Simplified analysis of acetaminophen glucuronide for quantifying gluconeogenesis and glycogenolysis using deuterated water

Measurement of acetaminophen glucuronide (AG) ²H enrichment from deuterated water (²H₂O) by ²H nuclear magnetic resonance (NMR) analysis of its monoacetone glucose (MAG) derivative provides estimation of gluconeogenic and glycogenolytic contributions to endogenous glucose production (EGP). However, AG derivatization to MAG is laborious and unsuitable for high-throughput studies. An alternative derivative, 5-O-acetyl monoacetone glucuronolactone (MAGLA), was tested. Eleven healthy subjects ingested ²H₂O to 0.5% body water enrichment and 500 mg of acetaminophen. Plasma glucose and urinary glucuronide positional ²H enrichments were measured by ²H NMR spectroscopy of MAG and MAGLA, respectively. A Bland–Altman analysis indicated agreement at the 95% confidence level between glucose and glucuronide estimates.     

An integrated (2)H and (13)C NMR study of gluconeogenesis and TCA cycle flux in humans

Hepatic glucose synthesis from glycogen, glycerol, and the tricarboxylic acid (TCA) cycle was measured in five overnight-fasted subjects by (1)H, (2)H, and (13)C NMR analysis of blood glucose, urinary acetaminophen glucuronide, and urinary phenylacetylglutamine after administration of [1,6-(13)C(2)]glucose, (2)H(2)O, and [U-(13)C(3)]propionate. This combination of tracers allows three separate elements of hepatic glucose production (GP) to be probed simultaneously in a single study: 1) endogenous GP, 2) the contribution of glycogen, phosphoenolpyruvate (PEP), and glycerol to GP, and 3) flux through PEP carboxykinase, pyruvate recycling, and the TCA cycle. Isotope-dilution measurements of [1,6-(13)C(2)] glucose by (1)H and (13)C NMR indicated that GP in 16-h-fasted humans was 10.7 +/- 0.9 micromol.kg(-1).min(-1). (2)H NMR spectra of monoacetone glucose (derived from plasma glucose) provided the relative (2)H enrichment at glucose H-2, H-5, and H-6S, which, in turn, reflects the contribution of glycogen, PEP, and glycerol to total GP (5.5 +/- 0.7, 4.8 +/- 1.0, and 0.4 +/- 0.3 micromol.kg(-1).min(-1), respectively). Interestingly, (13)C NMR isotopomer analysis of phenylacetylglutamine and acetaminophen glucuronide reported different values for PEP carboxykinase flux (68.8 +/- 9.8 vs. 37.5 +/- 7.9 micromol.kg(-1).min(-1)), PEP recycling flux (59.1 +/- 9.8 vs. 27.8 +/- 6.8 micromol.kg(-1).min(-1)), and TCA cycle flux (10.9 +/- 1.4 vs. 5.4 +/- 1.4 micromol.kg(-1).min(-1)). These differences may reflect zonation of propionate metabolism in the liver.     

Measurement of inter-glycosidic 13C-1H coupling constants in a cyclic β(1→2)-glucan by 13C-filtered 2D {1H,1H}ROESY

Summary A method for measuring three-bond ¹³C-¹H scalar coupling constants across glycosidic bonds in a cyclic (12)-glucan icosamer is presented. This oligosaccharide molecule, with its high degree of symmetry, represents a particular challenge for NMR spectroscopy to distinguish inter-residue from intra-residue heteronuclear coupling effects. Chemically equivalent H2 protons in adjacent glucosyl residues are distinguished on the basis of their different through-space, dipolar interactions with the anomeric protons (H1). The strong NOE contact between anomeric (H1) and aglyconic (H2) protons permits the selective observation of the inter-residue heteronuclear couplings ³J_C1H2 and ³J_C2H1 in a natural-abundance ¹³C-₁-half-filtered {¹H,¹H} ROESY experiment.Abbreviations COSY scalar correlated spectroscopy - NOE nuclear Overhauser effect - NOESY NOE spectroscopy - ROESY rotating-frame NOE spectroscopy     

Measurement of amino acid metabolism derived from [1-13C]glucose in the rat brain using13C magnetic resonance spectroscopy

