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1.
R Geyer H Geyer S Stirm G Hunsmann J Schneider U Dabrowski J Dabrowski 《Biochemistry》1984,23(23):5628-5637
The highly microheterogeneous, N-glycosidically linked oligosaccharides in the glycoproteins of Friend murine leukemia virus (as produced by Eveline cells) were liberated with endo-beta-N-acetylglucosaminidase H and by alkaline hydrolysis. They were fractionated (as desialylated oligosaccharitols) by gel filtration and by concanavalin A affinity chromatography, and the major fractions were analyzed by methylation-gas chromatography-mass spectrometry, by digestion with exoglycosidases, and, especially, by one- and two-dimensional proton nuclear magnetic resonance spectroscopy. Guidelines for qualitative and quantitative analysis of complex oligosaccharide mixtures by NMR were worked out and the results compared with those obtained by methylation analysis. It was found that these major fractions consist of bi-, tri-, and tetraantennary oligosaccharitols of the "complex" type (comprising a minority of species with N-acetyllactosamine repeating units), which are, in part, substituted by nonreducing terminal Gal alpha (1----3) and/or bisecting GlcNAc beta (1----4) residues. 相似文献
2.
Rudolf Geyer Silvia Diabaté Hildegard Geyer Hans-Dieter Klenk Heiner Niemann Stephan Stirm 《Glycoconjugate journal》1987,4(1):17-32
Fowl plague virus, strain Dutch, was metabolically labeled withd-[2-3H]mannose, or withd-[6-3H]glucosamine, and the small subunit (HA2; 0.8 mg in total) of the viral hemagglutinin was isolated by preparative sodium dodecylsulfate-polyacrylamide gel electrophoresis. After proteolytic digestion, the radioactive oligosaccharides were sequentially liberated from the glycopeptides by treatment with different endo--N-acetylglucosaminidases and with peptide:N-glycosidase or, finally, by hydrazinolysis. In this manner, four groups of glycans could be obtained by consecutive gel filtrations and were subfractionated by HPLC. The structures of the individual oligosaccharides were analyzed by micromethylation, by acetolysis or by digestion with exoglycosidases. The major species amongst the high mannose glycans at Ans-406 of the viral glycopolypeptide were found to be Man1-2Man1-3(Man1-2Man1-6)Man1-6(Man1-2Man1-2Man1-3)Man1-4GlcNac1-4GlcNAc and Man1-3(Man1-2Man1-6)Man1-6(Man1-2Man1-2Man1-3)Man1-4GlcNAc1-4GlcNAc, while the complex glycans at Asn-478 are predominantly GlcNAc1-2Man1-3(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc (lacking, in part, one of the outerN-acetylglucosamine residues) and GlcNAc1-2Man1-3(Gal1-4GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc.Abbreviation BSA
bovine serum albumin
- endo D (F,H)
endo--N-acetyl-d-glucosaminidase D (F,H)
- HA
hemagglutinin (HA1, large subunit of HA
- HA2
small subunit
- FPV
fowl plague virus
- PNGase F
peptide:N-glycosidase F
- SDS
sodium dodecylsulfate 相似文献
3.
