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.     

Conformations of nucleoside analogue 1-(2'-deoxy-beta-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide in different DNA sequence contexts

The concept of using a dynamic base-pairing nucleobase as a mode for degenerate recognition presents a unique challenge to analysis of DNA structure. Proton and phosphorus NMR studies are reported for two nine-residue DNA oligodeoxyribonucleotides, d(CATGGGTAC).d(GTACNCATG) (1) and d(CATGTGTAC).(GTACNCATG) (2), which contained 1-(2'-deoxy-beta-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide (N) in the center of the helix at position 14. The duplexes were compared to the canonical Watson-Crick duplexes, d(CATGGGTAC).d(GTACCCATG) (3) and d(CATGTGTAC).d(GTACACATG) (4). Two-dimensional NOESY spectra of 1-4 in H(2)O and D(2)O solutions collected at 5 degrees C allowed assignment of the exchangeable and nonexchangeable protons for all four oligodeoxyribonucleotides. Thermodynamic and circular dichroism data indicated that 1-4 formed stable, B-form duplexes at 5 degrees C. Two-dimensional (1)H-(31)P correlation spectra indicated that there were minor perturbations in the backbone only near the site of the triazole base. Strong NOESY cross-peaks were observed between the H5 and H1' of N14 in 1 and, unexpectedly, 2, which indicated that, in both duplexes, N14 was in the syn(chi)() conformation about the glycosidic bond. NOESY spectra of 1 and 2 recorded in 95% H(2)O, 5% D(2)O indicated that the imino proton of the base opposite N14, G5, or T5, formed a weak hydrogen bond with N14. These conformations place the polar carboxamide functional group in the major groove with motional averaging on the intermediate time scale.     

Two-dimensional 1H and 31P NMR spectra and restrained molecular dynamics structure of a covalent CPI-CDPI2-oligodeoxyribonucleotide decamer complex

CPI-CDPI2 is a synthetic analogue of CC-1065, which is a naturally occurring antitumor antibiotic. Assignment of the 1H NMR spectra of a CPI-CDPI2-oligodeoxyribonucleotide decamer, d-(CGCTTAAGCG)2, complex has been made by two-dimensional 1H/1H spectroscopy. The solution structure of the complex was calculated by an iterative hybrid relaxation matrix method combined with NOESY distance restrained molecular dynamics. Refinement proceeded in two steps in which the decamer was initially refined alone and then CPI-CDPI2 was added to the structure to allow initial estimates of drug-DNA contacts. A hybrid matrix/MD refinement was used to better take into account problems associated with spin diffusion. Thus the distances from the 2D NOESY spectra were calculated from the relaxation rate matrix which were evaluated from a hybrid NOESY volume matrix comprising elements from the experimental spectrum and those calculated from an initial structure. The hybrid matrix derived distances were then used in a restrained molecular dynamics procedure to obtain a new structure that better approximates the NOESY spectra. The resulting partially refined structure was then used to calculate an improved theoretical NOESY volume matrix which is once again merged with the experimental matrix until refinement is complete. The efficacy of CC-1065 has been attributed to its minor groove binding and alkylation to the N3 position of adenosine. CPI-CDPI2 appears to bind to the decamer in a similar manner. The effect of CPI-CDPI2 on the decamer's 1H and 31P spectrum was consistent with a minor groove binding motif with the drug alkylating at A17 with the CDPI rings oriented toward the 5'-end of the alkylated strand. In addition, the NMR data support one major adduct but also indicate the presence of a minor adduct. The latter could represent a drug alkylation of the DNA at a secondary site (or alternative orientation of the rings).     

Oxygen disruption of the 2[4Fe-4S] clusters in Clostridium pasteurianum ferredoxin shown by 1H-NMR.