To clarify the unique characteristics of amino acid metabolism derived from glucose in the central nervous system (CNS), we injected [1-¹³C]glucose intraperitoneally to the rat, and extracted the free amino acids from several kinds of tissues and measured the amount of incorporation of¹³C derived from [1-¹³C]glucose into each amino acid using¹³C-magnetic resonance spectroscopy (NMR). In the adult rat brain, the intensities of resonances from¹³C-amino acids were observed in the following order: glutamate, glutamine, aspartate, -aminobutyrate (GABA) and alanine. There seemed no regional difference on this labeling pattern in the brain. However, only in the striatum and thalamus, the intensities of resonances from [2-¹³C]GABA were larger than that from [2,3-¹³C]aspartate. In the other tissues, such as heart, kidney, liver, spleen, muscle, lung and small intestine, the resonances from GABA were not detected and every intensity of resonances from¹³C-amino acids, except¹³C-alanine, was much smaller than those in the brain and spinal cord. In the serum,¹³C-amino acid was not detected at all. When the rats were decapitated, in the brain, the resonances from [1-¹³C]glucose greatly reduced and the intensities of resonances from [3-¹³C]lactate, [3-¹³C]alanine, [2, 3, 4-¹³C]GABA and [2-¹³C]glutamine became larger as compared with those in the case that the rats were sacrificed with microwave. In other tissues, the resonances from [1-¹³C]glucose were clearly detected even after the decapitation. In the glioma induced by nitrosoethylurea in the spinal cord, the large resonances from glutamine and alanine were observed; however, the intensities of resonances from glutamate were considerably reduced and the resonances from GABA and aspartate were not detected. These results show that the pattern of¹³C label incorporation into amino acids is unique in the central nervous tissues and also suggest that the metabolic compartmentalization could exist in the CNS through the metabolic trafficking between neurons and astroglia.Abbreviations NMR nuclear magnetic resonance - GABA -aminobutyrate - GFAP glial fibrillary acidic protein Special issue dedicated to Dr. Bernard W. Agranoff.     

Metabolism of [1-13C]glucose in a synaptosomally enriched fraction of rat cerebrum studied by1H/13C magnetic resonance spectroscopy

This study explored the utility of¹H and¹³C magnetic resonance spectroscopy to study a standard synaptosomally enriched fraction (P2 pellet) made from rat cerebrum. The preparations contained high concentrations of N-acetylaspartate and -aminobutyric acid and low concentrations of glutamine, indicating that they were in fact rich in neuronal cytosol. The metabolic competence of the preparation was assessed by quantitative measurements of its ability to convert [1-¹³C]glucose into lactate, glutamate, aspartate, and other metabolites under well oxygenated conditions in 30 minutes. The minimum mean glycolytic rate was 0.8 mM glucose/min and the flow through the tricarboxylic acid cycle was equivalent to 0.2 mM glucose/min.Abbreviations ppm parts per million (chemical shift scale) - NMR nuclear magnetic resonance - GABA -aminobutyric acid - PBS phosphate-buffered normal saline solution - TSP 3-trimethylsilylpropionate During the performance of these studies Dr. A.P. Burlina was on leave from Instituto di Clinica delle Malattie Nervose e Mentali, University of Padua, Padua, Italy.     

Biosynthesis of theobroxide and its related compounds, metabolites of Lasiodiplodia theobromae

Administration of (13)C labeled acetates ([1-(13)C], [2-(13)C] and [1,2-(13)C(2)] to Lasiodiplodia theobromae showed the tetraketide origins of both theobroxide, a potato-tuber inducing substance [1, (1S, 2R, 5S, 6R)-3-methyl-7-oxa-bicyclo[4.1.0]hept-3-en-2,5-diol]) and its carbonyldioxy derivative [2, (1S, 4R, 5S, 6R)-7,9-dioxa-3-methyl-8-oxobicyclo [4.3.0]-2-nonene-4,5-diol]. The incorporation of acetate-derived hydrogen into 1 and 2 was studied using [2-(2)H(3), 2-(13)C]acetate. Three and one deuterium atoms were incorporated at one methyl and epoxy carbons, respectively. The observed loss of deuterium atoms from the methyl group suggests a considerable amount of exchange from the methyl group of [2-(2)H(3), 2-(13)C]acetate during biosynthesis of 1 and 2. Incorporation of [1-(13)C]- and [1,2-(13)C(2)]acetates indicates the carbonyl carbon of the carbonyldioxy derivative is derived from the carboxy carbon of the precursor.     

