1H-NMR conformational analysis of a high-affinity antigenic 11-residue peptide from the tryptophan synthase beta 2 subunit.

Two synthetic peptides from the beta 2 subunit of tryptophan synthase have been studied by 1H-NMR spectroscopy at 300 MHz. One peptide, His-Gly-Arg-Val-Gly-Ile-Tyr-Phe-Gly-Met-Lys (peptide 11; Ile, isoleucine) is antigenic and binds with a high affinity to a monoclonal antibody that recognizes the native beta 2 subunit. The second peptide, His-Gly-Arg-Val-Gly-Ile-Tyr-Phe (peptide 8) reacts very weakly with the antibody. The 1H-NMR spectra of the two peptides have been assigned from two-dimensional techniques in H2O, 2H2O and (2H6) dimethyl sulfoxide [(2H6)Me2SO]. The structure has been evaluated through analysis of nuclear Overhauser effects, coupling constants, amide-proton exchange rates and their temperature coefficients, and chemical shifts. In aqueous solvent, the C-terminal part of peptide 11 presents some structure centered around residues Phe-Gly-Met. The relationship between the structure found in peptide 11 and its antigenic nature is discussed.     

Structural features of N-glycans linked to royal jelly glycoproteins: structures of high-mannose type, hybrid type, and biantennary type glycans

The structures of N-glycans of total glycoproteins in royal jelly have been explored to clarify whether antigenic N-glycans occur in the famous health food. The structural feature of N-glycans linked to glycoproteins in royal jelly was first characterized by immunoblotting with an antiserum against plant complex type N-glycan and lectin-blotting with Con A and WGA. For the detail structural analysis of such N-glycans, the pyridylaminated (PA-) N-glycans were prepared from hydrazinolysates of total glycoproteins in royal jelly and each PA-sugar chain was purified by reverse-phase HPLC and size-fractionation HPLC. Each structure of the PA-sugar chains purified was identified by the combination of two-dimensional PA-sugar chain mapping, ESI-MS and MS/MS analyses, sequential exoglycosidase digestions, and 500 MHz 1H-NMR spectrometry. The immunoblotting and lectinblotting analyses preliminarily suggested the absence of antigenic N-glycan bearing beta1-2 xylosyl and/or alpha1-3 fucosyl residue(s) and occurrence of beta1-4GlcNAc residue in the insect glycoproteins. The detailed structural analysis of N-glycans of total royal jelly glycoproteins revealed that the antigenic N-glycans do not occur but the typical high mannose-type structure (Man(9 to approximately 4)GlcNAc2) occupies 71.6% of total N-glycan, biantennary-type structures (GlcNAc2Man3 GlcNAc2) 8.4%, and hybrid type structure (GlcNAc1 Man4GlcNAc2) 3.0%. Although the complete structures of the remaining 17% N-glycans; C4, (HexNAc3 Hex3HexNAc2: 3.0%), D2 (HexNAc2Hex5HexNAc2: 4.5%), and D3 (HexNAc3Hex4HexNAc2: 9.5%) are still obscure so far, ESI-MS analysis, exoglycosidase digestions by two kinds of beta-N-acetylglucosaminidase, and WGA blotting suggested that these N-glycans might bear a beta1-4 linkage N-acetylglucosaminyl residue.     

Assignments of the iminoproton resonances of Bombyx mori tRNA(UCCGly) and the comparison of its structure and stability with those of tRNA(GCCGly)

Most of the iminoproton resonances in the 1H-NMR spectrum of Bombyx mori tRNA(UCCGly) have been assigned by the sequential NOEs. Any peak which indicates the presence of the tertiary GC base pair between the D and T loops could not be detected. The effects of temperature and the addition of magnesium ions and spermine on the 1H-NMR spectrum of this tRNA were examined. From the temperature change, it was found that the acceptor stem and the D stem in Bombyx mori tRNA(UCCGly) are equally stable even in the absence of magnesium, which is different from tRNA(GCCGly) where the D stem is not so stable.     

