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).     

Two-dimensional NMR studies of Kazal proteinase inhibitors. 2. Sequence-specific assignments and secondary structure of reactive site modified turkey ovomucoid third domain

The solution structure of modified turkey ovomucoid third domain (OMTKY3*) was investigated by high-resolution proton NMR techniques. OMTKY3* was obtained by enzymatic hydrolysis of the scissile reactive site peptide bond (Leu18-Glu19) in turkey ovomucoid third domain (OMTKY3). All of the backbone proton resonances were assigned to sequence-specific residues except the NH's of Leu1 and Glu19, which were not observed. Over 80% of the side-chain protons also were assigned. The secondary structure of OMTKY3*, as determined from assigned NOESY cross-peaks and identification of slowly exchanging amide protons, contains antiparallel beta-sheet consisting of three strands (residues 21-25, 28-32, and 49-54), one alpha-helix (residues 33-44), and one reverse turn (residues 26-28). This secondary structure closely resembles that of OMTKY3 in solution [Robertson, A. D., Westler, W. M., & Markley, J. L. (1988) Biochemistry (preceding paper in this issue)]. On the other hand, changes in the tertiary structure of the protein near to and remote from the cleavage site are indicated by differences in the chemical shifts of numerous backbone protons of OMTKY3 and OMTKY3*.     

Proton resonance assignments of horse ferricytochrome c

Two-dimensional nuclear magnetic resonance spectroscopy (2D NMR) was used to obtain extensive resonance assignments in the 1H NMR spectrum of horse ferricytochrome c. Assignments were made for the main-chain and C beta protons of 102 residues (all except Pro-44 and Gly-84) and the majority of side-chain protons. As starting points for the assignment of the oxidized protein, a limited set of protons was initially assigned by use of 2D NMR magnetization transfer methods to correlate resonances in the oxidized form with assigned resonances in the reduced form [Wand, A. J., Di Stefano, D. L., Feng, Y., Roder, H., & Englander, S. W. (1989) Biochemistry (preceding paper in this issue)]. Given the complexity of the spectrum due to the size of this protein (104 residues) and its paramagnetic center, the initial search for side-chain spin systems in J-correlated spectra was successful only for the simplest side chains, but the majority of NH-C alpha H-C beta H subspin systems (NAB sets) could be identified at this stage. The subsequent search for sequential NOE connectivities focused on NAB sets, with use of previously assigned residues to place NOE-connected segments within the amino acid sequence. Selective proton labeling of either the slowly or the rapidly exchanging amide sites was used to simplify the spectra, and systematic work at two temperatures was used to resolve ambiguities in the 2D NMR spectra. These approaches, together with the use of magnetization transfer methods to correlate reduced and oxidized cytochrome c spectra, provide multiple cross-checks to verify assignments.     

A nuclear magnetic resonance study of axial ligation for the reduced states of chloroperoxidase,cytochrome P-450cam,and porphinatoiron(II) thiolate complexes

The reduced forms of cytochrome P-450_cam and chloroperoxidase were examined by proton NMR spectroscopy. The pH and temperature dependences of the proton NMR spectra of both ferrous enzymes are reported. A series of alkyl mercaptide complexes of both synthetic and natural-derivative iron(II) porphyrins was also examined. The proton NMR spectra of these complexes facilitated the assignment of resonances due to the axial ligand in the model compounds on the basis of their isotropic shifts and multiplicities. Comparison of model compound data with that for the reduced enzymes supports assignment of the methylene protons for the axial cysteinate of ferrous cytochrome P-450_cam and ferrous chloroperoxidase to proton NMR resonances at 279 and 200 ppm (pH 7.0, 298K), respectively. Differences in the active site structure of the two enzymes are further demonstrated by ¹⁵N-NMR spectroscopy of the cyanide complexes of the ferric forms.     

