Homonuclear 2D Research Articles

The detailed structure of tandem G·A mismatched base-pair motifs in RNA duplexes is context dependent

The solution structure of the RNA duplex (rGGGCU GA AGCCCU), containing tandem G·A mismatches has been determined by NMR spectroscopy and restrained molecular dynamics. A homonuclear 3D TOCSY-NOESY was used to derive 18 to 30 distance restraints per nucleotide, as well as all γ torsion angles and sugar puckers for the central UGAA part of the molecule. Using these constraints, together with cross-strand distances, involving exchangeable imino protons, and essentially all other torsion angles that can accurately be determined (i.e. β, ϵ) otherwise, the structure of the UGAA domain could be determined with high precision (r.m.s.d. 0.62 Å), without the aid of isotopically enriched RNA. The G·A base-pairs are of the sheared pairing type, with both nucleotides in the anti conformation, and hydrogen bonds between the guanine 2-amino and the adenine N7 and between the guanine N3 and the adenine 6-amino. Surprisingly the sugar of the guanosine of the G·A mismatch adopts a 2′- endo sugar pucker conformation. Comparison with other RNA structures, in which two such G·A base-pairs are formed reveals that this detailed structure depends on the identity of the base 5′ to the guanosine in the tandem G·A base-pairs. A geometrical model for the incorporation of sheared tandem G·A base-pairs in A-form helices is formulated, which explains the distinct different stacking properties and helical parameters in sequences containing tandem sheared G·A base-pairs.

Jenisseensosides C and D, biologically active acylated triterpene saponins from Silene jenisseensis

We previously reported the isolation and structure elucidation of a new trans- p-methoxycinnamoyl triterpene-saponin along with its cis- p-methoxycinnamoyl isomer as an inseparable mixture from the roots of Silene jenisseensis. In a continuing study on this plant, two additional new acylated triterpene-saponins were obtained as an inseparable mixture. Their structures have been established by chemical means and spectroscopic methods including 1D and 2D homonuclear and heteronuclear correlation NMR spectroscopy as 3- O-[β- d-galactopyranosyl-(1 → 2)-β- d-glucuronopyranosyl]-28- O-[{α- l-rhamnopyranosyl-(1 → 2)}- {4- O- trans- p-methoxycinnamoyl}-β- d-fucopyranosyl] quillaic acid and its cis-isomer, respectively. They showed a significant enhancement of the granulocyte phagocytosis in vitro. © 1997 Elsevier Science Ltd. All rights reserved

Asterlingulatosides C and D, cytotoxic triterpenoid saponins from Aster lingulatus.

A further investigation of Aster lingulatus has led to the isolation of two additional novel triterpene saponins, asterlingulatoside C [3-O-beta-D-glucopyranosyl-3 beta, 16 alpha-dihydroxyolean-12-en-28-oic acid 28-O-beta-D-xylopyranosyl-(1-->4)-alpha-L-rhamnopyranosyl-(1-->2)-alpha- L- arabinopyranoside] (1) and asterlingulatoside D [3-O-beta-D-glucopyranosyl-3 beta,16 alpha-dihydroxyolean-12-en-28-oic acid 28-O-beta-D-xylopyranosyl-(1-->3)-beta-D-xylopyranosyl-(1-->4)-alpha-L- rhamnopyranosyl-(1-->2)-alpha-L-arabinopyranoside] (2). Elucidation of the structures of 1 and 2 was mainly based on FABMS and 1D and 2D homonuclear and heteronuclear NMR techniques. Compounds 1 and 2 showed good inhibitory activity against DNA synthesis in human leukemia HL-60 cells with IC50 values of 8.8 and 6.1 microM, respectively.