Structural characterization of sialylated tetrasaccharides and pentasaccharides with blood group H and Le(x) activity isolated from bovine submaxillary mucin

In this study we have investigated the structures of a sialylated tetrasaccharide and two sialylated pentasaccharides released from bovine submaxillary mucin by alkaline borohydride treatment and isolated by high-performance liquid chromatography. The tetrasaccharide contained NeuGc, while one of the pentasaccharides contained NeuAc and the other contained NeuGc. All three oligosaccharides contained the core type-3 structure (GlcNAc beta 1----3GalNAcol). The structures, determined by a combination of one- and two-dimensional 1H-NMR spectroscopy at 270 MHz and methylation analysis involving gas-liquid chromatography/mass spectrometry, were as follows: [formula: see text]. The oligosaccharides occurred in the approximate molar ratios, 1.0:0.6:0.3. This is the first report of these oligosaccharides in bovine submaxillary mucin. 1H-NMR data for structures A1/2c and A1/2e, which are novel structures, are presented for the first time. Oligosaccharide A1/2e contains the blood-group-H type-2 antigenic determinant while oligosaccharide A1/2d contains the Lewis(x) determinant.     

1H-NMR assignment and secondary structure of a herpes simplex virus glycoprotein D-1 antigenic domain

The peptide alpha Ahx-Met-Ala-Asp-Pro-Asn-Arg-Phe-Arg-Gly-Lys-Asp-Leu-Pro-Val-Leu- Asp-Gln-Leu-Thr-Asp-Pro-Pro-alpha Ahx (epsilon Ahx = 6-aminohexanoyl), the antigenic sequence 11-32 from Herpes simplex virus glycoprotein D-1, has been synthesised. Its 1H-NMR spectrum has been assigned by a combination of two-dimensional techniques in H2O and 2H2O. Its secondary structure has been defined by nuclear Overhauser effects and amide proton exchange rates, and also to some extent chemical shifts, coupling constants and amide proton temperature coefficients. These latter parameters are shown to be less reliable as guides to secondary structure. The peptide has a helical (type I/III) turn at residues Pro-14-Asn-15 and helical structure at residues Lys-20-Val-24, in rapid equilibrium with random-coil structure. A beta-turn at residues Arg-18-Gly-19 may be present as a minor component. These locations of secondary structure correspond with previously determined regions of antigenic activity.     

A powerful method of sequential proton resonance assignment in proteins using relayed 15N-1H multiple quantum coherence spectroscopy

A powerful method of sequential resonance assignment of protein 1H-NMR spectra is presented and illustrated with respect to the DNA-binding protein ner from phage Mu. It is based on correlating proton-proton through-space and through-bond connectivities with the chemical shift of the directly bonded 15N atom. By this means, ambiguities arising from chemical shift degeneracy of amide proton resonances can be resolved. The experiments described involve combining the 1H-detected heteronuclear multiple quantum coherence correlation experiment with homonuclear nuclear Overhauser enhancement, J-correlated or Hartmann-Hahn experiments.     

N-linked oligosaccharides of cobra venom factor contain novel alpha(1-3)galactosylated Le(x) structures

Cobra venom factor (CVF), a nontoxic, complement-activating glycoprotein in cobra venom, is a functional analog of mammalian complement component C3b. The carbohydrate moiety of CVF consists exclusively of N-linked oligosaccharides with terminal alpha1-3-linked galactosyl residues, which are antigenic in human. CVF has potential for several medical applications, including targeted cell killing and complement depletion. Here, we report a detailed structural analysis of the oligosaccharides of CVF. The structures of the oligosaccharides were determined by lectin affinity chromatography, antibody affinity blotting, compositional and methylation analyses, and high-resolution (1)H-NMR spectroscopy. Approximately 80% of the oligosaccharides are diantennary complex-type, approximately 12% are tri- and tetra-antennary complex-type, and approximately 8% are oligomannose type structures. The majority of the complex-type oligosaccharides terminate in Galalpha1-3Galbeta1-4(Fucalpha1-3)GlcNAcbeta1, a unique carbohydrate structural feature abundantly present in the glycoproteins of cobra venom.     