Biosynthetically directed fractional 13C labeling facilitates identification of Phe and Tyr aromatic signals in proteins

Analysis of 2D [¹³C,¹H]-HSQC spectra of biosynthetic fractionally ¹³C labeled proteins is a reliable, straightforward means to obtain stereospecific assignments of Val and Leu methyl sites in proteins. Herein we show that the same fractionally labeled protein sample facilitates observation and identification of Phe and Tyr aromatic signals. This is the case, in part, because the fractional ¹³C labeling yields aromatic rings in which some of the ¹³C-¹³C J-couplings, present in uniformly labeled samples, are absent. Also, the number of homonuclear J-coupling partners differs for the -, - and -carbons. This enabled us to vary their signal intensities in distinctly different ways by appropriately setting the ¹³C constant-time period in 2D [¹³C,¹H]-HSQC spectra. We illustrate the application of this approach to an 18 kDa protein, c-VIAF, a modulator of apoptosis. In addition, we show that cancellation of the aromatic ¹³C CSA and ¹³C-¹H dipolar interactions can be fruitfully utilized in the case of the fractionally labeled sample to obtain high resolution ¹³C constant-time spectra with good sensitivity.     

Support of1H NMR assignments in proteins by biosynthetically directed fractional13C-labeling

Summary Biosynthetically directed fractional incorporation of¹³C into proteins results in nonrandom¹³C-labeling patterns that can be investigated by analysis of the¹³C–¹³C scalar coupling fine structures in heteronuclear¹³C–¹H or homonuclear¹³C–¹³C correlation experiments. Previously this approach was used for obtaining stereospecific¹H and¹³C assignments of the diastereotopic methyl groups of valine and leucine. In the present paper we investigate to what extent the labeling patterns are characteristic for other individual amino acids or groups of amino acids, and can thus be used to support the¹H spin-system identifications. Studies of the hydrolysates of fractionally¹³C-labeled proteins showed that the 59 aliphatic carbon positions in the 20 proteinogenic amino acids exhibit 16 different types of¹³C–¹³C coupling fine structures. These provide support for the assignment of the resonances of all methyl groups in a protein, which are otherwise often poorly resolved in homonuclear¹H NMR spectra. In particular, besides the individual methyl assignments in Val and Leu, unambiguous distinctions are obtained between the methyl groups of Ala and Thr, and between the - and -methyl groups of Ile. In addition to the methyl resonances, the CH₂ groups of Glu and Gln can be uniquely assigned because of the large coupling constant with the -carbon, and the identification of most of the other spin systems can be supported on the basis of coupling patterns that are common to small groups of amino acid residues.Abbreviations NOE nuclear Overhauser effect - fractional¹³C labeling biosynthetically directed fractional¹³C-labeling - TOCSY total correlation spectroscopy - ROESY rotating frame Overhauser enhancement spectroscopy - [¹³C,¹H]-COSY two-dimensional¹³C–¹H correlation spectroscopy - isotopomer isotope isomer - P22 c2 repressor c2 repressor of the salmonella phage P22 consisting of a polypeptide chain with 216 residues - P22 c2(1-76) N-terminal domain of the P22 c2 repressor with residues 1–76     

Time-saving methods for heteronuclear multidimensional NMR of (13C, 15N) doubly labeled proteins

Summary Heteronuclear 2D (¹³C, ¹H) and (¹⁵N, ¹H) correlation spectra of (¹³C, ¹⁵N) fully enriched proteins can be acquired simultaneously with virtually no sensitivity loss or increase in artefact levels. Three pulse sequences are described, for 2D time-shared or TS-HSQC, 2D TS-HMQC and 2D TS-HSMQC spectra, respectively. Independent spectral widths can be sampled for both heteronuclei. The sequences can be greatly improved by combining them with field-gradient methods. By applying the sequences to 3D and 4D NMR spectroscopy, considerable time savings can be obtained. The method is demonstrated for the 18 kDa HU protein.Abbreviations HMQC heteronuclear multiple-quantum coherence spectroscopy - HSQC heteronuclear single-quantum coherence spectroscopy - HSMQC heteronuclear single- and multiple-quantum coherence spectroscopy - NOESY nuclear Overhauser enhancement spectroscopy     

Selectively 13C-enriched DNA: 13C and 1H assignments of a triple helix by two-dimensional relayed HMQC experiments

Summary We present NMR studies of an intramolecular triple helix, the three strands of which have been linked by a hexaethylene glycol chain. To overcome the generally encountered difficulties of assignment in the homopyrimidine strands, the carbon C1 of the pyrimidines were selectively ¹³C-enriched. Assignments of the aromatic and sugar protons were obtained from NOESY-HMQC and TOCSY-HMQC spectra. We show that the recognition of a DNA duplex by a third strand via triplex formation is easily carried out in solution by observing the changes of the ¹H1–¹³C1 connectivities as a function of pH. Furthermore, the conformation of the sugars has been found to be C2-endo, on the basis of the coupling constant values directly measured in an HSQC spectrum.     