Automated NOESY interpretation with ambiguous distance restraints: the refined NMR solution structure of the pleckstrin homology domain from β-spectrin

We have used a novel, largely automated, calculation method to refine the NMR solution structure of the pleckstrin homology domain of β-spectrin. The method is called ARIA for Ambiguous Restraints for Iterative Assignment. The starting point for ARIA is an almost complete assignment of the proton chemical shifts, and a list of partially assigned NOEs, mostly sequential and secondary structure NOEs. The restraint list is then augmented by automatically interpreting peak lists generated by automated peak-picking. The central task of ARIA is the assignment of ambiguous NOEs during the structure calculation using a combination of ambiguous distance restraints and an iterative assignment strategy. In addition, ARIA calibrates ambiguous NOEs to derive distance restraints, merges overlapping data sets to remove duplicate information, and uses empirical rules to identify erroneous peaks. While the distance restraints for the structure calculations were exclusively extracted from homonuclear 2D experiments, ARIA is especially suited for the analysis of multidimensional spectra. Applied to the pleckstrin homology domain, ARIA generated structures of good quality, and of sufficiently high accuracy to solve the X-ray crystal structure of the same domain by molecular replacement. The comparison of the free NMR solution structure to the X-ray structure, which is complexed to d-myo-inositol-1,4,5-triphosphate, shows that the ligand primarily induces a disorder-order transition in the binding loops, which are disordered in the NMR ensemble but well ordered in the crystal. The structural core of the protein is unaffected, as evidenced by a backbone root-mean-square difference between the average NMR coordinates and the X-ray crystal structure for the secondary structure elements of less than 0.6 Å.

Characterization of a site-directed mutant of cytochrome b5 designed to alter axial imidazole ligand plane orientation.

Mutants of cytochrome b5 were designed to achieve reorientation of individual axial imidazole ligands. The orientation of the axial ligand planes is thought to modulate the reduction potential of bis(imidazole) axially ligated heme proteins. The A67V mutation achieved this goal through the substitution of a bulkier, hydrophobic ligand for a residue, in the sterically hindered hydrophobic heme binding pocket. Solution structures of mutant and wild-type proteins in the region of the mutation were calculated using restraints obtained from 1H and 15N 2D homonuclear and heteronuclear NMR spectra and 1H-15N 3D heteronuclear NMR spectra. More than 10 restraints per residue were used in the refinement of both structures. Average local rmsd for 20 refined structures was 0.30 A for the wild-type structure and 0.38 A for the A67V mutant. The transfer of amide proton resonance assignments from wild-type to the mutant protein was achieved through overlays of 15N-1H heteronuclear correlation spectra of the reduced proteins. Side chain assignments and sequential assignments were established using conventional assignment strategies. Calculation of the orientation of the components of the anisotropic paramagnetic susceptibility tensor, using methods similar to procedures applied to the wild-type protein, shows that the orientation of the in-plane components are identical in the wild-type and mutant proteins. However, the orientation of the z-component of the susceptibility tensor calculated for the mutant protein differs by 17 degrees for the A-form and by 11 degrees for the B-form from the orientation calculated for the wild-type protein. The rotation of the z-component of the susceptibility tensor (toward the delta meso proton) is in the same direction and is of the same magnitude as the rotation of the H63 imidazole ring induced by mutation.

Synthesis of tritium-labeled azaline B acetate

Reaction of azaline B acetate (1) with freshly prepared trifluoroacetyl hypoiodite in trifluoroacetic acid - methylene chloride solution under anhydrous conditions produced a complex mixture of products from which compounds 2 and 3, resulting from iodination at the 1′-and 8′-positions of the Ac-D Nal1 residue, were isolated by preparative reversed-phase HPLC chromatography. Mass spectral analysis and 1H, 2D DQF-COSY and homonuclear 2D J-resolved NMR experiments confirmed these structures. Reductive tritiation of 2 over 10% Pd/C in EtOH - DMF (1:9, v/v) with 50 Ci T2 followed by chromatographic purification and salt exchange gave 37.5 mCi of tritiated azaline B acetate (4) having >98% chemical and radiochemical purities and a specific radioactivity of 11.2 Ci / mmol. A single peak at 7.59 ppm was observed in the proton decoupled tritium NMR spectrum. © 1997 John Wiley & Sons, Ltd.

Application of homonuclear 3D NMR experiments and 1D analogs to study the conformation of sialyl Lewis(x) bound to E-selectin.

