New and old NMR experiments for the resonance assignment of complex oligosaccharides--application to a cyclodextrin derivative.

The complete assignment of the (1)H and (13)C sugar resonances in mono-3,6-anhydro-heptakis(2,3-O-methyl)-hexakis(6-O-methyl)-ß-cyclodextrin, an asymmetrically functionalized ß-cyclodextrin, was carried out by means of 2D NMR experiments. The TOCSY and the homonuclear multiple relay COSY spectra provided most of the (1)H assignments. The multiplicity edited HSQC and a set of F(1) selective HSQC-TOCSY and multiple relay HSQC-COSY spectra gave access to most of the (13)C chemical shifts. The latter were fully and accurately determined by means of a pair of complementary, highly folded HSQC-TOCSY spectra. The TOCSY-ROESY and ROESY-TOCSY spectra yielded the sequential assignment of the sugar units. A high resolution F(1) selective F(1) decoupled version of the TOCSY-ROESY experiment was recorded.

An alternative scheme for the multiplexed acquisition of 1D and 2D NMR spectra.

Spin system selective 1D (1)H, 2D DQF-COSY and 2D HSQC NMR spectra were recorded in order to fully assign the (1)H and (13)C 1D NMR spectra of an asymmetrical beta-cyclodextrin derivative. Instead of individually accessing the seven sugar anomeric protons by means of long multiplet selective pulses, only short region selective pulses were used. The simultaneously selected anomeric protons were differentiated by allowing their magnetization to evolve under the sole effect of the chemical shift interaction. In each experiment, the seven recorded spectra were linear combinations of the seven desired ones. The combination coefficients were measured and used to obtain almost perfectly separated sugar unit sub-spectra. This multiplexed acquisition scheme resulted in a time gain factor of about 2.

Exploring the molecular function of PIN1 by nuclear magnetic resonance.

PIN1 participates in the regulation of a number of signalling pathways in the cell involving protein phosphorylation/dephosphorylation. Its role seems to be an essential control level in addition to the protein phosphorylation by proline-directed kinases. Its cellular function includes regulation of the cell cycle by interaction with phosphorylated mitotic proteins such as Cdc25 and transcription factors such as p53. PIN1 was shown to be involved in the malignant transformation of cells in breast cancer, by up regulation of cyclinD1 and is thought to be involved in the development of the AD by regulating the function of phosphorylated Tau. We propose here to discuss the molecular function of PIN1 at the atomic level based on data from the recent literature and our own results obtained by the technique of Nuclear Magnetic Resonance. PIN1 specifically interacts with pThr/pSer-Pro motifs and is constituted by two domains: a WW N-terminal domain that binds pThr/pSer-Pro epitopes and a prolyl cis/trans isomerase C-terminal catalytic domain. An exception to this organisation is found in the plant PIN1 homologous enzymes, like PIN1At from Arabidopsis thaliana, that are constituted of the sole catalytic domain. The molecular function of PIN1, binding to and isomerization of pThr/pSer-Pro bonds, are thought to lead to several functional consequences. In a first mode of action, exemplified by its competition with the CKS protein, the interaction with PIN1 prevents interaction with other regulatory proteins, like ubiquitin-ligases that lead to degradation pathways. In a second mode of action, the idea is largely accepted that the local isomerization modifies the global conformation of the protein substrate and hence its intrinsic activity, although this has never been directly demonstrated. Finally, isomerization catalysis is thought to regulate the (de)phosphorylation of specific pThr/pSer-Pro motifs, exemplified by the stimulation of the dephosphorylation of pThr231 of Tau by the PP2A phosphatase.

Rotor synchronization of radiofrequency and gradient pulses in high-resolution magic angle spinning NMR.

We have investigated the extent to which rotor synchronization of radiofrequency pulses leads to spectral improvement in high-resolution magic angle spinning NMR experiments. Several pulse sequences were tested, and the effect was found to be maximal in homonuclear TOCSY spectra. The physicochemical nature of the sample plays a role in the phenomenon, as rotor synchronization allows the refocusing of residual anisotropic interactions. However, even in a liquid sample the effects were visible. Radial inhomogeneities of the radiofrequency field were identified as an important source of the problem.

In vivo detection of the cyclic osmoregulated periplasmic glucan of Ralstonia solanacearum by high-resolution magic angle spinning NMR.