A nuclear-Overhauser-enhancement study of the solution structure of a double-stranded DNA undecamer comprising a portion of the specific target site for the cyclic-AMP-receptor protein in the gal operon. Sequential resonance assignment

A 500-MHz 1H-NMR study on a double-stranded non-self-complementary DNA undecamer comprising a portion of the specific target site for the cyclic AMP receptor protein in the gal operon is presented. Using pre-steady-state nuclear Overhauser effect (NOE) measurements, all exchangeable imino, non-exchangeable base, methyl, and H1', H2' and H2" sugar proton resonances are assigned in a sequential manner. In addition, some of the H3' sugar proton resonances are also assigned and some of the exchangeable amino proton resonances identified. The relative magnitudes of the intranucleotide and internucleotide NOEs are indicative of a right-handed B-type conformation for the duplex undecamer in solution.     

1H-NMR study of endothelin, sequence-specific assignment of the spectrum and a solution structure

The solution conformation of the recently discovered bi-cyclic, 21 amino acid vasoconstrictor peptide, Endothelin I, has been examined by 1H-NMR in deuterated dimethyl sulphoxide. A full sequential assignment has been achieved. In addition, 19 long range NOEs were detected which were employed as distance constraints in molecular dynamics calculations to yield a possible solution structure for this new peptide.     

Structure of P401 (mast cell degranulating peptide) in solution

P401 (also known as mast cell degranulating protein, MCD) is a minor component of honeybee venom. Its primary structure is related to that of apamin. We have studied the structure of P401 in solution by high-resolution two-dimensional 1H-NMR spectroscopy. Almost all the backbone proton resonances have been assigned by sequential assignment strategy. Analysis of NOEs shows that P401 has a conformation very similar to that of apamin. N-terminal residues Ile-1-Cys-5 are in an extended conformation and residues His-13-Asn-22 on the C-terminus are in an alpha-helical structure. These two secondary structural elements are connected by two tight turns.     

Evidence for a complex structure of neutralization antigenic site I of poliovirus type 1 Mahoney

We have selected neutralization escape mutants by using a monoclonal antibody (nt-MAb) against a sequential epitope between amino acids 93 through 104 (neutralization antigenic site I) of poliovirus type 1 Mahoney. The majority of mutants were also resistant against five strain-specific nt-MAbs which recognized conformation-dependent epitopes, suggesting that the neutralization antigenic site I must be involved in the formation of such epitopes. An analysis of all mutants by the binding of nt-MAbs and by isoelectric focusing of VP1 allowed discrimination of five classes of mutants. Sequence analysis of mutant RNAs revealed point mutations and deletions in the antibody-binding site.     

Staged equilibrium of carbonic anhydrase unfolding in strong denaturants

It has been shown by 1H-NMR, circular dichroism, fluorescence and viscometry techniques that equilibrium unfolding of carbonic anhydrase B (a one-domain globular protein) in urea guanidine hydrochloride consists of two sequential stages. The first stage is connected with a decrease of intramolecular interactions, stabilizing the rigid tertiary structure and with the increase of mobility of aliphatic side chain groups. At the second stage the decrease of protein secondary structure and hydrophobic interactions take place as well as the increase of mobility of massive aromatic side chain groups.     

Sequence determination in oligosaccharides by relayed NOE experiments in the rotating frame

An hybrid experiment, composed of 1H-NMR total correlation spectroscopy (TOCSY) and rotating frame nuclear Overhauser enhancement spectroscopy (ROESY) steps, makes it possible to determine both the partial (disaccharide) sequences, and the sequential order, of whole linear and branched oligosaccharide chains.     

