Homonuclear three-dimensional NMR methods for the complete assignment of proton NMR spectra of oligosaccharides--application to Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc.

Two new homonuclear three-dimensional NMR techniques are described for the simplification of proton resonance assignment in oligosaccharides, namely HOHAHA-COSY and ROESY-COSY. The former technique is of value in the resonance assignment of gluco-configuration monosaccharide residues, whereas the latter is more suited to resonance assignment of galacto-configuration monosaccharide residues. The value of these techniques is illustrated by application to the proton resonance assignment of the pentasaccharide Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3 Gal beta 1-4Glc, a compound which exhibits a variety of assignment problems due to severe cross-peak overlap in conventional COSY or HOHAHA spectra.     

2D NMR investigation of the binding of the anticancer drug actinomycin D to duplexed dATGCGCAT: conformational features of the unique 2:1 adduct

One- and two-dimensional NMR studies on the oligomer dA1T2G3C4G5C6A7T8, with and without actinomycin D (ActD), were conducted. Analysis of the NMR data, particularly 2D NOE intensities, revealed that the free oligonucleotide is a duplex in a standard right-handed B form. At the ratio of 1 ActD/duplex (R = 1), 1D NMR studies indicate that two 1:1 unsymmetric complexes form in unequal proportions with the phenoxazone ring intercalated at a GpC site, in agreement with previous studies [Scott, E.V., Jones, R.L., Banville, D.L., Zon, G., Marzilli, L.G., & Wilson, W.D. (1988) Biochemistry 27, 915-923]. The 2D COSY data also confirm this interpretation since eight cytosine H6 to H5 and two ActD H8 to H7 cross-peaks are observed. At R = 2, both COSY and NOESY spectra confirm the formation of a unique 2:1 species with C2 symmetry. The oligomer remains in a right-handed duplex but undergoes extreme conformational changes both at and adjacent to the binding site. The deoxyribose conformation of T2, C4, and C6 shifts from primarily C2'-endo in the free duplex to an increased amount of C3'-endo in the 2:1 complex as revealed by the greater intensity of the base H6 to 3' NOE cross-peak relative to the intensity of the H6 to H2' NOE cross-peak. This conformational change widens the minor groove and should help alleviate the steric crowding of the ActD peptides. The orientation of the ActD molecules at R = 2 has the quinoid portion of the phenoxazone ring at the G3pC4 site and the benzenoid portion of the phenoxazone ring at the G5pC6 site on the basis of NOE cross-peaks from ActD H7 and H8 to G5H8 and C6H6. All base pairs retain Watson-Crick type H-bonding, unlike echinomycin complexes [e.g., Gao, X., & Patel, D.J. (1988) Biochemistry 27, 1744-1751] where Hoogsteen base pairs have been observed. In contrast to previous studies on ActD, we were able to distinguish the two peptide chains.(ABSTRACT TRUNCATED AT 400 WORDS)     

A complete proton and carbon-13 NMR analysis of fluocinonide, a fluorinated corticosteroid

This paper presents a complete analysis of the proton and carbon-13 NMR spectra of 21-acetoxy-6 alpha,9-difluoro-11 beta-hydroxy-16 alpha,17-(1-methylethylidene) bis-(oxy) pregna-1,4-diene-3,20-dione, a potent anti-inflammatory fluorosteroid. The 300 MHz proton spectrum was analyzed using a combination of the two-dimensional homonuclear chemical shift correlation (COSY) technique and one-dimensional NOE difference spectra. Exact coupling constants and chemical shifts were obtained by spectral simulation and iteration. The carbon-13 spectrum was assigned from the proton spectrum via a two-dimensional heteronuclear chemical shift experiment, and long-range fluorine-proton couplings were confirmed by a fully coupled heteronuclear COSY-type experiment.     