We investigate the mobility of the osmoregulated periplasmic glucans of Ralstonia solanacearum in the bacterial periplasm through the use of high-resolution (HR) NMR spectroscopy under static and magic angle spinning (MAS) conditions. Because the nature of periplasm is far from an isotropic aqueous solution, the molecules could be freely diffusing or rather associated to a periplasmic protein, a membrane protein, a lipid, or the peptidoglycan. HR MAS NMR spectroscopy leads to more reproducible results and allows the in vivo detection and characterization of the complex molecule.

High resolution magic angle spinning NMR in combinatorial chemistry.

Solid phase organic chemistry coupled with combinatorial methods promises to increase dramatically the diversity and number of small molecules available for medical and biological applications. However, optimizing the reaction conditions can be a time consuming step, especially since analytical tools to monitor reaction progress and detect impurities for solid phase chemistry are less developed than for solution chemistry. The use of high resolution magic angle spinning (HRMAS) NMR is described here as such an analytical tool. Whereas initial applications of molecular identification using deuterated organic solvents to swell the resins presented a significant gain in time over the cleave-and-analysis methods, the introduction of a differential diffusion filter has made immediate recording of spectra possible without any sample treatment. The applications of HRMAS NMR to different solid supports that are used in combinatorial chemistry will be described in terms of rapidity, robustness and sensitivity.

1H NMR study on the binding of Pin1 Trp-Trp domain with phosphothreonine peptides.

The recent crystal structure of Pin1 protein bound to a doubly phosphorylated peptide from the C-terminal domain of RNA polymerase II revealed that binding interactions between Pin1 and its substrate take place through its Trp-Trp (WW) domain at the level of the loop Ser(11)-Arg(12) and the aromatic pair Tyr(18)-Trp(29), and showed a trans conformation for both pSer-Pro peptide bonds. However, the orientation of the ligand in the aromatic recognition groove still could be sequence-specific, as previously observed in SH3 domains complexed by peptide ligands or for different class of WW domains (Zarrinpar, A., and Lim, W. A. (2000) Nat. Struct. Biol. 7, 611-613). Because the bound peptide conformation could also differ as observed for peptide ligands bound to the 14-3-3 domain, ligand orientation and conformation for two other biologically relevant monophosphate substrates, one derived from the Cdc25 phosphatase of Xenopus laevis (EQPLpTPVTDL) and another from the human tau protein (KVSVVRpTPPKSPS) in complex with the WW domain are here studied by solution NMR methods. First, the proton resonance perturbations on the WW domain upon complexation with both peptide ligands were determined to be essentially located in the positively charged beta-hairpin Ser(11)-Gly(15) and around the aromatic Trp(29). Dissociation equilibrium constants of 117 and 230 microm for Cdc25 and tau peptides, respectively, were found. Several intermolecular nuclear Overhauser effects between WW domain and substrates were obtained from a ligand-saturated solution and were used to determine the structures of the complexes in solution. We found a similar N to C orientation as the one observed in the crystal complex structure of Pin1 and a trans conformation for the pThr-Pro peptidic bond in both peptide ligands, thereby indicating a unique binding scheme for the Pin1 WW domain to its multiple substrates.

p13(SUC1) and the WW domain of PIN1 bind to the same phosphothreonine-proline epitope.

The WW domain of the human PIN1 and p13(SUC1), a subunit of the cyclin-dependent kinase complex, were previously shown to be involved in the regulation of the cyclin-dependent kinase complex activity at the entry into mitosis, by an unresolved molecular mechanism. We report here experimental evidence for the direct interaction of p13(SUC1) with a model CDC25 peptide, dependent on the phosphorylation state of its threonine. Chemical shift perturbation of backbone (1)H(N), (15)N, and (13)Calpha resonances during NMR titration experiments allows accurate identification of the binding site, primarily localized around the anion-binding site, occupied in the crystal structure of the homologous p9(CKSHs2) by a sulfate molecule. The epitope recognized by p13(SUC1) includes the proline at position +1 of the phosphothreonine, as was shown by the decrease in affinity for a mutated CDC25 phosphopeptide, containing an alanine/proline substitution. No direct interaction between the PIN1 WW domain or its catalytic proline cis/trans-isomerase domain and p13(SUC1) was detected, but our study showed that in vitro the WW domain of the human PIN1 antagonizes the binding of the p13(SUC1) to the CDC25 phosphopeptide, by binding to the same phosphoepitope. We thus propose that the full cyclin-dependent kinase complex stimulates the phosphorylation of CDC25 through binding of its p13(SUC1) module to the phosphoepitope of the substrate and that the reported WW antagonism of p13(SUC1)-stimulated CDC25 phosphorylation is caused by competitive binding of both protein modules to the same phosphoepitope.