The conformation of the sialyl Lewis(x) tetrasaccharide bound to E-selectin was previously determined from transfer NOE (trNOE) experiments in conjunction with a distance-geometry analysis. However, the orientation of the tetrasaccharide ligand in the binding site of E-selectin is still unknown. It can be predicted that the accurate quantitative analysis of all trNOEs, including those originating from spin diffusion, is one key to analyze the orientation of sialyl Lewis(x) in the binding pocket of E-selectin. Therefore, we applied homonuclear 3D NMR experiments and 1D analogs to obtain trNOEs that could not unambiguously be assigned from previous 2D trNOESY spectra, due to severe resonance-signal overlap. A 3D TOCSY-trNOESY experiment, a 1D TOCSY-trNOESY experiment, and a 1D trNOESY-TOCSY experiment of the sialyl Lewis(x)/E-selectin complex furnished new interglycosidic trNOEs and provided additional information for the interpretation of trNOEs that have been described before. A 2D trROESY spectrum of the sialyl Lewis(x)/E-selectin complex allowed one to identify the amount of spin-diffusion contributions to trNOEs. Finally, an unambiguous assignment of all trNOEs, and an analysis of spin-diffusion pathways, was obtained, creating a basis for a quantitative analysis of trNOEs in the sialyl Lewis(x)/E-selectin complex.

Structural study and serological characterisation of the O-specific polysaccharide of Hafnia alvei PCM 1185, another Hafnia O-antigen that contains [formula omitted

The O-specific polysaccharide of H. alvei strain PCM 1185 contains d-glucose, d-glucuronic acid, 2-acetamido-2-deoxy- d-glucose, and 3,6- dideoxy-3-[(R)-3- hydroxybutyramido]- d-glucose (Qui3NAcyl) in the ratios 2:1:1:1, as well as O-acetyl groups. On the basis of sugar and methylation analyses of the polysaccharide before and after chemical modifications ( O-deacetylation, carboxyl reduction, Smith degradation), as well as 1H and 13C NMR spectroscopy, including 1D sequential, selective spin-decoupling, 2D homonuclear, and 13C, 1H heteronuclear correlation spectroscopy (COSY), and 2D rotating-frame NOE spectroscopy, it was found that the polysaccharide has a pentasaccharide repeating unit with the following structure: ▪ with O-acetyl groups present in nonstoichiometric amounts, mainly at position 2 of GlcA and position 6 of GlcNAc or lateral Glc. Serological study showed that H. alvei strain PCM 1185 can be placed in a new serotype D and that an O-acetyl group can be a part of its epitope.

Homonuclear 1D and 2D NMR Experiments for the Observation of Solvent–Solute Interactions

Homonuclear 1D and 2D NMR Experiments for the Observation of Solvent–Solute Interactions

Automated sequence-specific NMR assignment of homologous proteins using the program GARANT

The program GARANT (General Algorithm for Resonance AssignmeNT) for automated sequence-specific NMR assignment of proteins is based on the mapping of peaks predicted from the amino acid sequence onto the peaks observed in multidimensional spectra [C. Bartels, P. Güntert, M. Billeter and K. Wüthrich (1996) J. Comput. Chem., manuscript submitted for publication]. In this paper we demonstrate the potential of GARANT for the assignment of homologous proteins when either the three-dimensional structure or the chemical shifts of the parent protein are known. In these applications, GARANT utilizes supplementary information either in the form of interatomic distances derived from the three-dimensional structure, in order to add nuclear Overhauser effects reflecting the tertiary structure to the list of expected peaks, or in the form of the chemical shifts of the parent protein, in order to obtain a better estimate of the positions of the expected peaks. The procedure is illustrated with three different proteins: (i) a mutant form of Tendamistat (74 residues), using homonuclear 2D (1)H NMR spectra and either the three-dimensional structure or the chemical shifts of the wild-type protein; (ii) the mutant Antp(C39S, W56S) homeodomain (68 residues), using homonuclear 2D (1)H NMR spectra and the three-dimensional structure of the Antp(C39S) homeodomain; and (iii) free cyclophilin A (165 residues), using heteronuclear 3D NMR spectra and the three-dimensional structure of a cyclophilin A-cyclosporin A complex. In these three systems nearly complete assignment of the polypeptide backbone resonances and assignment of over 80% of the amino acid side-chain resonances was obtained without manual intervention.