Principles of quantitative analysis of two-dimensional spectra of nuclear Overhauser effect in evaluation of protein and peptide conformation

To elucidate potentialities of two-dimensional homonuclear Overhauser effect (NOESY) spectra of peptides and proteins for their spatial structure determination, impact of experimental parameters and intrinsic properties of the investigated molecule on proton cross-peak volumes in NOESY spectra was analysed. Recommendations which could increase accuracy of cross-peak volume measurements were suggested. Influence of intrinsic properties of a molecule (spin-lattice relaxation times T1, correlation time tau C and surrounding protons) on the volume of cross-peak for particular protons was analyzed using a complete relaxation matrix of the (formula; see text) helix of gramicidin A. Nonselective relaxation time T1 of the protons was found to affect only slightly the results of cross-peak volumes computer simulation, whereas correlation time tau C and surrounding protons seriously influenced cross-peak volumes. Nevertheless, cross-peak volumes between NH, C alpha H and C beta H protons of a dipeptide fragment of the entire molecule could be accurately simulated using the relaxation matrix of the individual dipeptide. Thus local conformations (torsion angles phi, psi and chi 1) of amino acid residues could be deduced independently of one another and prior to the complete analysis of a molecular structure. The result can be obtained even in the presence of spin-diffusion at mixing times providing maximal volumes of cross-peaks in NOESY spectra.     

Complete assignment of the 500 MHz 1H-NMR spectra of bleomycin A2 in H2O and D2O solution by means of two-dimensional NMR spectroscopy

1H-NMR spectra of bleomycin A2 recorded at 500 MHz in D2O and H2O at 24 degrees C and 3 degrees C were investigated. Resonances of the individual spin systems were identified by using two-dimensional correlation spectroscopy (COSY), two-dimensional spin echo correlated spectroscopy (SECSY) and by the application of two-dimensional Nuclear Overhauser Effect spectroscopy (NOESY). Employment of these techniques allowed the assignment of 113 exchangeable and 59 non-exchangeable protons in the 1H NMR spectrum of bleomycin A2. By means of 2D NOE spectroscopy also interresidual connectivities could be observed. Comparison of the NOESY spectra at 3 degrees C and 24 degrees C suggest that at low temperatures the central party of the bleomycin A2 molecule tends to adopt an extended conformation.     

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)     

Two-dimensional 1H-NMR study of the 1-34 fragment of human parathyroid hormone

The complete assignment of resonances in the proton nmr spectrum of the 1-34 amino acid fragment of human parathyroid hormone [hPTH(1-34)], determined using a combination of one- and two-dimensional nmr techniques at 500 MHz, is described. In particular, homonuclear Hartmann-Hahn experiments, recorded in H2O and D2O, are used to resolve ambiguities in the connectivities between the highly overlapped resonances in the aliphatic region of the spectrum. One-dimensional multiple quantum filtering experiments are used to identify serine and phenylalanine spin systems. Analyses of the through-bond and through-space connectivities in the alpha H-NH fingerprint regions of the correlated spectroscopy (COSY) and nuclear Overhauser effect spectroscopy (NOESY) spectra lead to the assignment of resonances to specific amino acid residues in the polypeptide. Examination of the observed NOE cross peaks indicates that hPTH(1-34) has no detectable secondary structural elements in aqueous solution.     

Determination of local conformation of proteins from 1H-NMR spectroscopy data

A method is proposed to determine conformations of amino acid residues of the protein and effective correlation time tau c from cross-peak intensities in two-dimensional nuclear Overhauser enhancement (NOESY) spectra. The method consists in fitting complete relaxation matrix of dipeptide unit protons to experimental cross-peak intensities by varying phi, psi, chi torsional angles and tau c. To verify the method, NOESY spectra of basic pancreatic trypsin inhibitor (BPTI) were theoretically generated at mixing times tau m = 25-300 ms and tau c = 4 ns and used for local structure determination. The method works well with optimum for measurement of NOE intensities tau m 100-200 ms. As a result, the backbone phi, psi torsion angles were unambiguously determined at tau m = 100 ms for all but Gly residues of BPTI, and chi 1 angles were determined for the majority of side chains. The obtained dipeptide unit conformations are very close to the BPTI crystallographic structure: root mean square deviation (RMSD) of interproton distances within dipeptide units, on the average, is 0.08 A (maximal deviation 0.44 A), and RMSD of phi and psi angles are 18 and 9 degrees, respectively (maximal deviations are 44 and 22 degrees).     