Carbohydrates of influenza virus. Structural elucidation of the individual glycans of the FPV hemagglutinin by two-dimensional 1H n.m.r. and methylation analysis. 总被引:9,自引:1,他引:8
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W Keil R Geyer J Dabrowski U Dabrowski H Niemann S Stirm H D Klenk 《The EMBO journal》1985,4(10):2711-2720
The structures of the oligosaccharides of the hemagglutinin of fowl plague virus [influenza A/FPV/Rostock/34 (H7N1)] have been elucidated by one- and two-dimensional 1H n.m.r. spectroscopy at 500 MHz and by microscale methylation analysis. N-Glycosidic oligosaccharides of the oligomannosidic (OM) and of the N-acetyllactosaminic type have been found, the latter type comprising biantennary structures, without (A) or with (E) bisecting N-acetylglucosamine, and triantennary (C) structures. Analysis of the tryptic and thermolytic glycopeptides of the hemagglutinin allowed the allocation of these oligosaccharides to the individual glycosylation sites. Each attachment site contained a unique set of oligosaccharides. Asn12 contains predominantly structures C and E which are highly fucosylated. Asn28 contains OM and A structures that lack fucose and sulfate. Asn123 shows A that has incomplete antennae but is highly fucosylated and sulfated. Asn149 has fucosylated A and E. Asn231 shows fucosylated A and E with incomplete antennae. Asn406 has OM oligosaccharides. Asn478 has A and E with little fucose. Localization of the oligosaccharides on the three-dimensional structure of the hemagglutinin revealed that the oligomannosidic glycans are attached to glycosylation sites at which the enzymes responsible for carbohydrate processing do not have proper access. These observations demonstrate that an important structural determinant for the oligosaccharide side chains is the structure of the glycoprotein itself. In addition, evidence was obtained that the rate of glycoprotein synthesis also has an influence on carbohydrate structure. 相似文献
4.
The assignment of the 1H nuclear magnetic resonance (n.m.r.) spectrum of the protease inhibitor IIA from bull seminal plasma is described and documented. The assignments are based entirely on the amino acid sequence and on two-dimensional n.m.r. experiments at 500 MHz. Individual assignments were obtained at 18 degrees C and 45 degrees C for the backbone protons of all 57 amino acid residues, with the single exception of the N-terminal pyroglutamate amide proton. The amino acid side-chain resonance assignments are complete, with the exception of 17 long side-chains, i.e. Pro13, Met43 and all the Glu, Gln, Lys and Arg, where only one or two resonances of C beta H2 and in some cases C gamma H2 could be identified. The sequential assignments showed that the order of the two C-terminal residues in the previously established primary structure had to be changed; this was then confirmed by chemical methods. The chemical shifts for the assigned resonances at 18 degrees C and 45 degrees C are listed for an aqueous solution at pH 4.9. A preliminary characterization of the polypeptide secondary structure was obtained from the observed patterns of sequential connectivities. 相似文献
5.
G Pfeiffer U Dabrowski J Dabrowski S Stirm K H Strube R Geyer 《European journal of biochemistry》1992,205(3):961-978
The carbohydrate side chains of the thrombin-like serine protease ancrod from the venom of the Malayan pit viper Agkistrodon rhodostoma were liberated from tryptic glycopeptides by treatment with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F and fractionated by high-performance liquid chromatography. Glycans obtained were characterized by digestion with exoglycosidases, methylation analysis and, in part, by liquid secondary-ion mass spectrometry and 1H-NMR spectroscopy. The results reveal that this snake venom glycoprotein contains partially truncated di-, tri- and tetraantennary complex type N-glycans carrying Fuc(alpha 1-6) residues at the innermost N-acetylglucosamine and solely (alpha 2-3)-linked sialic acid substituents. As a characteristic feature, ancrod oligosaccharides comprise mainly sialylated Gal beta 3GlcNAc beta lactosamine antennae. Furthermore, a small proportion of the sugar chains were found to carry a NeuAc alpha 3GalNAc beta 4GlcNAc beta antenna exclusively linked to C-2 of Man(alpha 1-3) residues of the pentasaccharide core. Thus, many of the glycans found represent novel glycoprotein-N-glycan structures. 相似文献
6.
A conformational study of the opioid peptide dermorphin by one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy
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Dermorphin, a natural peptide opioid containing a D-Ala2 residue, has been studied in dimethyl sulfoxide (DMSO) solution by means of several one-dimensional and two-dimensional 1H nuclear magnetic resonance (NMR) methods at various fields from 80 to 600 MHz. The combined use of conventional NMR parameters and of nuclear Overhauser effect effects points to an essentially extended structure. This conformation may be, in part, the result of strong interaction of the amide groups with DMSO molecules. 相似文献
7.