4-Oxalocrotonate tautomerase, a 41-kDa homohexamer: backbone and side-chain resonance assignments, solution secondary structure, and location of active site residues by heteronuclear NMR spectroscopy.

4-Oxalocrotonate tautomerase (4-OT), a homohexamer consisting of 62 residues per subunit, catalyzes the isomerization of unsaturated alpha-keto acids using Pro-1 as a general base (Stivers et al., 1996a, 1996b). We report the backbone and side-chain 1H, 15N, and 13C NMR assignments and the solution secondary structure for 4-OT using 2D and 3D homonuclear and heteronuclear NMR methods. The subunit secondary structure consists of an alpha-helix (residues 13-30), two beta-strands (beta 1, residues 2-8; beta 2, residues 39-45), a beta-hairpin (residues 50-57), two loops (I, residues 9-12; II, 34-38), and two turns (I, residues 30-33; II, 47-50). The remaining residues form coils. The beta 1 strand is parallel to the beta 2 strand of the same subunit on the basis of cross stand NH(i)-NH(j) NOEs in a 2D 15N-edited 1H-NOESY spectrum of hexameric 4-OT containing two 15N-labeled subunits/hexamer. The beta 1 strand is also antiparallel to another beta 1 strand from an adjacent subunit forming a subunit interface. Because only three such pairwise interactions are possible, the hexamer is a trimer of dimers. The diffusion constant, determined by dynamic light scattering, and the rotational correlation time (14.5 ns) estimated from 15N T1/T2 measurements, are consistent with the hexameric molecular weight of 41 kDa. Residue Phe-50 is in the active site on the basis of transferred NOEs to the bound partial substrate 2-oxo-1,6-hexanedioate. Modification of the general base, Pro-1, with the active site-directed irreversible inhibitor, 3-bromopyruvate, significantly alters the amide 15N and NH chemical shifts of residues in the beta-hairpin and in loop II, providing evidence that these regions change conformation when the active site is occupied.

New Furanoid and seco-Labdanoid Diterpenes from Leonurus persicus

Six new labdane diterpenoids, leopersin C (1), 15-epi-leopersin C (2), leopersin D (3), leopersin E (4), leopersin F (5), and 7-epi-leopersin F (6) were isolated from the aerial parts of Leonurus persicus. Their structures were elucidated by extensive use of 1D and 2D homonuclear and heteronuclear shift-correlated 1 H- 13 C-NMR spectroscopic methods. Leopersin C (1) and 15-epi-leopersin C (2) were obtained as a C-15 epimeric mixture, and their structures were elucidated on this basis.

Solution structure of the E-domain of staphylococcal protein A.

The E-domain of staphylococcal protein A is one of five homologous IgG-binding domains designated E, D, A, B, and C that comprise the extracellular portion of protein A. The E-domain binds tightly to Fc fragments of IgG and binds certain Fv fragments with micromolar affinity. To explore further the structural features of Fc binding by protein A, and as a first step in developing a structural understanding of E-domain/Fv complex formation, we have determined the solution structure of the uncomplexed E-domain using 2D homonuclear and heteronuclear NMR spectroscopy. Complete 1H and 15N resonance assignments were obtained, and the structure was determined from 383 NOE-derived distance restrains, 34 phi and 19 chi 1 dihedral angle restraints, and 54 restraints for 27 H-bonds. 3JH alpha-H beta coupling constants and long-range NOEs involving Phe11 indicate the side chain exists in more than one conformation with differing chi 1 values. NOE restraints that were incompatible with chi 1 = -60 degrees were removed from one set of structure calculations, and those incompatible with chi 1 = 180 degrees were removed from a second set to allow Phe11 to explore both rotamer wells. Thus, two sets of 20 final structures, having no distance or dihedral angle restraint violations greater than 0.12 A or 1.6 degrees, respectively, represent the solution structure of the E-domain. Backbone atomic rms differences with respect to the mean coordinates for each set of 20 structures for residues 8-53 averaged 0.41 +/- 0.06 and 0.35 +/- 0.06 A. No significant differences in the overall structure result from the different orientations of Phe11. The solution structure of the E-domain consists of three alpha-helices that pack together to form a compact helical bundle. A detailed comparison between the E-domain ensembles and the previously determined structure for the B-domain in complex with Fc indicates that only the 180 degrees chi 1 rotamer of Phe11 is competent for binding; the -60 degrees chi 1 rotamer must reorient to 180 degrees to create a cavity that is filled by Ile253 from the CH2 domain of Fc in the Fc-bound complex.