Overcoming the overlap problem in the assignment of 1H NMR spectra of larger proteins by use of three-dimensional heteronuclear 1H-15N Hartmann-Hahn-multiple quantum coherence and nuclear Overhauser-multiple quantum coherence spectroscopy: application to interleukin 1 beta

The application of three-dimensional (3D) heteronuclear NMR spectroscopy to the sequential assignment of the 1H NMR spectra of larger proteins is presented, using uniformly labeled (approximately 95%) [15N]interleukin 1 beta, a protein of 153 residues and molecular mass of 17.4 kDa, as an example. The two-dimensional (2D) 600-MHz spectra of interleukin 1 beta are too complex for complete analysis, owing to extensive cross-peak overlap and chemical shift degeneracy. We show that the combined use of 3D 1H-15N Hartmann-Hahn-multiple quantum coherence (HOHAHA-HMQC) and nuclear Overhauser-multiple quantum coherence (NOESY-HMQC) spectroscopy, designed to provide the necessary through-bond and through-space correlations for sequential assignment, provides a practical general-purpose method for resolving ambiguities which severely limit the analysis of conventional 2D NMR spectra. The absence of overlapping cross-peaks in these 3D spectra allows the unambiguous identification of C alpha H(i)-NH(i+1) and NH(i)-NH(i+1) through-space nuclear Overhauser connectivities necessary for connecting a particular C alpha H(i)-NH(i) through-bond correlation with its associated through-space sequential cross-peak The problem of amide NH chemical shift degeneracy in the 1H NMR spectrum is therefore effectively removed, and the assignment procedure simply involves inspecting a series of 2D 1H-1H slices edited by the chemical shift of the directly bonded 15N atom. Connections between residues can be identified almost without any knowledge of the spin system types involved, though this type of information is clearly required for the eventual placement of the connected residues within the primary sequence.     

Sequential 1H NMR assignments and secondary structure of the kringle domain from urokinase.

The sequence-specific 1H NMR assignments of the 89-residue recombinant kringle domain from human urokinase are presented. These were achieved primarily by utilizing TOCSY and NOESY spectra in conjunction with COSY spectra recorded at 500 MHz and 600 MHz. Regular secondary structure elements have been derived from a qualitative interpretation of nuclear Overhauser enhancement, JNH alpha coupling constant, and amide proton exchange data. Two helices have been identified. One helix, involving Ser40-Gly46, corresponds to that reported for t-PA kringle 2 (Byeon et al., 1991), but does not exist in other kringles with known structures. The second helix, in the region Asn26-Gln33, is thus far unique to the urokinase kringle. Three antiparallel beta-sheets and three tight turns have also been identified, which correspond exactly to those identified in t-PA kringle 2 both in solution and in the crystalline state (de Vos et al., 1992). Despite the very different ligand binding properties of the urokinase kringle, NOE data indicate that the tertiary fold of the molecule conforms closely to that found for other kringles.     

113CD-1H spin-spin couplings in homonuclear 1H correlated spectroscopy of metallothionein. Identification of the cysteine 1H spin systems

Heteronuclear spin-spin couplings between 113Cd and C beta protons of the metal-bound cysteines were observed in phase-sensitive, double-quantum filtered, homonuclear two-dimensional correlated (COSY) 1H NMR spectra of 113Cd-metallothionein-2 from rabbit liver. Comparison of 113Cd- and 112Cd-metallothionein-2 spectra revealed that 19 1H spin systems show heteronuclear couplings to at least one 113Cd and were thus identified as 19 of the 20 cysteines in this protein. From a detailed analysis of the manifestations of heteronuclear couplings in the homonuclear 1H COSY spectra, two cysteines could be identified as 'bridging cysteines', with spin-spin couplings to two different 113Cd nuclei. The observed 113Cd-1H coupling constants vary between less than or equal to 5 Hz and 80 Hz.     