Two-dimensional NMR studies of Kazal proteinase inhibitors. 1. Sequence-specific assignments and secondary structure of turkey ovomucoid third domain

Two-dimensional proton NMR experiments have been used to sequentially assign resonances to all of the peptide backbone protons of turkey ovomucoid third domain (OMTKY3) except those of the N-terminal alpha-amino group whose signal was not resolved owing to exchange with the solvent. Assignments also have been made for more than 80% of the side-chain protons. Two-dimensional chemical shift correlated spectroscopy (COSY), relayed coherence transfer spectroscopy (RELAY), and two-dimensional homonuclear Hartmann-Hahn spectroscopy (HOHAHA) were used to identify the spin systems of almost half of the residues prior to sequential assignment. Assignments were based on two-dimensional nuclear Overhauser enhancements observed between adjacent residues. The secondary structure of OMTKY3 in solution was determined from additional assigned NOESY cross-peaks; it closely resembles the secondary structure determined by single-crystal X-ray diffraction of OMTKY3 in complex with Streptomyces griseus proteinase B [Fujinaga, M., Read, R.J., Sielecki, A., Ardelt, W., Laskowski, M., Jr., & James, M.N.G. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 4868-4872]. The NMR data provide evidence for three slowly exchanging amide protons that were not identified as hydrogen-bond donors in the crystal structure.     

Kinetics of hydrogen-deuterium exchange of tryptophan and tryptophan peptides in deutero-trifluoroacetic acid using proton magnetic resonance spectroscopy

Summary The methods which have been used for the observation and assignment of resonances in the NMR spectra of proteins are reviewed. One such method, the selective deuteration of the aromatic protons of tryptophyl residues, is studied by NMR spectroscopy in model compounds in this paper, and in proteins in the following paper.On the basis of a reassignment of the PMR spectrum of the aromatic protons of L-tryptophan, the relative rates of H-D exchange in deutero-trifluoroacetic acid (d-TFA) are H-2 > H-5 > H-6 > H-4 – H-7. The energies of activation for the first order exchange of both the H-2 and H-5 protons is 12 k.cal.mol^–1.The rate constant for exchange of the H-2 protons of tryptophyl residues in peptides is much greater than in the amino acid itself and 5–10 times that for exchange of the H-5 protons. This suggests that the method can be used to label tryptophyl residues in proteins rapidly and specifically.An invited article.     

1H nuclear magnetic resonance study of the histidine residues of insulin

Insulin has proved difficult to study by nuclear magnetic resonance spectroscopy because of its complex aggregation behaviour in solution and its insolubility between pH 4 and 7. Now for the first time it has been possible to assign the ¹H nuclear magnetic resonances of the H-2 histidine protons of residues B5 and B10 of bovine 2 Zn insulin and Zn-free insulin, and the B5 and A8 residues of hagfish insulin. As expected, the addition of Zn to Zn-free insulin causes virtually no change in the chemical shift or the rate of H-D exchange of the H-2 proton of histidine B5, which is not involved in Zn binding in the 2 Zn insulin hexamer. The rate of H-D exchange of the H-2 proton of histidine B10 is decreased markedly on Zn binding at this residue, but the chemical shift of the resonance remains virtually constant owing to the balancing of an upfield ring current shift of the ordered histidine residues by a downfield shift due to electron withdrawal from the ring nitrogen by the Zn binding.     

Synthèse et étude R.M.N. de disaccharides et trisaccharides dans la série du l-rhamnose

Various di- and tri-saccharides containing l-rhamnose were synthesized by condensation of 2,3,4-tri-O-acetyl- or 2,3,4-tri-O-benzoyl-α-l-rhamnopyranosyl bromide with an unblocked glycopyranoside. The determination of the anomeric configuration of l-rhamnose saccharides by n.m.r. is difficult because structure has a greater effect on the spectra than does configuration. The α and β configurations and the position of the substitution may be assigned from the chemical shifts of H-5 and CH₃. In all the compounds having a β configuration, a shielding of the methyl group and a deshielding of the H-5 proton have been observed as compared to the compounds having an α configuration. The H-5 proton and the methyl group of peracetylated, (1→3)-linked α-l derivatives always resonate at higher fields than the corresponding protons of (1→6)-linked α-l derivatives.     