The core structure of Citrobacter PCM 1487 lipopolysaccharide has been established using methylation analysis/mass spectrometry, chemical degradations and one- and two-dimensional 1H-NMR spectroscopy at 500 MHz. 1H-NMR assignments are given for all sugar components of the core oligosaccharide. In the formula shown below, the alternative locations of branch terminal heptose (LDHep) and diphosphorylethanolamine (PPEtN) residues are marked by dashed lines; dOclA stands for 3-deoxy-D-manno-octulosonic acid. (Formula: see text). The sample of the core oligosaccharide showed some microheterogeneity due to a slightly incomplete substitution by terminal N-acetylgalactosamine and a partial splitting of diphosphorylethanolamine residues. 相似文献
8.
Amide proton exchange in the alpha-amylase polypeptide inhibitor Tendamistat studied by two-dimensional 1H nuclear magnetic resonance 总被引:2,自引:0,他引:2
The individual amide proton exchange rates in Tendamistat at pH 3.0 and 50 degrees C were measured by using two-dimensional 1H nuclear magnetic resonance. Overall, it was found that the distribution of exchange rates along the sequence is dominated by the interstrand hydrogen bonds of the beta-sheet structures. The slowly exchanging protons in the core of the two beta-sheets were shown to exchange via an EX2 mechanism. Further analysis of the data indicates that different large-scale structure fluctuations are responsible for the exchange from the two beta-sheets, even though the three-dimensional structure of Tendamistat appears to consist of a single structural domain. 相似文献
9.
Phospholipid packing and conformation in small vesicles revealed by two-dimensional 1H nuclear magnetic resonance cross-relaxation spectroscopy.
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Two-dimensional 1H-NMR spectroscopy has been used to examine cross-relaxation in sonicated phospholipid vesicle systems. The observed pattern of proton cross-relaxation reveals several important features of these vesicle systems. For example, cross-relaxation rates on each monolayer of the vesicle system can be resolved and reflect the expected geometric packing constraints of the vesicle system. Small but significant magnetization-exchange is also seen to develop between the headgroup N-methyl resonance and the terminal methyl resonance. Spectra taken with deuterated lipids indicate that this exchange is not mediated by spin-diffusion down the length of the alkyl chains. Since spin-diffusion is the only process that is expected to facilitate magnetization-exchange over distances of 15-20 A, a close proximity of headgroup and terminal methyl protons in a fraction of the membrane lipid is indicated by these results. This could occur by events such as lipid interdigitation or alkyl chain bends that terminate lipid alkyl chain ends near the membrane surface. 相似文献
10.
A two-dimensional Fourier transform nuclear magnetic resonance study of the ribosomal protein E-L30 is reported. Five two-dimensional techniques, namely: nuclear magnetic resonance J-resolved spectroscopy, correlated spectroscopy, double quantum spectroscopy, relayed coherence transfer and nuclear Overhauser enhancement spectroscopy were used. Qualitative inspection of the spectra obtained by these techniques provided evidence that the E-L30 molecule has a well-defined structure in solution. This analysis indicated that, despite the fact that the protein is stable only at moderate temperatures and neutral pH, a structural analysis of the molecule would be feasible. A detailed analysis of the spectra permitted unambiguous discrimination between the spin systems of different amino acids, resulting in residue-specific resonance assignments. We were able to assign all resonances of all six threonine, four valine, five alanine, two histidine, two serine, one phenylalanine, one asparagine and one aspartic acid residue of E-L30. Complete resonance assignment was obtained for two glycine residues. Partial assignments became available for all six isoleucine, three glycine and one glutamine residue. These results form a sound basis for the structure determination of the protein described in the accompanying paper. 相似文献
11.