Structures of oligomannoside chains of alpha-mannosidase from porcine kidney

Lysosomal acid alpha-mannosidase from porcine kidney was found to contain mannose (4.8%), galactose (0.9%), fucose (0.5%), N-acetylglucosamine (3.1%), and mannose 6-phosphate (0.1%). Approximately 50% of the total hexose of the oligosaccharide chains could be released by endo-beta-N-acetylglucosaminidase-H (endo-H). They were predominantly neutral, oligomannoside-type oligosaccharides containing 5, 6, and 9 mannose residues, respectively, in the centesimal ratio of 36:25:34. 500-MHz 1H-NMR spectroscopy in conjunction with sequential exoglycosidase digestion of the reduced compounds revealed that each of the three fractions consisted of a single isomer only; the Man9 compound has the following structure: (Formula: see tex). The Man6-compound lacks Man residues D1, D2, and D3, while the Man5-compound lacks Man-C as well. In addition to the neutral ones, some (5%) phosphorylated oligomannoside-type oligosaccharides were obtained. The endo-H resistant glycopeptides were subjected to hydrazinolysis. Approximately 60% of the oligosaccharides released by hydrazine were found to be of rather small size; their composition can be represented asMan2-3GlcNAc[Fuc]0-1GlcNAcol. The remaining 40% consist of larger-size galactose-containing, N-acetyllactosamine-type oligosaccharides. Studies involving sequential exoglycosidase digestion and 500-MHz 1H-NMR spectroscopy performed on the highly purified small-sized compounds revealed the following four structures for the endo-H-resistant oligosaccharides: (Formula: see text).     

Localization of sequential antigenic determinants on bovine beta-casein with synthetic peptides and antisera from mouse, rabbit, and goat.

The antigenic determinants of bovine beta-casein (beta-CN) were localized by using twenty overlapping peptides encompassing the entire sequence of beta-CN and anti-beta-CN antisera from outbred mouse, rabbit and goat. The profile of the reactions was characteristic to the species, the dominant antigenic regions being 80-95, 143-158 and 195-209 in mouse, 1-16 in rabbit and 100-115 in goat. Regions 1-16, 100-115, 121-136 and 143-158 were antigenic in all three species. The number of antigenic regions recognized by goat was much fewer than that by mouse and rabbit, possibly because of the homology between bovine and goat beta-CN. A mixture of the twenty peptides could absorb about 50-60% of beta-CN specific antibodies from each species. Furthermore, the mouse and rabbit anti-beta-CN antibodies were also specific to the phosphorylated regions. We therefore conclude that the major antigenic determinants on beta-CN would be largely sequential and include the phosphorylated sites.     

Computer-aided assignment of the 1H-NMR spectrum of the viral-protein-genome-linked polypeptide from cowpea mosaic virus

The 1H-NMR spectrum of the viral-protein-genome-linked (VPg) polypeptide from cowpea mosaic virus, has been interpreted via application of 2-dimensional (2D) NMR techniques. The interpretation of the data was performed by a computer program called 'PROSPECT' (PROtein SPECTra), which detects the cross-peak patterns of the amino acid residues in the spectra, assigns these patterns to amino acid types, and finally performs the sequential assignments using the well-known 'sequential walks' obtained from the NOE spectrum. Due to the severe overlap of resonances in the NMR spectrum of the VPg polypeptide, several ways of performing these walks existed. The program detected six alternatives for the sequential assignments of backbone N alpha H-C beta H-C beta H moieties.     

The position of the LysN epsilon H2-grafted antigens along the sequential oligopeptide carrier, Ac-(Aib-Lys-Aib-Gly)n (SOCn-II), influences the antibody recognition: application to the Sm main autoimmune epitope

A sequential oligopeptide carrier of antigenic peptides is presented, incorporating two Aib residues in each repetitive moiety: Ac-(Aib-Lys-Aib-Gly)(n) (SOC(n) -II; n = 2-4). The conformational study, by (1)H-nmr, CD, and Fourier transform ir spectroscopy, indicated that the SOC(n) -II carrier displays a pronounced 3(10)-helix, compared to the Ac-(Lys-Aib-Gly)(n) (SOC(n) -I) carrier of the same approximately backbone length, previously reported. One of the dominant autoimmune epitopes of the Sm and U1RNP cellular components, the PPGMRPP sequence, was coupled to the Lys-N(epsilon)H(2) groups of the SOC(n) -II carrier and used as antigenic substrate for detecting anti-Sm/U1RNP autoantibodies in ELISA assays. Anti-Sm antibodies are highly specific for systemic lupus erythematosus, while anti-U1RNP are specific for mixed connective tissue disease. The anti-(PPGMRPP)(5)-SOC(n) -II ELISA was compared with the anti-(PPGMRPP)(n) -SOC(n) -I ELISA, provided that both antigenic substrates possess the same amount of the epitope replicates. The significance of the lysine positions along the oligopeptide backbone of the carrier for a favorable antibody recognition of the anchored antigens is also examined.     