Capsular polysaccharide produced by the thermophilic cyanobacterium Mastigocladus laminosus. Structural study of an undecasaccharide obtained by lithium degradation.

The capsular polysaccharide produced by the thermophilic cyanobacterium Mastigocladus laminosus has been subjected to a specific degradation with lithium in ethylenediamine. The released undecasaccharide attached to one unit of tetrahydroxycyclopentanecarboxylic acid has been characterized by a combination of 2D nuclear magnetic resonance spectroscopy, mass spectrometry, monosaccharidic composition and linkage analyses. From the overlap of the structure of this oligosaccharide with previously identified di-, tri- and pentasaccharides released by mild acid hydrolysis, the capsular polysaccharide was deduced to have a pentadecasaccharide repeating unit with the following structure:

Characterization of a multi-lipopeptides mixture used as an HIV-1 vaccine candidate.

A multi-component vaccine has been defined, which contains six different synthetic 24- to 32-amino acid lipopeptides derived from the sequence of HIV-1 proteins. The physicochemical properties of the lipopeptide components were compatible with multi-dimensional analysis, using RP-HPLC, Edman sequencing, electrospray mass spectrometry, and 2D-NMR. Detailed analysis of the impurity profiles led to the detection and evaluation of the relative proportions of most by-products: several contaminants resulted from the formation of acetylated fragments, transpeptidation reactions with succinimide or piperidide formation, or methionine and/or tryptophan mono-oxidations. The first batch to be produced underwent extensive pharmacotoxicological testings to confirm its safety; this vaccine candidate has now been used in phase I clinical trials. Despite the complexity of such multi-lipopeptide vaccines, our findings suggest the possibility of preparing a clear and precise assignment of by-products to toxicologically qualified impurities in the eventuality of a future production of several successive batches.

Glycerol's influence on the oxidized insulin B-chain conformation in relation to the selectivity variation of subtilisin: an nuclear magnetic resonance and simulated annealing study.

Glycerol, employed to mimic biological media with restricted water activity, has been shown to modify the activity of subtilisin BPN', an endopeptidase, towards the oxidized insulin B-chain, a well-studied substrate (FEBS Lett., 279 (1991) 123-131). Without minimizing the role of the microenvironment on the enzyme, we have studied the effect of glycerol addition on the structure of the enzyme substrate by homonuclear NMR spectroscopy and simulated annealing. Our results show that, in water, the oxidized insulin B-chain tertiary structure loses its central helix (residues B9-B19) and presents a folded structure with a flexible turn (residues B18-B24) in the beta-turn region of the insulin B-chain; whereas, in glycerol, the peptide is more rigid and is not folded. Moreover, in our experimental conditions, glycerol favors beta-strand secondary structure formation. Following these results, hypotheses about the differences observed in enzymatic activity on this substrate in glycerol have been postulated.

Identification of a sequence-dependent reversible acylation of tosylarginine in a peptidyl-resin reacted with isonicotinyl p-nitrophenylcarbonate.

In this paper, we report on the identification of an unexpected acylation that occurred on the solid phase when a peptide containing an unprotected lysyl and a tosyl-protected arginyl residue was treated with a large excess of isonicotinyl p-nitrophenylcarbonate. NMR and matrix-assisted laser desorption/ionisation -post-source decay analysis of the purified peptide demonstrated the presence of one extra isonicotinyloxycarbonyl (iNoc) group located on the omega nitrogen atom of the arginine which was adjacent to the Lys(iNoc). The desired peptide was obtained by quantitative removal of the unwanted iNoc group during a brief treatment with diluted aqueous hydrazine.

Structural characterization of the exocellular polysaccharides produced by Streptococcus thermophilus SFi39 and SFi12.