On the interaction of caffeine with nucleic acids: III. 1NMR studies of caffeine-5'-adenosine monophosphate and caffeine-poly(riboadenylate) interactions

1) The self-association of both caffeine (Cf) and 5'-adenosine monophosphate (AMP) in aqueous solution has been reinvestigated by ¹H NMR. The self-association process is characterized by an isodesmic model. The apparent self-association constants of the vertical stacking process are K_Cf= (10.6 ± 1.0) M^?1 and k_amp = (1.67 ± 0.17) M^?1. The arrangement of the monomeric units in the stacked aggregates is discussed in terms of isoshielding curves theoretically calculated by Giessner-Prettre and Pullman. Models are proposed which are consistent with these and further previous NMR data. 2) The interaction of Cf and AMP has been studied by ^?1 H NMR. The apparent association constant of the complex Cf-AMP is K_C-A = (7.3 ± 1.2) M^?1.Two models of the mutual arrangement of AMP and Cf in the complex are proposed on the basis of the calculated isoshielding curves considering both ring current and local atomic diamagnetic anisotropy effects. 3) The interaction of Cf and poly(riboadenylate), (rA)_n is indicated by a downfield shift of the H-8 line but an upfield shift of the H-2 line in the ¹H NMR spectra of (rA)_n. The concentration dependence of the ¹H NMR shifts of both Cf and (rA)_n can be explained by the existence of two binding mechanisms. We suggest (i) partial insertion of Cf between adjacent base residues of ordered single-stranded regions of (rA)_n and (ii) outside binding of Cf in form of monomeric Cf as well as of self-associated aggregates. The complex geometry of insertion proposed on the basis of the calculated isoshielding curves is characterized by a stronger overlapping of the Cf ring and the H-2 proton of (rA)_n as compared to the H-8 proton.     

Connectivity of proton and carbon spectra of the blue copper protein,plastocyanin, established by two-dimensional nuclear magnetic resonance

NMR studies of plastocyanin have centered on the ligands to the copper atom at the active site, particularly histidines-37 and -87. Heteronuclear (¹³C, ¹H) J-connectivity spectroscopy has enabled cross assignment of ¹H and ¹³C NMR resonances from the two copper-ligated histidines. In addition to providing assignments of the ¹³C resonances, the two-dimensional Fourier transform NMR results require the reversal of the original ¹H NMR assignments to the ring protons of histidine-37. The line widths of the ring protons of histidine-87 are field-dependent leading to determination of the reduced lifetime of the proton on the N_δ atom (about 400 μs).     

The solution structure of desulforedoxin,a simple iron-sulfur protein

Desulforedoxin is a simple dimeric protein isolated from Desulfovibrio gigas containing a distorted rubredoxin-like center with one iron coordinated by four cysteinyl residues (7.9?kDa with a 36-amino-acid monomer). ¹H NMR spectra of the oxidized Dx(Fe³⁺) and reduced Dx(Fe²⁺) forms were analyzed. The spectra show substantial line broadening due to the paramagnetism of iron. However, very low-field-shifted resonances, assigned to Hβ protons, were observed in the reduced state and their temperature dependence analyzed. The active site of Dx was reconstituted with zinc, and its solution structure was determined using 2D NMR methods. This diamagnetic form gave high-resolution NMR data enabling the identification of all the amino acid spin systems. Sequential assignment and the determination of secondary structural elements was attempted using 2D NOESY experiments. However, because of the symmetrical dimer nature of the protein standard, NMR sequential assignment methods could not resolve all cross peaks due to inter- and intra-chain effects. The X-ray structure enabled the spatial relationship between the monomers to be obtained, and resolved the assignment problems. Secondary structural features could be identified from the NMR data; an antiparallel β-sheet running from D5 to V18 with a well-defined β-turn around cysteines C9 and C12. The section G22 to T25 is poorly defined by the NMR data and is followed by a turn around V27-C29. The C-terminus ends up near residues V6 and Y7. Distance geometry (DG) calculations allowed families of structures to be generated from the NMR data. A family of structures with a low target function violation for the Dx monomer and dimer were found to have secondary structural elements identical to those seen in the X-ray structure. The amide protons for G4, D5, G13, L11 NH and Q14 NHε amide protons, H-bonded in the X-ray structure, were not seen by NMR as slowly exchanging, while structural disorder at the N-terminus, for the backbone at E10 and for the section G22–T25, was observed. Comparison between the Fe and Zn forms of Dx suggests that metal substitution does not have an effect on the structure of the protein.     