Cross-correlated spin relaxation effects in methyl <Superscript>1</Superscript>H CPMG-based relaxation dispersion experiments: Complications and a simple solution

Artifacts associated with the measurement of methyl ¹H single quantum CPMG-based relaxation dispersion profiles are described. These artifacts arise due to the combination of cross-correlated spin relaxation effects involving intra-methyl ¹H–¹H dipolar interactions and imperfections in ¹H refocusing pulses that are applied during CPMG intervals that quantify the effects of chemical exchange on measured transverse relaxation rates. As a result substantial errors in extracted exchange parameters can be obtained. A simple work-around is presented where the ¹H chemical shift difference between the exchanging states is extracted from a combination of ¹³C single quantum and ¹³C–¹H multiple quantum dispersion profiles. The approach is demonstrated with an application to a folding/unfolding reaction involving a G48M mutant Fyn SH3 domain.     

1H, 15N, 13C and 13CO assignments and secondary structure determination of basic fibroblast growth factor using 3D heteronuclear NMR spectroscopy

Summary The assignments of the ¹H, ¹⁵N, ¹³CO and ¹³C resonances of recombinant human basic fibroblast growth factor (FGF-2), a protein comprising 154 residues and with a molecular mass of 17.2 kDa, is presented based on a series of three-dimensional triple-resonance heteronuclear NMR experiments. These studies employ uniformly labeled ¹⁵N- and ¹⁵N-/¹³C-labeled FGF-2 with an isotope incorporation >95% for the protein expressed in E. coli. The sequence-specific backbone assignments were based primarily on the interresidue correlation of C, C and H to the backbone amide ¹H and ¹⁵N of the next residue in the CBCA(CO)NH and HBHA(CO)NH experiments and the intraresidue correlation of C, C and H to the backbone amide ¹H and ¹⁵N in the CBCANH and HNHA experiments. In addition, C and C chemical shift assignments were used to determine amino acid types. Sequential assignments were verified from carbonyl correlations observed in the HNCO and HCACO experiments and C correlations from the carbonyl correlations observed in the HNCO and HCACO experiments and C correlations from the HNCA experiment. Aliphatic side-chain spin systems were assigned primarily from H(CCO)NH and C(CO)NH experiments that correlate all the aliphatic ¹H and ¹³C resonances of a given residue with the amide resonance of the next residue. Additional side-chain assignments were made from HCCH-COSY and HCCH-TOCSY experiments. The secondary structure of FGF-2 is based on NOE data involving the NH, H and H protons as well as ³J_H n _H coupling constants, amide exchange and ¹³C and ¹³C secondary chemical shifts. It is shown that FGF-2 consists of 11 well-defined antiparallel -sheets (residues 30–34, 39–44, 48–53, 62–67, 71–76, 81–85, 91–94, 103–108, 113–118, 123–125 and 148–152) and a helix-like structure (residues 131–136), which are connected primarily by tight turns. This structure differs from the refined X-ray crystal structures of FGF-2, where residues 131–136 were defined as -strand XI. The discovery of the helix-like region in the primary heparin-binding site (residues 128–138) instead of the -strand conformation described in the X-ray structures may have important implications in understanding the nature of heparin-FGF-2 interactions. In addition, two distinct conformations exist in solution for the N-terminal residues 9–28. This is consistent with the X-ray structures of FGF-2, where the first 17–19 residues were ill defined.     

The solution conformation of sialyl-α(2→6)-lactose studied by modern NMR techniques and Monte Carlo simulations