Triterpenoid saponins from Bellis sylvestris. 2. Structures of partially deacylated saponins.

Four saponins have been isolated from the herbal parts of Bellis sylvestris and the same, together with three further compounds, from the underground parts. The structures were elucidated by electrospray ionization mass spectrometry, including tandem mass spectrometry, and by 1D and 2D homonuclear and heteronuclear NMR spectroscopy. Two of the compounds are new saponins with the structures 3-O-beta-D-glucopyranosyl-2 beta, 3 beta, 23-trihydroxyolean-12-en-28-oic acid 28-O-beta-D-glucopyranosyl (1-->3)-[alpha-L-rhamnopyranosyl (1-->2)]-beta-D- glucopyranoside and 3-O-beta-D-glucopyranosyl-2 beta, 3 beta,16 alpha, 23-tetrahydroxyolean-12-en- 28-oic acid 28-O-alpha-L-rhamnopyranosyl (1-->3)-beta-D-xylopyranosyl(1-->4)-alpha-L- rhamnopyranosyl(1-->2)-[(E)-2-butenoic acid (1-->3)]-beta-D-fucopyranoside. Taxonomically these data provide significant evidence of a link between the Bellis genus and Bellium bellidioides.

New Polyphenol Glycosides from Ramonda myconi

Two new polyphenol glycosides, myconoside (1) and hispidulin-8-C-β-d-galactopyranoside (2), and the known disaccharide sucrose have been isolated and identified from a MeOH extract of Ramonda myconi leaves. Structures were determined mainly using homonuclear and inverse-detected 2D NMR experiments.

1D analogues of homonuclear 2D NMR by DANTE-selective pulses

One-dimensional (1D) analogues of two-dimensional NMR experiments are discussed as a method of saving instrument time and data storage space. Two-dimensional NMR experiments can be converted to their 1D analogues by replacing a hard pulse with a selective pulse and by replacing all variable delays with fixed delays. The experiments described can be performed on spectrometers that do not have pulsed-field gradients or pulse-shaping capabilities. Examples of some 1D analogues are presented using DANTE pulses for selective excitation. © 1996 John Wiley & Sons, Inc.

Complete Spectral Assignments and Microstructures of Photopolymerized Acrylonitrile/Methacrylic Acid Copolymers by NMR Spectroscopy

Copolymers of acrylonitrile/methacrylic acid were prepared by photopolymerization using the uranyl ion as a photosensitizer. The comonomer reactivity ratios, determined by both Kelen-Tudos (KT) and nonlinear error in variables (EVM) methods are r A = 0.135 ± 0.04 and r M = 3.618 ± 0.49. The microstructure was obtained in terms of the distribution of A- and M-centered triad sequences from 13 C-{ 1 H} NMR spectra of the copolymers. Homonuclear 2D TOCSY NMR was used to simplify the complex 1 H spectra of AIM copolymers in terms of configurational/conformational sequences. The triad concentration calculated from Monte Carlo simulations (MC) gave good agreement with the triad concentration determined from NMR spectroscopy. MC simulation was also used to study the effect of the degree of polymerization on triad fractions.