MSP-1 malaria pseudopeptide analogs: biological and immunological significance and three-dimensional structure

Merozoite Surface Protein-1 (MSP-1) has been considered as a malaria vaccine candidate. It is processed during the Plasmodium falciparum invasion process of red blood cells (RBCs). A conserved MSP-1 C-terminal peptide was identified as a high-activity erythrocyte-binding peptide (HAEBP) termed 1585. Since conserved HAEBPs are neither antigenic nor immunogenic we decided to assess the significance of a single peptide bond replacement in 1585. Thus, two pseudopeptides were obtained by introducing a Y[CH2-NH] reduced amide isoster into the 1585 critical binding motif. The pseudopeptides bound to different HLA-DR alleles, suggesting that backbone modifications affect MHC-II binding patterns. Pseudopeptide-antibodies inhibit in vitro parasite RBC invasion by recognizing MSP-1. Each pseudopeptide-induced antibody shows distinct recognition patterns. 1H-NMR studies demonstrated that isoster bonds modulate the pseudopeptides' structure and thus their immunological properties, therefore representing a possible subunit malaria vaccine component.     

Somatic antigens of the Brucella genus. The structure of the O-specific polysaccharide chain of Brucella melitensis lipopolysaccharide

The phenol-phase soluble antigenic lipopolysaccharide was isolated from Brucella melitensis, strain 565, by the routine phenol/water procedure followed by chromatography on Sepharose 4B. After mild acid hydrolysis and chromatography on Sephadex G-50, the lipopolysaccharide yielded a linear O-specific polysaccharide built up from 1,2-linked 4,6-dideoxy-4-formamido-alpha-D-mannopyranosyl units. The structure of the polysaccharide was deduced mainly from the nuclear magnetic resonance and methylation analyses. The phenol-soluble lipopolysaccharide, isolated from commercial vaccine strain B. abortus 19-BA, on mild hydrolysis afforded material, 13C and 1H-NMR spectra of which were identical to those of the O-specific polysaccharide from B. melitensis 565.     

1H-NMR study of neurotoxin B-IV from the marine worm Cerebratulus lacteus. Solution properties, sequence-specific resonance assignments, secondary structure and global fold.

Sequence-specific resonance assignments are reported for the 500-MHz 1H-NMR spectrum of the 55-residue neurotoxin B-IV, isolated from the heteronemertine worm Cerebratulus lacteus. A range of two-dimensional homonuclear correlated and NOE spectra was used in making these assignments, which include NH, C alpha H and C beta H resonances, as well as most resonances from longer-chain spin systems, with the exception of the ten Lys residues, where spectral overlap prevented complete, unambiguous assignments. The secondary structure of B-IV was identified from the pattern of sequential (i, i + 1) and medium range (i, i + 2/3/4) NOE connectivities and the location of slowly exchanging backbone amide protons. Two helices are present, incorporating residues 13-26 and 33-49, and the C-terminal five residues form a helix-like structure. A type-I reverse turn, involving residues 28-31 is present in a small loop linking the two major helices, and the N-terminus appears to be unordered at 27 degrees C, although it may adopt a more ordered conformation at lower temperatures. These elements of secondary structure, together with the four disulfide bonds in the protein, provide sufficient information to define the global fold of the molecule in solution. The pH and temperature dependence of the toxin have been investigated by 1H-NMR and the pKa values of several ionisable sidechains determined.