1H-NMR study on the tautomerism of the imidazole ring of histidine residues: II. Microenvironments of histidine-12 and histidine-119 of bovine pancreatic ribonuclease a

The NMR titration curves of proton chemical shifts were observed for the C₂ protons of histidine residues in intact bovine pancreatic RNAase A (EC 3.1.27.5) and carboxyalkylated RNAase A. By comparing the methyl region of NMR spectra, the 250–340 nm region of circular dichfoic spectra, and the NMR titration curves of tyrosine ring protons among intact and modified RNAase A, it was ascertained that the carboxyalkylation of histidine residues at position 12 or 119 did not make any appreciable conformational changes to RNAase A. With the pK values determined for intact and modified RNAase A, the microscopic pK values and molar ratios of tautomers were estimated for His-12 and His-119 by means of the procedure described in the preceding paper. The estimated microscopic pK values of tautomers were 6.2 for the N₁-H tautomer of His-12, more than 8 for the N₃-H tautomer of His-12, 7.0 for the N₁-H tautomer of His-119, and 6.4 for the N₃-Hf tautomer of His-119, respectively. These values were interpreted in terms of the microscopic environments surrounding the histidine residues. The microscopic structure estimated in the present study was discussed, comparing it with those from X-ray crystallography and hydrogen-tritium (or hydrogen-deuterium) exchange technique.     

1H NMR (500 MHz) identification of aromatic residues of gene 32 protein involved in DNA binding by use of protein containing perdeuterated aromatic residues and by site-directed mutagenesis

Preparation of gene 32 protein containing perdeuterated tyrosyl and phenylalanyl residues has allowed the resolution of separate 1H NMR signals for the Tyr and Phe residues of the protein by NMR difference spectra. Upfield shifts in the chemical shifts of a number of aromatic protons previously observed to accompany deoxyoligonucleotide complex formation with gene 32 protein [Prigodich, R. V., Casas-Finet, J., Williams, K. R., Konigsberg, W., & Coleman, J. E. (1984) Biochemistry 23, 522-529] can be assigned to five Tyr and two Phe residues that must form part of the DNA binding domain. Site-directed mutation of Tyr-115 to Ser-115 results in the disappearance of a set of 2,6 and 3,5 tyrosyl protons that are among those moved upfield by oligonucleotide complex formation. These findings suggest that the amino acid sequence from Tyr-73 to Tyr-115 which contains six of the eight Tyr residues of the protein forms part of the DNA binding surface.     

NMR spectroscopy of hydroxyl protons in aqueous solutions of peptides and proteins

Summary Hydroxyl groups of serine and threonine, and to some extent also tyrosine are usually located on or near the surface of proteins. NMR observations of the hydroxyl protons is therefore of interest to support investigations of the protein surface in solution, and knowledge of the hydroxyl NMR lines is indispensable as a reference for studies of protein hydration in solution. In this paper, solvent suppression schemes recently developed for observation of hydration water resonances were used to observe hydroxyl protons of serine, threonine and tyrosine in aqueous solutions of small model peptides and the protein basic pancreatic trypsin inhibitor (BPTI). The chemical shifts of the hydroxyl protons of serine and threonine were found to be between 5.4 and 6.2 ppm, with random-coil shifts at 4°C of 5.92 ppm and 5.88 ppm, respectively, and those of tyrosine between 9.6 and 10.1 ppm, with a random-coil shift of 9.78 ppm. Since these spectral regions are virtually free of other polypeptide¹H NMR signals, cross peaks with the hydroxyl protons are usually well separated even in homonuclear two-dimensional¹H NMR spectra. To illustrate the practical use of hydroxyl proton NMR in polypeptides, the conformations of the side-chain hydroxyl groups in BPTI were characterized by measurements of nuclear Overhauser effects and scalar coupling constants involving the hydroxyl protons. In addition, hydroxyl proton exchange rates were measured as a function of pH, where simple first-order rate processes were observed for both acid- and base-catalysed exchange of all but one of the hydroxyl-bearing residues in BPTI. For the conformations of the individual Ser, Thr and Tyr side chains characterized in the solution structure with the use of hydroxyl proton NMR, both exact coincidence and significant differences relative to the corresponding BPTI crystal structure data were observed.[/p]     