Submicromole structure elucidation: Cryptolepicarboline—a novel dimeric alkaloid from Cryptolepis sanguinolenta

AbstractCryptolepis sanguinolenta (Lindl.) Schlechter (Asclepiadaceae), a shrub indigenous to West Africa, has been employed by traditional healers in the treatment of various fevers, including malaria. Column chromatography of the alkaloid fraction obtained from a defatted ethanolic extract of the roots of the plant afforded the previously characterized alkaloids cryptolepine, quindoline and cryptospirolepine. Continued extensive column chromatography, followed by preparative high‐performance liquid chromatography, resulted in the isolation of nine new alkaloids, including approximately 100 μg of a novel indoloquinoline‐β‐carboline dimeric alkaloid, cryptolepicarboline. The structure was elucidated through the use of a combination of spectroscopic techniques which included UV, Fourier transform, electron impact mass spectrometry, and 500 MHz NMR spectroscopy. NMR data included 1D 1H and 13C reference spectra, the latter recorded in 40 μl of DMSO‐d6 using a new Varian heteronuclear Nano‐probe and in 140 μl of DMSO‐d6 using a Nalorac carbon‐optimized microprobe. These 13C NMR spectra allow the first direct comparison of these two probe formats. Homonuclear 2D NMR data acquired included both COSY and ROESY spectra. The homonuclear 2D experiments were variously performed in both a homonuclear Nano‐probe and micro‐inverse detection probes. Heteronuclear shift correlation experiments included HMQC, HMBC and IDR (inverted direct response)‐HMQC‐TOCSY spectra, which were uniformly performed using a 500 MHz Nalorac micro inverse‐detection probe.

Detailed 1H- and 13C-Nmr Investigations of Some Diterpenes Isolated from Leonurus persicus

Five new labdane diterpenoids, leopersin A [1], 8-deacetoxyleopersin A [2], leopersin B [3], 15-epi-leopersin B [4], and 4β-hydroxymethylptegaleopsin [5] were isolated from the aerial parts of Leonurus persicus, together with the known compounds pregaleopsin [7], its rearrangement product, galeopsin [8], and the optical antipode of (+)-leosibiricin [9]. Leopersin B [3] and 15-epi-leopersin B [4] were obtained as a C-15 epimeric mixture and their structures were elucidated on the basis of spectroscopic and chemical evidence. 4β-Hydroxymethylpregaleopsin [5] was isolated as its p-bromobenzoate derivative [6] and its absolute configuration established by single-crystal X-ray crystallographic analysis. Complete nmr assignments for the known compounds were made using extensive 1D and 2D homonuclear and heteronuclear 1 H- 13 C-nmr spectroscopic methods. For pregaleopsin [7], the 1 H- and 13 C-nmr data were reassigned, primarily on the basis of results of 2D nmr INADEQUATE measurements.

Structural and electronic similarity but functional difference in methylmalonyl-CoA mutase between coenzyme B12 and the analog 2',5'-dideoxyadenosylcobalamin.

The cofactor analog 2',5'-dideoxyadenosylcobalamin (ddAdoCbl) differs from the natural cofactor coenzyme B12 [5'-deoxyadenosylcobalamin (dAdoCbl)] by lacking only one oxygen atom. The 1H and 13C NMR spectra of ddAdoCbl have been assigned unambiguously by homonuclear and heteronuclear 2D NMR techniques. The 1H, 13C, and 31P chemical shift values for ddAdoCbl were compared with those of another organocobalamin, namely dAdoCbl. This assessment shows that the analog is very similar both electronically and structurally to the natural cofactor. The effectiveness of ddAdoCbl as a cofactor for both the human and Propionibacterium shermanii methylmalonyl-CoA mutases was compared with that of the natural cofactor. ddAdoCbl was found to be a competitive inhibitor with respect to dAdoCbl. Similar binding affinities to both enzymes were found for both the ddAdoCbl analog and the natural cofactor. However, in the presence of ddAdoCbl, the rate of conversion of methylmalonyl-CoA to succinyl-CoA was only 1-2% of that seen with the natural cofactor. There were no changes with time in the visible absorption spectrum of the bound cofactor analog in the presence of substrate, suggesting that the Co-C bond was not cleaved. The CD (circular dichroism) spectra of dAdoCbl and ddAdoCbl are very similar, consistent with the NMR results. The CD spectral changes upon binding to P. shermanii methylmalonyl-CoA mutase are large compared to those reported on the binding of dAdoCbl to ethanolamine ammonia lyase. Furthermore, the CD spectra of both enzyme-bound cobalamins are very similar, suggesting that similar changes in the conformation or structure in these cobalamins occur on binding to the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)