Barthwal R Awasthi P Monica Kaur M Sharma U Srivastava N Barthwal SK Govil G 《Journal of structural biology》2004,148(1):34-50
The 5' d-TpG 3' element is a part of DNA sequences involved in regulation of gene expression and is also a site for intercalation of several anticancer drugs. Solution conformation of DNA duplex d-TGATCA containing this element has been investigated by two-dimensional NMR spectroscopy. Using a total of 12 torsional angles and 121 distance constraints, structural refinement has been carried out by restrained molecular dynamics (rMDs) in vacuum up to 100 ps. The structure is characterized by a large positive roll at TpG/CpA base pair step and large negative propeller twist for AT and TA base pairs. The backbone torsional angle, gamma(O5'-C5'-C4'-C3'), of T1 residue adopts a trans-conformation which is corroborated by short intra nucleotide T1H6-T1H5' (3.7A) distance in nuclear overhauser effect spectroscopy (NOESY) spectra while the backbone torsional angle, beta(P-O5'-C5'-C4'), exists in trans as well as gauche state for T1 and C5 residues. There is evidence of significant flexibility of the sugar-phosphate backbone with rapid inter-conversion between two different conformers at TpG/CpA base pair step. The base sequence dependent variations and local structural heterogeneity have important implications in specific recognition of DNA by ligands. 相似文献
12.
Barthwal R Monica Awasthi P Srivastava N Sharma U Kaur M Govil G 《Journal of biomolecular structure & dynamics》2003,21(3):407-423
Solution conformation of self-complementary DNA duplex d-CGATCG, containing 5' d-CpG 3' site for intercalation of anticancer drug, daunomycin and adriamycin, has been investigated by nuclear magnetic resonance (NMR) spectroscopy. Complete resonance assignments of all the protons (except some H5'/H5" protons) have been obtained following standard procedures based on double quantum filtered correlation spectroscopy (dQF COSY) and two-dimensional nuclear Overhauser effect (NOE) spectra. Analysis of sums of coupling constants in one-dimensional NMR spectra, cross peak patterns in dQF COSY spectra and inter proton distances shows that the DNA sequence assumes a conformation close to the B-DNA family. The deoxyribose sugar conformation is in dynamic equilibrium with predominantly S-type conformer and a minor N-type conformer with N<-->S equilibrium varying with temperature. At 325 K, the mole fraction of the N-conformer increases for some of the residues by approximately 9%. Using a total of 10 spin-spin coupling constants and 112 NOE intensities, structural refinement has been carried out using Restrained Molecular Dynamics (rMD) with different starting structures, potential functions and rMD protocols. It is observed that pseudorotation phase angle of deoxyribose sugar for A3 and T4 residues is approximately 180 degrees and approximately 120 degrees, respectively while all other residues are close to C2'endo-conformation. A large propeller twist (approximately -18 degrees) and smallest twist angle (approximately 31 degrees) at A3pT4 step, in the middle of the sequence, a wider (12 A) and shallower (3.0 A) major groove with glycosidic bond rotation as high anti at both the ends of hexanucleotide are observed. The structure shows base-sequence dependent variations and hence strong local structural heterogeneity, which may have implications in ligand binding. 相似文献
13.
The conformation at the dA-dT junction in d-(AAAAATTTTT)2 was investigated by using a variety of phase-sensitive two-dimensional nuclear magnetic resonance experiments at 500 MHz for detailed studies of the deoxyribose ring puckers. Conformational constraints were collected from two-dimensional nuclear Overhauser enhancement spectra recorded with short mixing times and from quantitative simulations of the cross-peaks in two-dimensional correlated spectra. Overall, the decamer duplex adopts a conformation of the B-DNA type, and for dA4 and dA5 the pseudorotation phase angle P is in the standard range 150-180 degrees. The deoxyribose puckers for the other nucleotides deviate significantly from the standard B-DNA structure. Spectrum simulations assuming either static deviations from standard B-DNA or a simple two-state dynamic equilibrium between the C2'-endo and C3'-endo forms of the deoxyribose were used to analyze the experimental data. It was thus found that the ring pucker for dT6 deviates from the regular C2'-endo form of B-DNA by a static distortion, with the pseudorotation phase angle P in the range 100-130 degrees, and a similar value of P is indicated for dT7. For the peripheral base pairs dynamic distortions of the C2'-endo form of the deoxyribose were found. In agreement with recent papers on related duplexes containing (dA)n tracts, we observed prominent nuclear Overhauser effects between adenine-2H and deoxyribose-1'H, which could be largely due to pronounced propeller twisting as observed in the crystal structures of (dA)n-containing compounds. 相似文献
14.
