Investigation of the relationship between tyrosyl residues and the adenosine 5'-monophosphate binding site of rabbit liver fructose-1,6-biphosphatase as studied by chemical modification and nuclear magnetic resonance spectroscopy

The effects of AMP, fructose 6-phosphate (Fru-6-P), fructose 2,6-bisphosphate (Fru-2,6-P2), and paramagnetic ions on the aromatic region of the proton nuclear magnetic resonance (NMR) spectrum of rabbit liver fructose-1,6-bisphosphatase have been investigated at 300 MHz. Two well resolved peaks in this region of the NMR spectrum are assigned to the protons from the aromatic ring of a tyrosyl residue of the enzyme by chemical modification with tetranitromethane and by nuclear Overhauser effects. Nitration of the tyrosyl residue causes desensitization of the enzyme to AMP inhibition as well as the loss of activity. In the presence of AMP during the modifications, 1 tyrosyl residue could be protected, presumably the one observed by NMR. Binding of AMP, an allosteric inhibitor of the enzyme, to rabbit liver fructose-1,6-bisphosphatase leads to an upfield shift of the tyrosyl proton signals in the NMR spectrum. No chemical shift or line broadening could be detected in the presence of the paramagnetic manganous ion, Fru-2,6-P2, or Fru-6-P. The negative intramolecular nuclear Overhauser effect from the ribose H2' proton to the adenine H8 proton of AMP suggested that AMP binds to the enzyme with an anti conformation about the glycosidic bond. The failure to observe intermolecular nuclear Overhauser effects between the tyrosyl residue and the protons of AMP indicates that the distances between them are greater than 4 A. On the basis of these observations, it is suggested that the AMP-related tyrosyl residue may be close to the AMP binding site, but it is not directly involved in ligand binding. Rather, the protection of this tyrosyl residue by AMP as observed by chemical modification experiments may well be due to a conformational change that results from covalent modification of the enzyme.     

Proton nuclear magnetic resonance studies of intact native bovine parathyroid hormone

Native intact bovine PTH was studied by proton nuclear magnetic resonance (NMR) techniques, at pH 3.5 and pH 6.3. The 1H-NMR spectra had good resolution and many multiplet structures were observed. Assignment of the NMR resonances corresponding to specific amino acids was approached using 1H chemical shifts, coupling constants, and pH dependence in the one-dimensional spectra and the 1H-1H connectivities revealed in two-dimensional homonuclear correlated spectroscopy (COSY) experiments. All the aromatic proton resonances were assigned. Two histidine residues had lower pK than the other two. The methyl groups of two residues were moved significantly downfield: using COSY and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) correlations, these were assigned to an alanine residue close to both Trp-23 and Tyr-43, and a valine residue in close spatial proximity to Trp-23. The NOESY spectrum also showed cross-peaks between the residues of the upfield valine-leucine-isoleucine methyl envelope. Many of the H alpha protons moved upfield as the pH was increased. These results indicate that intact native PTH exists in a preferred conformation in solution at pH 6.5. Our studies have provided new information on the three-dimensional spatial proximity of several amino acids along the polypeptide chain. The observed interactions are consistent with the currently accepted model suggesting that the hormone has two separate structural domains associated with the amino- and carboxy-terminal regions of the molecule respectively. The potential implications of this model for the expression of biological activity are discussed.     

Proton magnetic resonance study of kringle 1 from human plasminogen. Insights into the domain structure

The aromatic H NMR spectrum of the kringle 1 domain from human plasminogen has been investigated by proton Overhauser experiments, acid-base titration, and two-dimensional chemical shift correlated spectroscopy. Spin-echo and pH response experiments lead to the identification of the N-terminal Tyr-3 phenol ring signals. The connectivities among the tryptophanyl aromatic protons have been established and sets of singlet-doublet-triplet resonances stemming from each of the two indole groups sorted according to their common side chain origin. Similarly, the four histidyl singlets have been identified and paired per imidazole group. From their pH responses, it is indicated that a histidyl (His31) and a tryptophanyl (Trp-II) residue are placed in the neighborhood of carboxyl groups. The high-field chemical shifts observed for proton resonances of the ligand epsilon-aminocaproic acid upon binding to kringle 1 indicate that the ligand-binding site is rich in aromatic components. Overhauser experiments reveal that Leu46 is surrounded by a cluster of interacting aromatic side chains, which includes Trp25, Phe36, His41, Trp62, and Tyr64, and define a hydrophobic region contiguous to the kringle lysine-binding site. Relative internuclear distances have been estimated for aromatic H-atoms in the vicinity of Leu46 by reference to one of the latter's CH3 sigma, sigma' groups. Some of the connectives have previously been found for Leu46 in kringle 4 which further supports the idea of a common structure for the homologous domains.     