The ferredoxin from Clostridium pasteurianum, which contains two [4Fe-4S] clusters, was investigated in its oxidized and reduced states by two-dimensional (2D) (1)H-(1)H nuclear Overhauser enhancement spectroscopy (NOESY). Comparison of the data from the oxidized ferredoxin with those published previously revealed the same NOE connectivities. No previous (1)H-(1)H NOESY study of the fully reduced ferredoxin has previously been published. However, it was possible to compare our results with those of a 2D exchange spectroscopy investigation of half-reduced C. pasteurianum ferredoxin. The present results with reduced C. pasteurianum ferredoxin confirm many of the (1)H peaks and NOE interactions reported earlier, revise others, and locate resonances previously undetected. When the ferredoxin was slightly exposed to oxygen, several of the hyperfine shifted resonances were irreversibly influenced. A resonance at 34 ppm in the (1)H NMR spectra of both redox states is indicative of oxygen exposure. These results indicate the importance of keeping the ferredoxin strictly anaerobic during purification and solvent exchange.     

Oxygenated iron bleomycin. A short-lived intermediate in the reaction of ferrous bleomycin with O2.

The reaction of Fe(II) . bleomycin with O2 to yield Fe(III) . bleomycin has been resolved into two kinetic events by stopped-flow spectrophotometry. The first event is first order with respect to both bleomycin and O2 and may be regarded as a second order reaction (k = 6.1 x 10(3) M-1s-1 at 2 degrees C). The first product has no EPR spectrum. The optical spectrum resembles those of Fe(II) . bleomycin complexes with CO, NO, and ethyl isocyanide. We propose that the first product is an Fe(II) . bleomycin . O2 complex. The second kinetic event is first order with respect to the first accumulated product (k = 0.11 s-1 at 2 degrees C) and independent of oxygen concentration. The product of this reaction is indistinguishable from Fe(III) . bleomycin by optical and EPR spectroscopy.     

Structure of a neutral O-specific polysaccharide of the bacterium Providencia alcalifaciens O5.

A neutral polysaccharide containing D-galactose, 2-acetamido-2-deoxy-D-glucose, and 3-acetamido-3,6-dideoxy-D-glucose (Qui3NAc) in the ratios 2:1:1 was obtained by mild acid degradation of lipopolysaccharide of the bacterium Providencia alcalifaciens O5 followed by gel chromatography and ion-exchange chromatography or treatment with anhydrous hydrogen fluoride. On the basis of full acid hydrolysis, methylation, and 1H- and 13C-NMR spectroscopy, including two-dimensional correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), H-detected heteronuclear 1H,13C single-quantum coherence (HSQC), and nuclear Overhauser effect spectroscopy (NOESY), the following structure of the linear tetrasaccharide repeating unit of the polysaccharide was established:     

High-resolution mono- and multidimensional magic angle spinning 1H nuclear magnetic resonance of membrane peptides in nondeuterated lipid membranes and H2O.

High-speed (14 kHz) solid-state magic angle spinning (MAS) 1H NMR has been applied to several membrane peptides incorporated into nondeuterated dilauroyl or dimyristoylphosphatidylcholine membranes suspended in H2O. It is shown that solvent suppression methods derived from solution NMR, such as presaturation or jump-return, can be used to reduce water resonance, even at relatively high water content. In addition, regioselective excitation of 1H peptide resonances promotes an efficient suppression of lipid resonances, even in cases where these are initially two orders of magnitude more intense. As a consequence, 1H MAS spectra of the peptide low-field region are obtained without interference from water and lipid signals. These display resonances from amide and other exchangeable 1H as well as from aromatic nonexchangeable 1H. The spectral resolution depends on the specific types of resonance and membrane peptide. For small amphiphilic or hydrophobic oligopeptides, resolution of most individual amide resonance is achieved, whereas for the transmembrane peptide gramicidin A, an unresolved amide spectrum is obtained. Partial resolution of aromatic 1H occurs in all cases. Multidimensional 1H-MAS spectra of membrane peptides can also be obtained by using water suppression and regioselective excitation. For gramicidin A, F2-regioselective 2D nuclear Overhauser effect spectroscopy (NOESY) spectra are dominated by intermolecular through-space connectivities between peptide aromatic or formyl 1H and lipid 1H. These appear to be compatible with the known structure and topography of the gramicidin pore. On the other hand, for the amphiphilic peptide leucine-enkephalin, F2-regioselective NOESY spectra mostly display cross-peaks originating from though-space proximities of amide or aromatic 1H with themselves and with aliphatic 1H. F3-regioselective 3D NOESY-NOESY spectra can be used to obtain through-space correlations within aliphatic 1H. Such intrapeptide proximities should allow determination of the conformation of the peptide in membranes. It is suggested that high-speed MAS multidimensional 1H NMR of peptides in nondeuterated membranes and in H2O can be used for studies of both peptide structure and lipid-peptide interactions.     