Attenuation of pathogenicity of fowl plague virus by recombination with other influenza A viruses nonpathogenic for fowl: nonexculsive dependence of pathogenicity on hemagglutinin and neuraminidase of the virus. 总被引:7,自引:3,他引:4
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A number of antigenic hybrids of influenza A viruses were produced possessing either the hemagglutinin or the neuraminidase of fowl plague virus and the corresponding antigen derived from another influenza A virus. Other recombinants were obtained carrying both surface antigens of fowl plague virus but differing from the parent in certain biological properties. None of the recombinants isolated were pathogenic for adult chickens. Most recombinants obtained after crosses between reciprocal recombinants carrying both fowl plague virus surface antigens were also apathogenic for chickens. Using the same parent recombinants for double infection some of the progeny "back-recombinants" were pathogenic, whereas others were not. From these results it is concluded that the surface components do not by themselves determine the pathogenicity of influenza A viruses. 相似文献
15.
E A de Jong J P van Duynhoven B J Harmsen R N Konings C W Hilbers 《Journal of molecular biology》1989,206(1):119-132
Two-dimensional nuclear magnetic resonance techniques were used to obtain residue- and sequence-specific assignments in the 1H spectrum of the single-stranded DNA-binding protein encoded by gene V of the filamentous phage IKe (IKe GVP). The residue-specific assignments are based on the analysis of J-correlated spectra, i.e. correlated spectroscopy and homonuclear-Hartmann-Hahn total correlated spectroscopy. Complete assignments of side-chain spin systems, e.g. long side-chains, were, to a major part, derived from two-dimensional spectra obtained by means of the latter technique. Sequence-specific residue assignments were obtained for the two neighbouring residues V41 and Y42, and the amino acid sequence segment encompassing residues S17 through I29. The structure of this segment, a beta-loop, was deduced from the interresidue nuclear Overhauser effect pattern. Residues S17 through V19 and P26 through I29 form an anti-parallel beta-ladder segment, whereas residues Q21 to K25 constitute the loop region. The beta-loop is expected to project into the solution and is intimately involved in binding to single-stranded DNA; it is therefore designated the "DNA-binding wing". By analogy with the structure of the DNA-binding wing deduced from IKe GVP, a similar structure is proposed for the corresponding domain of the gene V protein encoded by the filamentous phage Ff for which, from X-ray diffraction studies, a three-dimensional structure has been deduced. Essential differences appear to exist between the DNA-binding domain in the X-ray structure and that proposed in this paper. Possible reasons for these differences are discussed. 相似文献
16.
1H nuclear magnetic resonance studies of an integral membrane protein: subunit c of the F1F0 ATP synthase 总被引:1,自引:0,他引:1
M F Moody P T Jones J A Carver J Boyd I D Campbell 《Journal of molecular biology》1987,193(4):759-774
The membrane-traversing subunit c parallel from the F0 part of the ATP synthase molecule has been studied in chloroform/methanol by high-resolution 1H n.m.r. Various one-dimensional and two-dimensional techniques have been used for assignment purposes, some NOE connectivities were established and some 3JHN alpha coupling constants were measured from spin--echo experiments. The effects of varying pH, solvent composition, lanthanide concentration and temperature have been investigated. Evidence is presented that the molecule has extensive alpha-helical segments, and the hairpin structure suggested by other groups is supported by our n.m.r. data. Only one ionizable group, assigned to the C-terminal carboxyl, is observed to titrate in the pH range 2 to 10; so the conserved residue, Asp61, which binds dicyclohexylcarbodiimide, presumably has (at least in this solvent system) an abnormally high pK value. 相似文献
17.