Complete assignment of the imino protons of Escherichia coli valine transfer RNA: two-dimensional NMR studies in water

The imino proton spectrum of Escherichia coli valine tRNA has been studied by two-dimensional nuclear Overhauser effect spectroscopy (NOESY) in H2O solution. The small nuclear Overhauser effects from the imino proton of an internal base pair to the imino protons of each nearest neighbor can be observed as off-diagonal cross-peaks. In this way most of the sequential NOE connectivity trains for all the helices in this molecule can be determined in a single experiment. AU resonances can be distinguished from GC resonances by the AU imino NOE to the aromatic adenine C2-H, thus leading to specific base-pair assignments. In general, the NOESY spectrum alone is not capable of assigning every imino proton resonance even in well-resolved tRNA spectra. Multiple proton peaks exhibit more than two cross-peaks, resulting in ambiguous connectivities, and coupling between protons with similar chemical shifts produces cross-peaks that are incompletely resolved from the diagonal. The sequence of the particular tRNA determines the occurrence of the latter problem, which can often be solved by careful one-dimensional experiments. The complete imino proton assignments of E. coli valine tRNA are presented.     

The use of two-dimensional correlated spectroscopy to obtain new assignments in the high-resolution 1H nuclear magnetic resonance spectrum of the biantennary complex oligosaccharide isolated from human serum transferrin by hydrazinolysis

The asialo biantennary complex type oligosaccharide from human serum transferrin was isolated by hydrazinolysis, a method which results in the quantitative release of the intact oligosaccharide free of all amino acids. The 1H-NMR chemical shifts of the previously assigned anomeric and H-2 protons from the peripheral residues of the glycopeptide are identical to the corresponding values for the reduced oligosaccharide. The chemical shift of GlcNAc-1 H-1 proton in the reduced oligosaccharide was assigned by selective deuteration. Proton J connectivities were determined using two-dimensional 1H-1H correlated high resolution NMR spectroscopy. Twelve new assignments were made within the central envelope of the NMR spectrum and a further six were tentatively proposed. The ability to assign proton resonances in this way should allow further conformational studies of the oligosaccharide using nuclear Overhauser effects between the relevant assigned protons on different saccharide residues (Homans, S.W., Dwek, R.A., Fernandes, D.L. and Rademacher, T.W. (1982) FEBS Lett. 150, 503-506).     

Total assignments, including four aromatic residues, and sequence confirmation of the decapeptide tyrocidine A using difference double resonance. Qualitative nuclear overhauser effect criteria for beta turn and antiparallel beta-pleated sheet conformations.

The complete assignments of all the proton magnetic resonance signals from each NH-CalphaH-CbetaH2 moiety in a complex peptide containing several residues of the same type has not yet been achieved without specific or stereospecific isotopic enrichment. We report the sequencing and proton magnetic resonance spectral assignments, including those of 4 aromatic residues, of tyrocidine A, an analog of the decapeptide gramicidin S. Two complementary methods, proton-proton nuclear Overhauser enhancements and scalar decoupling, evaluated by two distinct forms of difference double resonance, were used. All chemical shifts, scalar coupling constants, and [1H:1H] nuclear Overhauser enhancements for the backbone protons are reported. The [1H:1H] nuclear Overhauser enhancements are consistent with tyrocidine A possessing a beta-I turn/beta-II' turn/antiparallel beta-pleated sheet conformation. In addition to the previously proposed nuclear Overhauser enhancement criteria for beta turns and antiparallel beta sheets, another criterion for identifying the antiparallel beta sheet is demonstrated; namely, the nuclear Overhauser enhancement between 2 CalphaH protons of the central resisdues, in this case the Phe7CalphaH and Orn2CalphaH.     