Structure of an acidic O-specific polysaccharide of Proteus mirabilis O5.

The following structure of the O-specific polysaccharide of Proteus mirabilis O5 was established by 1H and 13C NMR spectroscopy at 500 MHz, including two-dimensional COSY, TOCSY, NOESY, and H-detected 1H, 13C heteronuclear multiple-quantum coherence (HMQC) experiments: [formula: see text] where O-acetylation of alpha-D-GlcNAc at both positions is nonstoichiometric.     

Structure determination of the O-antigenic polysaccharide from the enteroinvasive Escherichia coli O136.

The structure of the O-antigen polysaccharide of the lipopolysaccharide from the enteroinvasive Escherichia coli O136 has been elucidated. The composition of the repeating unit was established by sugar and methylation analysis together with 1H and 13C NMR spectroscopy. Two-dimensional nuclear Overhauser effect spectroscopy (NOESY) and heteronuclear multiple-bond correlation experiments were used to deduce the sequence. The absolute configuration for the nonulosonic acid (NonA) could be determined using spin-spin coupling constants, 13C chemical shifts and NOESY. The anomeric configuration of the NonA was determined via vicinal and geminal 13C,1H coupling constants. The structure of the repeating unit of the polysaccharide from E. coli O136 is as follows, in which beta-NonpA is 5,7-diacetamido-3,5,7, 9-tetradeoxy-Lglycero-beta-Lmanno-nonulosonic acid: -->4)-beta-NonpA-(2-->4)-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->     

Proton nuclear magnetic resonance studies on huwentoxin-I from the venom of the spiderSelenocosmia huwena: 1. Sequence-specific1H-NMR assignments

The complete sequence-specific assignments of resonances in the¹H-NMR spectrum of huwentoxin-I from the Chinese bird spider,Selenocosmia huwena, is described. A combination of two-dimensional NMR experiments including 2D-COSY, 2D-NOESY, and 2D-TOCSY has been employed on samples of the toxin dissolved in D₂O and in H₂O for assignment purposes. Protons belonging to spin systems for each of the 33 amino acids were identified. The sequence-specific assignments were facilitated by the identification ofd _N connectivities on the fingerprint regions of the COSY and NOESY spectra and were supported by the identification ofd _NN andd _N connectivities in the TOCSY and NOESY spectra. These studies provide a basis for the determination of the solution-phase conformation of this toxin.Abbreviations HWTX-I huwentoxin-I - 2D two-dimensional - COSY 2D homonuclear correlation spectroscopy - NOE nuclear Overhauser enhancement - NOESY 2D nuclear Overhauser enhancement spectroscopy - TOCSY 2D total correlation spectroscopy - TPPI time-proportional phase incrementation - TSP sodium 3-(trimethyl-silyl)propionate-d4 - EDTA ethylenediaminetetraacetic acid     

Sequential NMR resonance assignment and secondary structure of ferrocytochrome c553 from Desulfovibrio vulgaris Hildenborough.