500-MHz 1H NMR studies of ragweed allergen Ra5

The solution conformation of short ragweed allergen Ra5, a protein of 45 amino acid residues cross-linked with four disulfide bridges, has been investigated by 1H NMR spectroscopy at 500 MHz. The aromatic region, which contains resonances from three tyrosines and two tryptophans, has been partially assigned. Two tyrosines titrate with a pK of 10.2; a third tyrosine is buried under the tryptophan resonances, and its pK could not be determined. The two tryptophans reside in different microenvironments; the resonances of one are very similar to those found in random coil structures while the other has dramatically shifted peaks. Nuclear Overhauser effect (NOE) difference spectroscopy is used to define two distinct spin-diffusion systems for the aromatic residues and to further identify several methyl-containing amino acids involved in these systems. Assignments in the methyl region are based on selective decoupling, chemical shifts, NOE difference spectra, and 2-D J-resolved and 2-D J-correlated spectroscopy (COSY) methodology. A unique ring-current-shifted methyl doublet in the Ra5 spectrum titrates into the bulk methyl region with a pK of 10.2. Examination of the COSY map suggests that this resonance belongs to either leucine-1 or isoleucine-38. Chemical removal of the N-terminal leucine did not affect the ring-current-shifted methyl. Therefore, this unique resonance has been assigned to the methyl of isoleucine-38.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complete 1H-NMR spectral assignments for globotriaosyl-Z- and isoglobotriaosyl-E-ceramide

Two-dimensional scalar-correlated (COSY) 1H-NMR spectra of the title compounds, and phase-sensitive COSY spectrum of lactosylceramide, have been fully assigned and some spectral reassignments for related structures suggested. Glycosylation-induced shifts, and shielding by Z- and E-ceramide residues are discussed.     

Analysis of the aliphatic 1H-NMR spectrum of plasminogen kringle 4. A comparative study of human, porcine, bovine and chicken homologs

The aliphatic 1H-NMR spectrum of the kringle 4 domain of human plasminogen has been studied via two-dimensional chemical shift correlated (COSY) and nuclear Overhauser correlated (NOESY) experiments at 300 MHz and 620 MHz. A number of aliphatic proton spin systems have been identified and several definite assignments have been made. This was mainly achieved by comparison of the human kringle 4 spectrum with spectra of the porcine, bovine and chicken homologs and also with that of the kringle 1 from human plasminogen on which we have reported previously. The three valyl and two leucyl residues of human kringle 4 have been assigned. The eleven threonyl spin systems have been identified via a RELAYED-COSY experiment and Thr17 has been assigned. The three alanyl spin systems have been identified and assigned. Six seryl spin systems have been identified and the signals from the seven glycyl residues of human kringle 4 have been located with Gly45 assigned. Furthermore, 24 AMX spin systems have been mapped in the COSY spectrum of human kringle 4 and H alpha-H beta,beta' spin systems of Tyr2, Tyr41, Tyr50, Tyr74, Trp25 and Trp62 have been assigned. From the spectrum of a deglycosylated chicken homolog, the epsilon-methyl singlets of Met28 and Met48 have been assigned. Finally, ligand effects on selected aliphatic resonances were observed which could be analyzed in terms of residues likely to neighbor the kringle lysine-binding site.     

A New Diterpene Glycoside in Aster ageratoides Turcz.

A new natural diterpene glycoside was isolated from Aster ageratoides Turcz. Its structure was elucidated by means of IR, MS (FAB, EI), NMR (1H-, 13C-, DEPT), 2D-NMR (1H-1H COSY, 13C-1 H COSY, J-resolved, CoLoC) spectra and chemical method as 16β, 17-dihydroxy- (—) -kauran- 19-oic acid-β-D-glucopyanosyl ester.     