Two gamma-bends in the backbone conformation of [D-Ala2]-leucine enkephalin in solution

The solution conformation of [D-Ala2]-leucine enkephalin in its zwitterionic form in DMSO-d6 has been monitored by one- and two-dimensional proton magnetic resonance spectroscopy at 500 MHz. The resonances from the labile amide protons and the nonlabile protons have been assigned from the shift correlated spectroscopy. The chemical shift of the amide and C-alpha protons are found to vary with temperature but in opposite directions, except the C-alpha proton of the terminal tyrosine residue. This behavior has been explained by the shifting of equilibrium between the zwitterionic and neutral forms of the [D-Ala2]-leucine enkephalin and probably conformational changes accompanying temperature variation. The low values of the temperature coefficients of leucine and glycine amide protons indicate that these protons are either intramolecularly hydrogen bonded or solvent shielded. The observation of sequential cross peaks in the nuclear Overhauser effect spectra obtained at various mixing times, tau m (200-900 ms), indicate an extended backbone, which does not corroborate with the presence of a folded structure, i.e., beta-bend type structure. The estimate of interproton distances in conjunction with the low values of temperature coefficients of the leucine and glycine amide protons and vicinal coupling constants 3JHN-C alpha H have been rationalized by the predominance of two gamma-bends in the backbone conformation of [D-Ala2]-leucine enkephalin. The gamma-bend around the D-Ala residue has phi = 80 degrees and psi = 270 degrees, while the one around Phe it has phi = 285 degrees and psi = 90 degrees.     

Nuclear magnetic resonance studies of the binding of trimethoprim to dihydrofolate reductase.

The resonances of the aromatic protons of trimethoprim [2,4-diamino-5-(3',4',5'-trimethoxybenzyl)pyrimidine] in its complexes with dihydrofolate reductases from Lactobacillus casei and Escherichia coli cannot be directly observed. Their chemical shifts have been determined by transfer of saturation experiments and by difference spectroscopy using [2',6'-2H2]trimethoprim. The complex of 2,4-diamino-5-(3',4'-dimethoxy-5'-bromobenzyl)pyrimidine with the L. casei enzyme has also been examined. At room temperature, the 2',6'-proton resonance of bound trimethoprim is very broad (line width great than 30 Hz); with the E. coli enzyme, the resonance sharpens with increasing temperature so as to be clearly visible by difference spectroscopy at 45 degrees C. This line broadening is attributed to an exchange contribution, arising from the slow rate of "flipping" about the C7-C1' bond of bound trimethoprim. The transfer of saturation measurements were also used to determine the dissociation rate constants of the complexes. In the course of these experiments, a decrease in intensity of the resonance of the 2',6'-proton resonance of free trimethoprim on irradiation at the resonance of the 6 proton of free trimethoprim was observed, which only occurred in the presence of the enzyme. This is interpreted as a nuclear Overhauser effect between two protons of the bound ligand transferred to those of the free ligand by the exchange of the ligand between the two states. The chemical shift changes observed on the binding of trimethoprim to dihydrofolate reductase are interpreted in terms of the ring-current shift contributions from the two aromatic rings of trimethoprim and from that of phenylalanine-30. On the basis of this analysis of the chemical shifts, a model for the structure of the enzyme-trimethoprim complex is proposed. This model is consistent with the (indirect) observation of a nuclear Overhauser effect between the 2',6' and 6 protons of bound trimethoprim.     

The use of two-dimensional correlated spectroscopy to obtain new assignments in the high-resolution 1H nuclear magnetic resonance spectrum of the biantennary complex oligosaccharide isolated from human serum transferrin by hydrazinolysis

The asialo biantennary complex type oligosaccharide from human serum transffrrin was isolated by hydrazinolysis, a method which results in the quantitative release of the intact oligosaccharide free of all amino acids. The ¹H-NMR chemical shifts of the previously assigned anomeric and H-2 protons from the peripheral residues of the glycopeptide are identical to the corresponding values for the reduced oligosaccharide. The chemical shift of GlcNAc-1 H-1 proton in the reduced oligosaccharide was assigned by selective deuteration. Proton J connectivities were determined using two-dimensional ¹H-¹H correlated high resolution NMR spectroscopy. Twelve new assignments were made within the central envelope of the NMR spectrum and a further six were tentatively proposed. The ability to assign proton resonances in this way should allow further conformational studies of the oligosaccharide using nuclear Overhauser effects between the relevant assigned protons on different saccharide residues (Homans, S.W., Dwek, R.A., Fernandes, D.L. and Rademacher, T.W. (1982) FEBS Lett. 150, 503–506).     