Two-dimensional nuclear magnetic resonance spectroscopy was used to assign the proton resonances of ferrocytochrome c553 from Desulfovibrio vulgaris Hildenbourough at 37 degrees C and pH = 5.9. Only a few side-chain protons were not identified because of degeneracy or overlap. The spin systems of the 79 amino acids were identified by DQF-COSY and HOHAHA spectra in H2O and D2O. Sequential assignments were obtained from NOESY connectivities between adjacent amide, C alpha H, and C beta H protons. From sequential NH(i)----NH(i + 1) and long-range C alpha H(i)----NH(i + 3) connectivities, four stretches of helices were identified (2----8, 34----46, 53----59, 67----77). Long-range NOE between residues in three different helices provide qualitative information on the tertiary structure, in agreement with the general folding pattern of cytochrome c. The heme protons, including the propionate groups, were assigned, and the identification of Met 57 as sixth heme ligand was established. The dynamical behavior of the ring protons of the six tyrosines was analyzed in detail in terms of steric hindrance. The NMR data for ferrocytochrome c553 are consistent with the X-ray structure for the homologous cytochrome from D. vulgaris Miyazaki. On the basis of the secondary structure element and of observed chemical shift due to the heme ring current, a structural alignment of eukaryotic and prokaryotic cytochromes c is proposed.     

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.     

海南捕鸟蛛毒素-I(HNTX-I)的~1H-NMR信号归属和二级结构分析(英文)

海南捕鸟蛛毒素-I(HNTX-I)是从海南捕鸟蛛(Ornithoctonus hainana)的粗毒中纯化的一种新型神经毒素。应用二维1H-NMR.技术研究HNTX-I的溶液结构特点,通过分析水和重水中的DOF-COSY、TOCSY和NOESY谱,识别出HNTX-I全部33个氨基酸残基自旋体系;通过NOESY谱中的dαN、dβN、dNN和Dαδ联系完成了序列专一的谱峰归属,从而确认了HNTX-I所有的主链质子和大于96％的侧链质子的化学位移。并通过分析3JNH-CaH耦合常数、序列间的NOE联系以及慢氢交换质子等,确定HNTX-I的二级结构主要是由三股反平行的β-折迭组成(Lys7-Cys9,Tyr20-Asn23和Trp28-Val31),这些结构特点与已经探明结构的其它蜘蛛毒素的基本相同。这些结果为完全解析HNTX-I的溶液三维结构奠定了基础。     

海南捕鸟蛛毒素-Ⅰ(HNTX-Ⅰ)的1H-NMR信号归属和二级结构分析

海南捕鸟蛛毒素-Ⅰ(HNTX-Ⅰ)是从海南捕鸟蛛(Ornithoctonus hainana)的粗毒中纯化的一种新型神经毒素.应用二维1H-NMR技术研究HNTX-Ⅰ的溶液结构特点,通过分析水和重水中的DQF-COSY、TOCSY和NOESY谱,识别出HNTX-Ⅰ全部33个氨基酸残基自旋体系;通过NOESY谱中的dαN、dβN、dNN和dαδ联系完成了序列专一的谱峰归属,从而确认了HNTX-Ⅰ所有的主链质子和大于96%的侧链质子的化学位移.并通过分析3JNH-CαH耦合常数、序列间的NOE联系以及慢氢交换质子等,确定HNTX-Ⅰ的二级结构主要是由三股反平行的β-折迭组成(Lys7-Cys9,Tyr20-Asn23和Trp28-Val31),这些结构特点与已经探明结构的其它蜘蛛毒素的基本相同.这些结果为完全解析HNTX-Ⅰ的溶液三维结构奠定了基础.     

Structure of a new acidic O-antigen of Proteus vulgaris O22 containing O-acetylated 3-acetamido-3,6-dideoxy-D-glucose.

The acidic O-specific polysaccharide of Proteus vulgaris O22 was studied using 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, NOESY, and H-detected 1H, 13C heteronuclear multiple-quantum coherence (HMQC) experiments, and the following structure for the branched pentasaccharide repeating unit was established: [sequence: see text] where Qui3NAc is 3-acetamido-3,6-dideoxyglucose, O-acetylation of QuiNAc at position 4 is stoichiometric and at position 2 nonstoichiometric. Serological relationships of P. vulgaris O22 with some other Proteus strains were substantiated on the level of the O-antigen structures.     