Determination of the functionally important heme peripheral vinyl group orientation in paramagnetic hemoprotein by 2D NMR

2D NMR spectroscopies have been successfully used to characterize the heme peripheral vinyl groups in paramagnetic hemoprotein in spite of the difficulties from the rapid paramagnetic relaxation and the low digital resolution of the 2D NMR map. The scalar coupling network system among the vinyl protons is clearly identified in the COSY spectra from its characteristic cross-peak pattern and the dipolar coupling connectivities of the vinyl proton resonances with other heme side-chain proton resonances not only provide the specific assignment of vinyl beta-proton resonances but also allow the determination of the vinyl group orientation with respect to the heme plane.     

Innovations in generation and analysis of 2D [(13)C,(1)H] COSY NMR spectra for metabolic flux analysis purposes.

2D [(13)C,(1)H] COSY NMR is used by the metabolic engineering community for determining (13)C-(13)C connectivities in intracellular compounds that contain information regarding the steady-state fluxes in cellular metabolism. This paper proposes innovations in the generation and analysis of these specific NMR spectra. These include a computer tool that allows accurate determination of the relative peak areas and their complete covariance matrices even in very complex spectra. Additionally, a method is introduced for correcting the results for isotopic non-steady-state conditions. The proposed methods were applied to measured 2D [(13)C,(1)H] COSY NMR spectra. Peak intensities in a one-dimensional section of the spectrum are frequently not representative for relative peak volumes in the two-dimensional spectrum. It is shown that for some spectra a significant amount of additional information can be gained from long-range (13)C-(13)C scalar couplings in 2D [(13)C,(1)H] COSY NMR spectra. Finally, the NMR resolution enhancement by dissolving amino acid derivatives in a nonpolar solvent is demonstrated.     

1H NMR study of the solution structure of the self-complementary dodecanucleotide d(TGCA)3.

The deoxyoligonucleotide d(TGCA)3 is a candidate for exhibiting unusual conformations. Its 1H NMR spectrum under low salt conditions has been obtained at 400 MHz and assigned using two-dimensional NMR techniques. The sugar puckers and glycosidic torsions have been determined by inspecting the relative intensities of the intranucleotide NOEs and COSY crosspeaks. At low electrolyte concentration (100 mM NaCl) the molecule exists as a right-handed duplex with twelve Watson-Crick base-pairs and deoxyribose moieties assuming the O1'-endo to C1'-exo pucker.     

Complete Assignment of the 500 MHz 1H-NMR Spectra of Bleomycin A2 in H2O and D2O Solution by means of Two-dimensional NMR Spectroscopy

Abstract

¹H-NMR spectra of bleomycin A2 recorded at 500 MHz in D₂O and H₂O at 24°C and 3°C were investigated. Resonances of the individual spin systems were identified by using two-dimensional correlated spectroscopy (COSY), two-dimensional spin echo correlated spectroscopy (SECSY) and by the application of two-dimensional Nuclear Overhauser Effect spectroscopy (NOESY). Employment of these techniques allowed the assignment of 13 exchangeable and 59 non-exchangeable protons in the ¹H NMR spectrum of bleomycin A2. By means of 2D NOE spectroscopy also interresidual connectivities could be observed. Comparison of the NOESY spectra at 3°C and 24°C suggest that at low temperatures the central part of the bleomycin A2 molecule tends to adopt an extended conformation.     

Improved spectral resolution in cosy 1H NMR spectra of proteins via double quantum filtering

A double quantum filter is inserted into a two-dimensional correlated (COSY) 1H NMR experiment to obtain phase-sensitive spectra in which both cross peak and diagonal peak multiplets have anti-phase fine structure, and in which the cross peaks and the major contribution to the diagonal peaks have absorption lineshapes in both dimensions. The elimination of the dispersive character of the diagonal peaks in phase-sensitive, double quantum-filtered COSY spectra allows identification of cross peaks lying immediately adjacent to the diagonal, which represents a significant improvement over the conventional COSY experiment.