A proton nuclear magnetic resonance and nuclear overhauser effect (NOE) study of human plasma prealbumin, including the development and application to spectral assignment of a combined ring current shift and NOE prediction program

Human prealbumin, a homotetrameric protein with an Mr of approximately 55,000 has been characterised by proton magnetic resonance spectroscopy. The pH dependences of the chemical shifts of the 4 histidine residues/subunit are unremarkable; variable temperature studies show that the protein tertiary and quaternary structure is stable to at least 80 degrees C. Assignment of a number of residues was aided by the development of a computer program, PARSNIP (Programme to Analyse Ring current Shifts and nuclear Overhauser effects (NOEs) In Proteins), which employs high resolution x-ray crystal structure atomic co-ordinates (Blake, C.F., and Oatley, S.J. (1977) Nature 268, 115-120, in the case of prealbumin) and several different predictive formalisms to calculate structure-dependent ring current-induced shifts of aromatic and methyl group protons. The program combines these with distance measurements to give plots of expected NOE difference profiles resulting from irradiation at a discrete frequency. The routine should prove applicable to many proteins whose structures have been determined but which are too large for the application of sequential residue assignment techniques. Three well resolved high field shifted methyl groups resonating at 0.18, 0.28, and 0.34 ppm are assigned to Ala-120, Leu-111, and Val-122, respectively. NOEs between these signals and the aromatics, interaromatic effects, and effects involving some of the considerable number of downfield shifted alpha-protons, have been used to identify substituents on several other residues.     

Comparative study of conformational behaviour of leucine and methionine enkephalinamides by1H-nuclear magnetic resonance spectroscopy

The conformational proclivity of leucine and methionine enkephalinamides in deuterated dimethyl sulphoxide has been investigated using proton magnetic resonance at 500 MHz. The resonances from the spin system of the various amino acid residues have been assigned from the 2-dimensional correlated spectroscopy spectra. The temperature variation of the amide proton shifts indicates that none of the amide proton is intramolecularly hydrogen-bonded or solvent-shielded. The analysis of vicinal coupling constants,³J_{HN.C 2}H,along with temperature coefficients and the absence of characteristic nuclear Overhauser effect cross peaks between the NH protons reveal that there is no evidence of the chain folding in these molecules. However, the observation of nuclear Overhauser effect cross peaks between the NH and the C^αH of the preceding residue indicates preference for extended backbone conformation with preferred side chain orientations particularly of Tyr and Phe in both [Leu⁵]- and [Met⁵]-enkephalinamides.     

A study of the interactions between residues in the C-terminal half of calmodulin by one and two-dimensional NMR methods and computer modelling

Assignments of the six sets of aromatic ring protons and four high-field-shifted methyl group protons of the C-terminal fragment of calmodulin, residues 78-148, was achieved by a combination of one and two-dimensional NMR spectroscopic methods. A full spectral analysis of the aromatic region in terms of chemical shifts and scalar coupling constants was achieved and confirmed by spectral simulation. A three-dimensional structural model of the C-terminal fragment was constructed by interactive computer graphics techniques and combined with nuclear Overhauser enhancements to propose sequence assignments for all aromatic and high-field-shifted methyl groups. This computer-generated three-dimensional model was generally supported by the fact that it qualitatively accounted for many of the ring-current-shifted proton resonances and the intraresidue and interresidue nuclear Overhauser enhancements.     

The 1H-NMR assignments of the aromatic resonances in complexes of Lactobacillus casei dihydrofolate reductase and the origins of their chemical shifts

All the aromatic proton resonances in the 500-MHz NMR spectra of Lactobacillus casei dihydrofolate reductase have been assigned for several of its complexes with inhibitors. For the complexes with methotrexate and trimethoprim this was achieved by using a combination of NMR techniques in conjunction with a selectively deuterated protein designed to simplify the spectra such that nuclear Overhauser effect (NOE) connections could be detected with greater ease and certainty. By correlating these NOE data with crystal structure data on related complexes it was possible to assign all the aromatic resonances and to extend these assignments to spectra of other complexes of dihydrofolate reductase. The conformation-dependent chemical shifts observed for many of the resonances could be explained qualitatively, but not quantitatively, in terms of ring-current shifts. The discrepancies between calculated ring-current shifts and the observed conformation-dependent shifts could not in general be accounted for satisfactorily in terms of carbonyl-group anisotropic shielding contributions calculated using presently available models. In the case of the H delta 1, delta 2 protons of Phe30 some of the discrepancy probably results from a difference in the conformation of the Phe ring between the solution and crystal states.     