Two-dimensional magnetization exchange spectroscopy of Anabaena 7120 ferredoxin. Nuclear Overhauser effect and electron self-exchange cross peaks from amino acid residues surrounding the 2Fe-2S* cluster

Hyperfine 1H NMR signals of the 2Fe-2S* vegetative ferredoxin from Anabaena 7120 have been studied by two-dimensional (2D) magnetization exchange spectroscopy. The rapid longitudinal relaxation rates of these signals required the use of very short nuclear Overhauser effect (NOE) mixing times (0.5-20 ms). The resulting pattern of NOE cross-relaxation peaks when combined with previous 1D NOE results [Dugad, L. B., La Mar, G. N., Banci, L., & Bertini, I. (1990) Biochemistry 29, 2263-2271] led to elucidation of the carbon-bound proton spin systems from each of the four cysteines ligated to the 2Fe-2S* cluster in the reduced ferredoxin. Additional NOE cross peaks were observed that provide information about other amino acid residues that interact with the iron-sulfur cluster. NOE cross peaks were assigned tentatively to Leu27, Arg42, and Ala43 on the basis of the X-ray coordinates of oxidized Anabaena 7120 ferredoxin [Rypniewski, W.R., Breiter, D.R., Benning, M.M., Wesenberg, G., Oh, B.-H., Markley, J.L., Rayment, I., & Holden, H. M. (1991) Biochemistry 30, 4126-4131]. Three chemical exchange cross peaks were detected in magnetization exchange spectra of half-reduced ferredoxin and assigned to the 1H alpha protons of Cys49 and Cys79 [both of whose sulfur atoms are ligated to Fe(III)] and Arg42 (whose amide nitrogen is hydrogen-bonded to one of the inorganic sulfurs of the 2Fe-2S* cluster). The chemical exchange cross peaks provide a means of extending assignments in the spectrum of reduced ferredoxin to assignments in the spectrum of the oxidized protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

SOACS index: an easy NMR-based query for glycan retrieval

¹H NMR is now a standard method to determine de novo primary sequence of all sorts of glycans. These last 30 years, tens of thousands of oligosaccharide sequences have been elucidated by NMR spectroscopy in conjunction with other physico-chemical methods including mass spectrometry and gas chromatography. Most of these sequences are now compiled and available in several web databases recently unified in publicly available GlycomeDB, along with sets of experimental data. However, because the search for an exact sequence exclusively based on proton chemical shifts is sometimes delicate for NMR non-specialists, we worked out a new type of query, named SOACS, which allows the easy retrieval of existing sequences. This query is based on the readily distinguished ¹H chemical shifts from any ¹H NMR spectrum, and was designed to be usable to the widest scientist community.     

1H and 15N NMR characterization of free and bound states of an amphiphilic peptide interacting with calmodulin.

A peptide of 17 amino acid residues Ac-L-K-W-K-K-L-L-K-L-L-K-K-L-L-K-L-G-NH2, designed to form an amphiphilic basic alpha-helix [DeGrado, W.F., Prendergast, F. G., Wolfe, H. R., Jr., & Cox, J. A. (1985) J. Cell. Biochem. 29, 83-93], was labeled with 15N at positions 1, 7, 9, and 10. Homo- and heteronuclear NMR techniques were used to characterize the conformational changes of the peptide when it binds to calmodulin in the presence of Ca2+ ions. The spectrum of the free peptide in aqueous solution at pH 6.3 and 298 K was completely assigned by a combined application of several two-dimensional proton NMR methods. Analysis of the short- and medium-range NOE connectivities and of the secondary chemical shifts indicated that the peptide populates, to a significant extent, an alpha-helix conformational state, in agreement with circular dichroism measurements under similar physicochemical conditions. 15N-edited 1D spectra and 15N(omega 2)-half-filtered two-dimensional NMR experiments on the peptide in a 1:1 complex with calmodulin allowed assignment of half of the amide proton resonances and three C alpha H resonances of the bound peptide. The observed NOE connectivities between the peptide backbone protons are indicative of a stable helical secondary structure spanning at least the fragment L1-K11. The equilibrium and dynamic NMR parameters of the bound peptide are discussed in terms of a molecular interaction model.     