Structure of the O-specific polysaccharide of Vibrio cholerae O9 containing 2-acetamido-2-deoxy-D-galacturonic acid

The O-specific polysaccharide (OPS) was isolated by mild-acid degradation of the lipopolysaccharide of Vibrio cholerae O9 and studied by carboxyl reduction, sugar and methylation analyses, Smith degradation, and two-dimensional NMR spectroscopy, including COSY, TOCSY, NOESY, and H-detected 1H,(13)C HMQC experiments. The following structure of the pentasaccharide-repeating unit of the OPS was established:     

NOE and two-dimensional correlated 1H-NMR spectroscopy of cytochrome c' from Chromatium vinosum.

1H two-dimensional (nuclear Overhauser effect spectroscopy (NOESY) and two-dimensional correlated spectroscopy (COSY) spectra of cytochrome c' from Chromatium vinosum have been obtained. The protein is of medium size (Mr 28,000), essentially high spin (S = 5/2) although some quantum mechanical spin admixing with S = 3 2 may be present. Under these circumstances NOESY cross peaks have been revealed between geminal protons (alpha-CH2 propionate and beta-CH2 protons of the bound histidine) and between alpha-CH2 propionate protons and the heme methyl groups. COSY maps have confirmed the geminal nature of the proton pairs, even with a linewidth as large as 900 Hz; the J value is about 12 Hz. This assignment has rationalized on a sound basis the biochemical behavior of this protein with pH and has showed the utility of this kind of spectroscopy for the other cytochromes c' structures and analogous systems.     

Structure and cross-reactivity of the O-antigen of Proteus vulgaris O8.

A high-molecular-mass O-specific polysaccharide was obtained by mild acid degradation of Proteus vulgaris O8 lipopolysaccharide followed by gel permeation chromatography. Studies of the polysaccharide by sugar and methylation analyses and 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, NOESY, and H-detected 1H, 13C heteronuclear multiple-quantum coherence (HMQC) experiments, demonstrated the presence of a tetrasaccharide repeating unit having the following structure: [sequence: see text] The role of an epitope associated with the alpha-L-FucpNAc-(1-->3)-D-GlcpNAc disaccharide in serological cross-reactivity of P. vulgaris O8 is discussed.     

Structure of the O-specific polysaccharide of the bacterium Proteus vulgaris O23

An acidic O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of the bacterium Proteus vulgaris O23 (strain PrK 44/57) and found to contain 2-acetamido-2-deoxy-D-galactose, 2-acetamido-2-deoxy-D-glucose, and D-galacturonic acid. Based on 1H- and 13C-NMR spectroscopic studies, including two-dimensional correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), and 1H,13C heteronuclear multiple-quantum coherence (HMQC) experiments, the following structure of the branched tetrasaccharide repeating unit of the polysaccharide was established: [figure], where the degree of O-acetylation of the terminal GalA residue at position 4 is about 80%. A structural similarity of the O-specific polysaccharides of P. vulgaris O23 and P. mirabilis O23 is discussed.     

Structure of an acidic O-specific polysaccharide of the bacterium Providencia alcalifaciens O7

An acidic O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of the bacterium Providencia alcalifaciens O7 and purified by gel chromatography followed by anion-exchange chromatography. On the basis of full acid hydrolysis, methylation, carboxyl reduction, selective cleavage with anhydrous hydrogen fluoride, and 1H- and 13C-NMR spectroscopy, including two-dimensional 1H,1H homonuclear and H-detected 1H,13C heteronuclear correlation spectroscopy and nuclear Overhauser effect spectroscopy (NOESY), the following structure of the linear tetrasaccharide repeating unit of the polysaccharide was established: [figure], where Rhap2Ac is 2-O-acetylrhamnopyranose.