Sequence-specific recognition of DNA. Nuclear magnetic resonance assignments and structural comparison of wild-type and mutant lambda OR3 operator DNA

The resonances of all the base protons and most of the sugar protons in both strands of the 17 base-pair OR3 operator of the phage lambda, and of the vC3 single base-pair mutant, have been assigned using two-dimensional nuclear magnetic resonance methods. The chemical shift and nuclear Overhauser effect data for these two DNA sequences reveal no structural perturbation at sites distal to the mutation, neither are there significant changes in structure immediately surrounding the altered base-pair in the mutant sequence. These results are consistent with the model proposed by Ohlendorf et al. (1982), based on crystallographic data on the cro protein, for the OR3-cro protein interaction. The data from these solution studies are examined and discussed in the light of this model. This work demonstrates that nuclear magnetic resonance chemical shifts and nuclear Overhauser effect intensities provide a method for comparing the solution structures of DNA molecules. From the resolution available in the spectra of the 17 base-pair operators studied, it is clear that DNA duplexes of up to 30 or more base-pairs can be studied using phase-sensitive methods.     

Assignment of the non-exchangeable proton resonances and solution conformation of the dodecanucleotide-d(CAATCCGGATTG)

The non-exchangeable base and sugar protons of the dodecanucleotide d(CAATCCGGATTG) have been assigned by the combined use of one and two-dimensional NMR spectroscopy. The sequential assignments of the base and sugar protons have been obtained using two dimensional nuclear Overhauser effect and homonuclear shift correlated spectra. By analysis of the nuclear Overhauser effect data it was determined that the dodecanucleotide assumes a right handed B-type helix in the aqueous medium used in this study.     

Four-dimensional 13C/13C-edited nuclear Overhauser enhancement spectroscopy of a protein in solution: application to interleukin 1 beta

A four-dimensional 13C/13C-edited NOESY experiment is described which dramatically improves the resolution of protein NMR spectra and enables the straightforward assignment of nuclear Overhauser effects involving aliphatic and/or aromatic protons in larger proteins. The experiment is demonstrated for uniformly (greater than 95%) 13C-labeled interleukin 1 beta, a protein of 153 residues and 17.4 kDa, which plays a key role in the immune response. NOEs between aliphatic and/or aromatic protons are first spread out into a third dimension by the 13C chemical shift of the carbon atom attached to the originating proton and subsequently into a fourth dimension by the 13C chemical shift of the carbon atom attached to the destination proton. Thus, each NOE cross peak is labeled by four chemical shifts. By this means, ambiguities in the assignment of NOEs that arise from chemical shift overlap and degeneracy are completely removed. Further, NOEs between protons with the same chemical shifts can readily be detected providing their attached carbon atoms have different 13C chemical shifts. The design of the pulse sequence requires special care to minimize the level of artifacts arising from undesired coherence transfer pathways, and in particular those associated with "diagonal" peaks which correspond to magnetization that has not been transferred from one proton to another.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nmr studies of an extrahelical cytosine in an A·T rich region of a deoxyribodecanucleotide

One-dimensional and two-dimensional (2D) nmr experiments were carried out on an oligonucleotide duplex that contains an unpaired cytosine, d(GCGAAC AAGCG)·d(CGCTTTTCGC), which will be referred to as the C-bulge decamer. Evidence from one-dimensional nuclear Overhauser effect (NOE) experiments on the exchangeable protons indicates that the unpaired cytosine is extrahelical. This conclusion is also supported by numerous cross-peaks in the 2D NOE spectroscopy (NOESY) spectrum of the nonexchangeable protons. The assignments for all of the resonances, with the exception of the H5′ and H5″ resonances, have been made through the use of 2D NOESY, correlated spectroscopy (COSY), and relayed COSY experiments. The temperature dependence of the C(H6) resonance chemical shifts indicates that the unpaired cytosine shows unusual behavior compared to other cytosines in the duplex. A comparison of chemical shifts for all, the assigned resonances of the duplexes with and without the unpaired cytosine suggests that the majority of the structural perturbation is localized in the A·T tract surrounding the unpaired base. The behavior of the imino resonances as a function of temperature also indicates that the perturbation to the duplex is localized and destabilizes the A·T base pairs adjacent to the unpaired base. © 1993 John Wiley & Sons, Inc.     