Phase-sensitive two-dimensional nuclear magnetic resonance (n.m.r.) experiments have been used to obtain extensive proton resonance assignments for the carbon monoxide complex of sperm whale myoglobin. Multiple quantum experiments were particularly important in the assignment procedure. The assignments are the most complete yet reported for a protein of such high molecular weight (approximately 18,000) and make possible new and comprehensive studies of the structure and dynamics of carbonmonoxymyoglobin in solution. Assignments for seven of the histidine residues are reported, including the critical proximal and distal histidines. Most of these are at variance with the assignments already in the literature. The present n.m.r. data indicate that histidines 24 (B5) and 119 (GH1) are hydrogen bonded to each other and, in contrast to neutron diffraction data, show that His24 does not protonate at pH greater than 5. The aromatic rings of all the phenylalanine and tyrosine residues undergo rapid flips about the ring axis. The side-chains of Leu89 (F4) and Phe138 (H15), which border a large hydrophobic cavity, are particularly mobile. 相似文献
18.
The conformation of the peanut inhibitor B-III was investigated by 1H NMR at 500 MHz. Resonances for about 30% of the residues were assigned and/or identified by conventional and two-dimensional 1H NMR measurements. The conformation of the modified inhibitor B-IIIRS, obtained by the cleavage of the peptide bonds of B-III at the first reactive site, Arg(10)-Arg(11), and second reactive site, Arg-(37)-Ser(38), was also investigated. Edman degradation was used to identify resonances of the residues adjoining the scissile sites. A comparison of the behavior of the resonances corresponding to Ala(12), Tyr(15), Phe(16), and Thr(37) in B-III and B-IIIRS indicated that the only conformational differences between the two proteins were those from the S-S loops containing the two reactive sites. From the similar behavior of Leu(7), Phe(24), His(26), Ala(29), five valine, and four threonine resonances in both proteins, it is suggested that the S-S loops that do not contain a scissile bond (the core region of the inhibitor) were rigid. Thus, no conformational change of the core region was observed upon cleavage of the reactive sites. 相似文献
19.
Solution structure of the kringle 4 domain from human plasminogen by 1H nuclear magnetic resonance spectroscopy and distance geometry 总被引:1,自引:0,他引:1
Kringle 4 is an autonomous structural and folding domain within the proenzyme plasminogen. Homologous domains are found throughout the blood clotting and fibrinolytic proteins. In this paper, we present the almost complete assignment of the 1H nuclear magnetic resonance (n.m.r.) spectrum of the kringle 4 domain of human plasminogen. A detailed structural analysis has been completed. The sequential pattern of nuclear Overhauser enhancements indicated little regular secondary structure but rather a series of turns and loops connecting beta-strands. A small stretch of antiparallel beta-sheet was identified between the residues 61 to 63 and 71 to 73 and the close proximity of other strands was determined from two-dimensional nuclear Overhauser enhancement spectra. Slowly exchanging amide (NH) resonances were found to be associated with residues of the beta-sheet and neighbouring strands that support the hydrophobic core of the domain. A total of 526 interproton distance constraints and two hydrogen bonds were specified as input to the distance geometry program DISGEO. Tertiary structures were produced that were consistent with the n.m.r. data. The structures were compared with that of our earlier model based on n.m.r. studies and with that of prothrombin fragment 1 determined crystallographically. 相似文献
20.
Iu S Krivoshein V A Shatrov Iu L Krivorutchenko I B Andronovskaia 《Mikrobiologicheekij zhurnal》1990,52(3):51-54
Immunogenic properties of influenza virus hemagglutinin, isolated by new detergents O-14 (desintegron-O) and B-14 (desintegron-B) have been studied. Hemagglutinin isolated by desintegron-O has been found to be more immunogenic than virions. It has been shown that hemagglutinin isolated by desintegron-B induces a lower humoral immune response than the influenza virus. 相似文献