Summary We present a comprehensive strategy for detailed characterization of the solution conformations of oligosaccharides by NMR spectroscopy and force-field calculations. Our experimental strategy generates a number of interglycosidic spatial constraints that is sufficiently large to allow us to determine glycosidic linkage conformations with a precision heretofore unachievable. In addition to the commonly used {¹H,¹H} NOE contacts between aliphatic protons, our constraints are: (a) homonuclear NOEs of hydroxyl protons in H₂O to other protons in the oligosaccharide, (b) heteronuclear {¹H,¹³C} NOEs, (c) isotope effects of O¹H/O²H hydroxyl groups on¹³C chemical shifts, and (d) long-range heteronuclear scalar coupling across glycosidic bonds.We have used this approach to study the trisaccharide sialyl-(26)-lactose in aqueous solution. The experimentally determined geometrical constraints were compared to results obtained from force-field calculations based on Metropolis Monte Carlo simulations. The molecule was found to exist in 2 families of conformers. The preferred conformations of the (26)-linkage of the trisaccharide are best described by an equilibrium of 2 conformers with angles at –60° or 180° and of the 3 staggered rotamers of the angle with a predominantgt conformer. Three intramolecular hydrogen bonds, involving the hydroxyl protons on C8 and C7 of the sialic acid residue and on C3 of the reducing-end glucose residue, contribute significantly to the conformational stability of the trisaccharide in aqueous solution. Supplementary material available from the corresponding author: Table containing values for the dihedral angles , , , , and for bond angles , for the six lowest-energy conformations of sialyl-(26)-lactose (1 page).     

L-[1-13C] phenylalanine breath test results reflect the activity of phenylalanine hydroxylase in carbon tetrachloride acute injured rat liver

L-[1-13C] phenylalanine breath tests (PheBTs) have been used to determine the hepatocyte functional capacity of patients. This study investigated the relationship between the PheBT parameter 13C excretion rate constant (PheBT-k) and activity of the phenylalanine metabolic rate-limiting enzyme phenylalanine hydroxylase (PAH) in rat liver. We noted that the time-course curves of 13C excretion presented as a single peak, which appeared 2 min after administration of L-[1-13C] phenylalanine (13C-Phe). 13C excretion during exhalation can be divided into a slow phase and a rapid phase. The PheBT-k in rats with carbon tetrachloride acute liver injury was.significantly lower than that of control rats. The rapid phase 13C disposition constants of the acute liver injured rats did not differ from that of the controls. The peak value of 13C abundance in the breath of the acute liver injured rats was markedly higher than that of the control group. Total liver PAH activity in the acute liver injured rats was significantly lower than that in the control group. PheBT-k was highly correlated with the total activity of liver PAH (r = 0.92, P < 0.001). The present findings indicate that PheBT results reflect PAH activity levels. The PheBT-k parameter is a sensitive index that can be used to evaluate PAH function in the liver. In addition we demonstrated that the rodent model used in this study is a valuable tool for basic research studies of the breath test.     

13C-detected HN(CA)C and HMCMC experiments using a single methyl-reprotonated sample for unambiguous methyl resonance assignment

Methyl groups provide an important source of structural and dynamic information in NMR studies of proteins and their complexes. For this purpose sequence-specific assignments of methyl 1H and 13C resonances are required. In this paper we propose the use of 13C-detected 3D HN(CA)C and HMCMC experiments for assignment of methyl 1H and 13C resonances using a single selectively methyl protonated, perdeuterated and 13C/15N-labeled sample. The high resolution afforded in the 13C directly-detected dimension allows one to rapidly and unambiguously establish correlations between backbone HN strips from the 3D HN(CA)C spectrum and methyl group HmCm strips from the HMCMC spectrum by aligning all possible side-chain carbon chemical shifts and their multiplet splitting patterns. The applicability of these experiments for the assignment of methyl 1H and 13C resonances is demonstrated using the 18.6 kDa B domain of the Escherichia coli mannose transporter (IIBMannose).     

Multinuclear NMR and molecular modelling investigations on the structure and equilibria of complexes that form in aqueous solutions of Ca and gluconate

Complexation of d-gluconate (Gluc⁻) with Ca²⁺ has been investigated via ¹H, ¹³C and ⁴³Ca NMR spectroscopy in aqueous solutions in the presence of high concentration background electrolytes (1 M ? I ? 4 M (NaCl) ionic strength). From the ionic strength dependence of its formation constant, the stability constant at 6 ? pH ? 11 and at I → 0 M has been derived (). The protonation constant of Gluc⁻ at I = 1 M (NaCl) ionic strength was also determined and was found to be log K_a = 3.24 ± 0.01 (¹³C NMR) and log K_a = 3.23 ± 0.01 (¹H NMR). It was found that ¹H and ¹³C NMR chemical shifts upon complexation (both with H⁺ and with Ca²⁺) do not vary in an unchanging way with the distance from the Ca²⁺/H⁺ binding site. From 2D ¹H-⁴³Ca NMR spectra, simultaneous binding of Ca²⁺ to the alcoholic OH on C2 and C3 was deduced. Molecular modelling results modulated this picture by revealing structures in which the Gluc⁻ behaves as a multidentate ligand. The five-membered chelated initial structure was found to be thermodynamically more stable than that derived from a six-membered chelated initial structure.     