External protons destabilize the activated voltage sensor in hERG channels

Extracellular acidosis shifts hERG channel activation to more depolarized potentials and accelerates channel deactivation; however, the mechanisms underlying these effects are unclear. External divalent cations, e.g., Ca²⁺ and Cd²⁺, mimic these effects and coordinate within a metal ion binding pocket composed of three acidic residues in hERG: D456 and D460 in S2 and D509 in S3. A common mechanism may underlie divalent cation and proton effects on hERG gating. Using two-electrode voltage clamp, we show proton sensitivity of hERG channel activation (pKa = 5.6), but not deactivation, was greatly reduced in the presence of Cd²⁺ (0.1 mM), suggesting a common binding site for the Cd²⁺ and proton effect on activation and separable effects of protons on activation and deactivation. Mutational analysis confirmed that D509 plays a critical role in the pH dependence of activation, as shown previously, and that cooperative actions involving D456 and D460 are also required. Importantly, neutralization of all three acidic residues abolished the proton-induced shift of activation, suggesting that the metal ion binding pocket alone accounts for the effects of protons on hERG channel activation. Voltage-clamp fluorimetry measurements demonstrated that protons shifted the voltage dependence of S4 movement to more depolarized potentials. The data indicate a site and mechanism of action for protons on hERG activation gating; protonation of D456, D460 and D509 disrupts interactions between these residues and S4 gating charges to destabilize the activated configuration of S4.     

Differences between the electronic environments of reduced and oxidized Escherichia coli DsbA inferred from heteronuclear magnetic resonance spectroscopy.

DsbA is the strongest protein disulfide oxidant yet known and is involved in catalyzing protein folding in the bacterial periplasm. Its strong oxidizing power has been attributed to the lowered pKa of its reactive active site cysteine and to the difference in thermodynamic stability between the oxidized and the reduced form. However, no structural data are available for the reduced state. Therefore, an NMR study of DsbA in its two redox states was undertaken. We report here the backbone 1HN, 15N, 13C(alpha) 13CO, 1H(alpha), and 13Cbeta NMR assignments for both oxidized and reduced Escherichia coli DsbA (189 residues). Ninety-nine percent of the frequencies were assigned using a combination of triple (1H-13C-15N) and double resonance (1H-15N or 1H-13C) experiments. Secondary structures were established using the CSI (Chemical Shift Index) method, NOE connectivity patterns, 3(J)H(N)H(alpha) and amide proton exchange data. Comparison of chemical shifts for both forms reveals four regions of the protein, which undergo some changes in the electronic environment. These regions are around the active site (residues 26 to 43), around His60 and Pro 151, and also around Gln97. Both the number and the amplitude of observed chemical shift variations are more substantial in DsbA than in E. coli thioredoxin. Large 13C(alpha) chemical shift variations for residues of the active site and residues Phe28, Tyr34, Phe36, Ile42, Ser43, and Lys98 suggest that the backbone conformation of these residues is affected upon reduction.     

Through-bond correlation of imino and aromatic resonances in 13C-,15N-labeled RNA via heteronuclear TOCSY

Summary Novel HCCNH TOCSY NMR experiments are presented that provide unambiguous assignment of the exchangeable imino proton resonances by intranucleotide through-bond connectivities to the (assigned) nonexchangeable purine H8 and pyrimidine H6 protons in uniformly ¹⁵N-, ¹³C-labeled RNA oligonucleotides. The HCCNH TOCSY experiments can be arranged as a two-dimensional experiment, correlating solely GH8/UH6 and GH1/UH3 proton resonances (HCCNH), of as three-dimensional experiments, in which additional chemical shift labeling either by GN1/UN3 (HCCNH) or by GC8/UC6 (HCCNH) chemical shifts is introduced. The utility of these experiments for the assignment of relatively large RNA oligonucleotides is demonstrated for two different RNA molecules.To whom correspondence should be addressed.