. The absence of symmetry in the domain implies that recognition does not exploit the symmetry of the binding site. A conserved hydrophobic pocket containing the side-chains of Tyr123 and Ile125 on the positively charged beta-sheet face is a candidate for the region of contact with the methyl-groups of the modified cytosine residues.
Subject(s)
Chromosomal Proteins, Non-Histone , CpG Islands , DNA Methylation , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , DNA/chemistry , DNA/metabolism , Repressor Proteins , Amino Acid Sequence , Binding Sites , Conserved Sequence , DNA/genetics , DNA-Binding Proteins/genetics , Methyl-CpG-Binding Protein 2 , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Conformation , Protein Folding , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Solutions , Structure-Activity RelationshipABSTRACT
A segment of complement receptor type 1 (CR1) corresponding to modules 15-17 was overexpressed as a functionally active recombinant protein with N-glycosylation sites ablated by mutagenesis (referred to as CR1 approximately 15-17(-)). A protein consisting of modules 15 and 16 and another corresponding to module 16 were also overexpressed. Comparison of heteronuclear nuclear magnetic resonance (NMR) spectra for the single, double, and triple module fragments indicated that module 16 makes more extensive contacts with module 15 than with module 17. A combination of NMR, differential scanning calorimetry, circular dichroism, and tryptophan-derived fluorescence indicated a complex unfolding pathway for CR1 approximately 15-17(-). As temperature or denaturant concentration was increased, the 16-17 junction appeared to melt first, followed by the 15-16 junction, and module 17 itself; finally, modules 15 and 16 became denatured. Modules 15 and 16 adopted an intermediate state prior to total denaturation. These results are compared with a previously published study [Clark, N. S., Dodd, I, Mossakowska, D. E., Smith, R. A. G., and Gore, M. G. (1996) Protein Eng. 9, 877-884] on a fragment consisting of the N-terminal three CR1 modules which appeared to melt as a single unit.
Subject(s)
Peptide Fragments/chemistry , Receptors, Complement 3b/chemistry , Amino Acid Sequence , Calorimetry, Differential Scanning , Circular Dichroism , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Peptide Fragments/biosynthesis , Peptide Fragments/genetics , Peptide Fragments/isolation & purification , Pichia/genetics , Protein Conformation , Protein Folding , Receptors, Complement 3b/biosynthesis , Receptors, Complement 3b/genetics , Receptors, Complement 3b/isolation & purification , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Solutions , Spectrometry, Fluorescence , ThermodynamicsABSTRACT
beta-Lactoglobulin (beta-Lg) is the major whey protein in ruminant milk and has been implicated in the irreversible denaturation of milk proteins and its associated poor processing behavior during heat treatment. In order to help understand this behavior, as well as to facilitate other studies into the relationship between the molecular structure and its behavior in solution, we have prepared and purified 15N-labeled and 13C/15N-double-labelled beta-Lg in sufficient quantities to permit a full determination of the structure and dynamics using heteronuclear NMR spectroscopy. The overexpression of the labeled protein using the Pichia pastoris system proceeds with good yield but requires the removal of significant quantities of copurifying carbohydrate which otherwise interfere with the NMR experiments. At pH 2, the resulting material gives triple resonance NMR spectra of good quality that are consistent with a monomeric, globular protein rich in beta-sheet.
Subject(s)
Lactoglobulins/chemistry , Lactoglobulins/genetics , Pichia/genetics , Amino Acid Sequence , Animals , Carbon Isotopes , Cattle , Dimerization , Gene Expression , Hydrogen-Ion Concentration , Lactoglobulins/isolation & purification , Magnetic Resonance Spectroscopy , Nitrogen Isotopes , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purificationABSTRACT
Although beta-lactoglobulin (beta-LG) has been studied extensively for more than 50 years, its physical properties in solution are not yet understood fully in terms of its three-dimensional (3D) structure. For example, despite a recent high-resolution crystal structure, it is still not clear why the two common variants of bovine beta-LG which differ by just two residues have different aggregation properties during milk processing. We have conducted solution-state NMR studies on a recombinant form of the A variant of beta-LG at low pH conditions where the protein is partially unfolded and exists as a monomer rather than a dimer. Using a 13C, 15N-labelled sample, expressed in Pichia pastoris, we have employed the standard combination of 3D heteronuclear NMR techniques to obtain near complete assignments of proton, carbon and nitrogen resonances. Using a novel pulse sequence we were able to obtain additional assignments, in particular those of methyl groups in residues preceding proline within the sequence. From chemical shifts and on the basis of inter-residue NOEs, we have inferred the secondary structure and topology of monomeric beta-LG A. It includes eight antiparallel beta-strands arranged in a barrel, flanked by an alpha-helix, which is typical of a member of the lipocalin family. A detailed comparison with the crystal structure of the dimeric form (for a mixture of A and B variants) at pH 6.5 reveals a close resemblance in both secondary structure and overall topology. Both forms have a ninth beta-strand which, at the higher pH, forms part of the dimer interface. These studies represent the first full NMR assignment of beta-LG and will form the basis for a complete characterisation of the solution structure and dynamics of this protein and its variants.
Subject(s)
Lactoglobulins/chemistry , Protein Folding , Protein Structure, Secondary , Amino Acid Sequence , Animals , Carbon Isotopes , Cattle , Crystallography, X-Ray/methods , Hydrogen , Molecular Sequence Data , Nitrogen Isotopes , Nuclear Magnetic Resonance, Biomolecular/methods , Protein Denaturation , Recombinant Proteins/chemistryABSTRACT
A pulsed field gradient version of the sensitivity-enhanced 2D TOCSY experiment is proposed which yields high-quality spectra with improved sensitivity and a minimum of two scans per t1 increment. For rapid acquisition of 1D TOCSY spectra, the 1D DPFGSE-TOCSY experiment was modified to include phase-encoded multiple-selective excitation followed by a simple spectral editing. Combination of these two building blocks is used in a sensitivity-enhanced 2D analog of the 3D TOCSY-TOCSY experiment which provides an efficient tool for resolving severely overlapped signals of oligomers in short experimental time.
Subject(s)
Image Enhancement , Magnetic Resonance Spectroscopy , Analgesics, Opioid/chemistry , Artifacts , Image Processing, Computer-Assisted , Oligopeptides/chemistry , Sensitivity and Specificity , SoftwareABSTRACT
An enhanced version of the X (omega1) half-filtered TOCSY experiment for measurement of long-range heteronuclear coupling constants is proposed which yields high-quality spectra with substantially increased sensitivity and resolution. The modified method features gradient-enhanced X filtering sequences, broadband homonuclear decoupling during t1, optional 1JXH scaling in the F1 domain, and gradient coherence selection in combination with the sensitivity-enhanced protocol for the TOCSY transfer. These modifications extend the applicability of the method--coupling constants can be measured accurately for natural abundance samples at low concentrations and for compounds yielding complex spectra. Computer-aided analysis of E.COSY-type multiplets is applied for the determination of heteronuclear long-range coupling constants.