NMR analysis of DNA junctions: imino proton NMR studies of individual arms and intact junction

The NMR resonances from the hydrogen-bonded guanine and thymine imino protons of base pairs in the four separate complexes forming the arms of a stable DNA four-arm junction have been assigned by using sequential nuclear Overhauser effects connecting protons in adjacent pairs. Comparison of the spectra of these individual duplex arms with that of the intact four-stranded junction suggests that base pairing occurs at the site of branching. The presence of new resonances in the spectrum of the junction can be inferred from comparison of the junction spectrum with the simulated spectra of the four individual arms. In addition, upfield shifts of the ring protons in the base pairs at the penultimate positions in the complex are observed, consistent with a change in the structure at the site of branching. These studies represent the first stage of a detailed analysis of the structure and dynamics of a DNA junction.     

An NMR structural study of nickel-substituted rubredoxin

The Ni(II) and Zn(II) derivatives of Desulfovibrio vulgaris rubredoxin (DvRd) have been studied by NMR spectroscopy to probe the structure at the metal centre. The βCH₂ proton pairs from the cysteines that bind the Ni(II) atom have been identified using 1D nuclear Overhauser enhancement (NOE) difference spectra and sequence specifically assigned via NOE correlations to neighbouring protons and by comparison with the published X-ray crystal structure of a Ni(II) derivative of Clostridium pasteurianum rubredoxin. The solution structures of DvRd(Zn) and DvRd(Ni) have been determined and the paramagnetic form refined using pseudocontact shifts. The determination of the magnetic susceptibility anisotropy tensor allowed the contact and pseudocontact contributions to the observed chemical shifts to be obtained. Analysis of the pseudocontact and contact chemical shifts of the cysteine Hβ protons and backbone protons close to the metal centre allowed conclusions to be drawn as to the geometry and hydrogen-bonding pattern at the metal binding site. The importance of NH–S hydrogen bonds at the metal centre for the delocalization of electron spin density is confirmed for rubredoxins and can be extrapolated to metal centres in Cu proteins: amicyanin, plastocyanin, stellacyanin, azurin and pseudoazurin.     

Quantification of DNA structure from NMR data: conformation of d-ACATCGATGT

Resonance assignments of nonexchangeable base and sugar protons have been obtained in double-helical d-ACATCGATGT by using two-dimensional correlated spectroscopy (COSY) and nuclear Overhauser enhancement spectroscopy (NOESY). The exchangeable imino protons have been assigned on the basis of their chemical shifts. The characteristic phase-sensitive multiplet patterns of the intrasugar cross-peaks in the omega 1-scaled COSY spectrum have been used to estimate several scalar coupling constants (J). The information on the J values combined with the intranucleotide COSY cross-peak intensities has been used to identify sugar puckers of individual nucleotide units. In most cases, the deoxyribofuranose rings are found to adopt a conformation close to O4'-endo. Spin diffusion has been monitored from the buildup of the normalized volumes of NOE cross-peaks in NOESY spectra as a function of mixing time. A set of 52 intranucleotide and internucleotide proton-proton distances have been estimated by using low mixing time NOESY spectra (tau m = 40 and 80 ms). The estimated intrasugar proton-proton distances rule out possibilities of existence of a fast equilibrium between C2'-endo and C3'-endo conformations. Intranucleotide proton-proton distances combined with the knowledge of sugar puckers have been used to fix the glycosidic bond torsion angle (chi). For this purpose, simulated distance contours depicting the dependence of intranucleotide proton-proton distances on pseudorotational phase angle (P) and glycosidic bond torsion angle (chi) have been used. Further, the proton homonuclear (J, delta) spectrum has been used to monitor the 31P-1H heteronuclear couplings, which are preserved in the omega 2 projection.(ABSTRACT TRUNCATED AT 250 WORDS)