NMR and modelling studies of disaccharide conformation

Long-range heteronuclear coupling constants were measured across the glycosidic linkages for a series of eight alpha- or beta-linked disaccharides in aqueous solution. Multiple 13C site-selective excitation experiments using 1H decoupling in conjunction with pulsed field gradient-enhanced spectroscopy were used to determine 3J(C,H) values. These were subsequently compared with the respective couplings calculated, using a Karplus relationship, from molecular dynamics simulations with the explicit inclusion of water.     

Assimilate partitioning affects 13C fractionation of recently assimilated carbon in maize

Coupling ¹³C natural abundance and ¹⁴C pulse labelling enabled us to investigate the dependence of ¹³C fractionation on assimilate partitioning between shoots, roots, exudates, and CO₂ respired by maize roots. The amount of recently assimilated C in these four pools was controlled by three levels of nutrient supply: full nutrient supply (NS), 10 times diluted nutrient supply (DNS), and deionised water (DW). After pulse labelling of maize shoots in a ¹⁴CO₂ atmosphere, ¹⁴C was traced to determine the amounts of recently assimilated C in the four pools and the δ¹³C values of the four pools were measured. Increasing amounts of recently assimilated C in the roots (from 8% to 10% of recovered ¹⁴C in NS and DNS treatments) led to a 0.3‰ ¹³C enrichment from NS to DNS treatments. A further increase of C allocation in the roots (from 10% to 13% of recovered ¹⁴C in DNS and DW treatments) resulted in an additional enrichment of the roots from DNS to DW treatments by 0.3‰. These findings support the hypothesis that ¹³C enrichment in a pool increases with an increasing amount of C transferred into that pool. δ¹³C of CO₂ evolved by root respiration was similar to that of the roots in DNS and DW treatments. However, if the amount of recently assimilated C in root respiration was reduced (NS treatment), the respired CO₂ became 0.7‰ ¹³C depleted compared to roots. Increasing amounts of recently assimilated C in the CO₂ from NS via DNS to DW treatments resulted in a 1.6‰ δ¹³C increase of root respired CO₂ from NS to DW treatments. Thus, for both pools, i.e. roots and root respiration, increasing amounts of recently assimilated C in the pool led to a δ¹³C increase. In DW and DNS plants there was no ¹³C fractionation between roots and exudates. However, high nutrient supply decreased the amount of recently assimilated C in exudates compared to the other two treatments and led to a 5.3‰ ¹³C enrichment in exudates compared to roots. We conclude that ¹³C discrimination between plant pools and within processes such as exudation and root respiration is not constant but strongly depends on the amount of C in the respective pool and on partitioning of recently assimilated C between plant pools. Section Editor: H. Lambers     

HNCAN pulse sequences for sequential backbone resonance assignment across proline residues in perdeuterated proteins

A TROSY-based triple-resonance pulse scheme is described which correlates backbone ¹H and ¹⁵N chemical shifts of an amino acid residue with the ¹⁵N chemical shifts of both the sequentially preceding and following residues. The sequence employs ¹ J _NC and ² J _NC couplings in two sequential magnetization transfer steps in an `out-and-back' manner. As a result, N,N connectivities are obtained irrespective of whether the neighbouring amide nitrogens are protonated or not, which makes the experiment suitable for the assignment of proline resonances. Two different three-dimensional variants of the pulse sequence are presented which differ in sensitivity and resolution to be achieved in one of the nitrogen dimensions. The new method is demonstrated with two uniformly ²H/¹³C/¹⁵N-labelled proteins in the 30-kDa range.     

Estimates of methyl 13C and 1H CSA values (Δσ) in proteins from cross-correlated spin relaxation

Simple pulse schemes are presented for the measurement of methyl ¹³C and ¹H CSA values from ¹H–¹³C dipole/¹³C CSA and ¹H–¹³C dipole/¹H CSA cross-correlated relaxation. The methodology is applied to protein L and malate synthase G. Average ¹³C CSA values are considerably smaller for Ile than Leu/Val (17 vs 25 ppm) and are in good agreement with previous solid state NMR studies of powders of amino acids and dipeptides and in reasonable agreement with quantum-chemical DFT calculations of methyl carbon CSA values in peptide fragments. Small averaged ¹H CSA values on the order of 1 ppm are measured, consistent with a solid state NMR determination of the methyl group ¹H CSA in dimethylmalonic acid.