We investigated the structures of the exopolysaccharides (EPSs) produced by Streptococcus thermophilus SFi39 and SFi12. Both polymers were found to have molecular masses of greater than 2 x 10(6) Da. The SFi39 EPS consisted of D-glucose and D-galactose in a molar ratio of 1:1, whereas the SFi12 EPS was composed of D-galactose, L-rhamnose, and D-glucose in a molar ratio of 3:2:1. Methylation analysis of and nuclear magnetic resonance spectra recorded from the native polysaccharide, as well as oligosaccharides released by partial acid hydrolysis, allowed the complete structural determination of the SFi39 EPS, which consists of the following tetrasaccharide repeating unit: [formula: see text] Similar spectra recorded only from the native polysaccharide were sufficient to allow the structural determination of the SFi12 EPS, which consists of the following hexasaccharide repeating unit: [formula: see text] This study shows that the texturizing properties of different S. thermophilus ropy strains are based on the production of EPSs exhibiting chemical similarities but structural differences.

Characterization of a new family of toxin-like peptides from the venom of the scorpion Leiurus quinquestriatus hebraeus. 1H-NMR structure of leiuropeptide II.

To extend our knowledge about the structural features of short scorpion toxins, the ion-exchange fractions obtained from Leiurus quinquestriatus hebraeus venom were investigated by plasma desorption mass spectrometry in order to select low molecular mass polypeptides. Three toxin-like peptides with molecular mass close to 3 kDa, named leiuropeptides I, II and III, were purified and found devoid of any significant toxicity against mammals and insects. Their amino acid sequences revealed a cysteine pattern analogous to that of short-chain scorpion toxins. The solution structure of leiuropeptide II was determined by 2D 1H-NMR spectroscopy and indicated the presence of a helix accommodating a proline, connected to a two-standard beta-sheet by three disulfide bonds. The overall fold of leiuropeptide II is found to be similar to that of leiurotoxin I, a 31-residue toxin present in the same scorpion venom which acts on K+ channels. In order to rationalize the absence of toxicity, the electrostatic potential of leiuropeptide II was compared to that of leiurotoxin I. The peptide is characterized by a large negative zone around Glu4, Asp5 and Asp8 residues, beginning in the neighbourhood of the beta-turn and extending along the helix. In the same area, leiurotoxin I exhibits a positive surface, around Arg6 and Arg13 basic residues, which are essential for its receptor affinity.

Sulfated lewis X determinants as a major structural motif in glycans from LS174T-HM7 human colon carcinoma mucin.

This article describes oligosaccharide structures of mucin isolated from nude mouse xenograft tumors produced by LS174T-HM7 cells, a subline of the human colon carcinoma LS174T with higher metastatic tendency and higher mucin production. A striking feature of the oligosaccharides of the LS174T-HM7 xenograft tumor mucin was a predominance of sulfated Lewis X determinants: HSO3-Galbeta1-4(Fucalpha1-3)GlcNAc. In addition to one previously known saccharide with one sulfated Lewis X determinant, the HM7 xenograft tumor mucin contained multiple novel structures containing one, two, or three sulfated Lewis X determinants. This determinant, known to act as a selectin ligand, has been found previously in minor saccharide components of human milk as well as mucins, but never before as a predominant structure in one mucin source.

Diagnosis of a case of acute chloroquine poisoning using 1H NMR spectroscopy: characterisation of drug metabolites in urine.

Analysis of biological fluids by proton nuclear magnetic resonance spectroscopy (1H NMR) is a promising tool in clinical biology. We have used this method for a rapid toxicological screening in the case of a suicide attempt. A urine sample was analysed at 300 and 600 MHz by 1D and 2D sequences (J-resolved and TOCSY) in a short experimental time. Quantification was realized by peak integration of the 1D spectrum. The results showed the presence of chloroquine and its major metabolite monodesethylchloroquine at concentrations of 462 and 140 mg/L, respectively. Ethanol was also detected in the spectrum. It can be concluded that 1H NMR provides many advantages as a tool for clinical diagnosis in a case of acute intoxication.

Measurement of three-bond coupling constants in the osmoregulated periplasmic glucan of Burkholderia solanacearum.

The cyclic osmoregulated periplasmic glucan produced by Burkholderia solanacearum contains 13 glucose units, all beta-(1-2) linked except for one alpha-(1-6) linkage. We report here the measurement of the 3J(C1-H2') and 3J(H1-C2') coupling constants, characterizing the glycosidic linkages, through the use of a 13C/12C double half-filtered NOESY experiments. The values obtained give information about the (phi, psi) angles of the different linkages. The results presented from an important step towards a detailed experimental model of the cyclic glucan, which might allow us to clarify its biological role and establish whether the cavity of these molecules is compatible with the capability of complexing host molecular signals.