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G Lippens

Publications and source records attributed to G Lippens.

31 records · Page 2Linked to original sources
PubMed1997

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.

Amino Acid Sequence↗
PubMed1996

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.

Burkholderia↗
PubMed1996

Cell-associated glucans of Burkholderia solanacearum and Xanthomonas campestris pv. citri: a new family of periplasmic glucans.

The cell-associated glucans produced by Burkholderia solanacearum and Xanthomonas campestris pv. citri were isolated by trichloroacetic acid treatment and gel permeation chromatography. The compounds obtained were characterized by compositional analysis, matrix-assisted laser desorption ionization mass spectrometry, and high-performance anion-exchange chromatography. B. solanacearum synthesizes only a neutral cyclic glucan containing 13 glucose residues, and X. campestris pv. citri synthesizes a neutral cyclic glucan containing 16 glucose residues. The two glucans were further purified by high-performance anion-exchange chromatography. Methylation analysis revealed that these glucans are linked by 1,2-glycosidic bonds and one 1,6-glycosidic bond. Our 600-MHz homonuclear and 1H-13C heteronuclear nuclear magnetic resonance experiments revealed the presence of a single alpha-1,6-glycosidic linkage, whereas all other glucose residues are beta-1,2 linked. The presence of this single alpha-1,6 linkage, however, induces such structural constraints in these cyclic glucans that all individual glucose residues could be distinguished. The different anomeric proton signals allowed complete sequence-specific assignment of both glucans. The structural characteristics of these glucans contrast with those of the previously described osmoregulated periplasmic glucans.

Burkholderia↗
PubMed1995

Synthesis, three-dimensional structure, and specific 15N-labelling of the streptococcal protein G B1-domain.

The 55-amino-acid B1-domain of the streptococcal protein G shows a high binding affinity to IgG isolated from a wide range of mammalian species. Since the B1-domain forms an extremely stable globular folding unit containing the major secondary structure elements and is devoid of proline residues and disulfide bridges, it is also a useful tool for protein folding and stability studies. Its small size makes this protein an ideal candidate for production by chemical synthesis, allowing incorporation of non-natural amino acids with the possibility of assessing the influence of such residues on both the functional and structural characteristics of proteins. In this study, we employed three successive chemical syntheses of the B1-domain in order to define the optimal conditions of coupling and protection. The stepwise solid-phase methodology using the tertbutyloxycarbonyl/benzyl strategy was used for this purpose. First, the sequence assembly difficulties were evaluated. After analyzing of the problems found during assembly, a second optimized synthesis was performed leading to formation of a synthetic B1-domain with a higher yield; the synthetic B1-domain was completely functional in its binding properties to IgG. Three orthogonal purification steps (gel-permeation, reverse-phase and ion-exchange HPLC) were required to obtain a sample suitable for structural analysis by high-resolution NMR. This study led to the conclusion that the synthetic B1-domain adopts a three-dimensional structure identical to that of the molecule obtained by recombinant techniques [Gronenborn, A.M., Filpula, D. R., Essig, N. Z., Achari, A., Whitlow, M., Wingfield, P. T. & Clore, G. M. (1991) Science 253, 657-661]. To demonstrate the usefulness of the chemical approach for the specific introduction of labelled amino acids in the primary structure, fourteen alpha-15N-labelled amino acids were incorporated at selected critical positions during the third synthesis. This analog is the first in a series of molecules planned to study in detail the folding dynamics of the B1-domain.

Amino Acid Sequence↗
PubMed1995

Native like structure and stability of apo AI in a n-propanol/water solution as determined by 13C NMR.

To elucidate the molecular details of the conformation of apolipoprotein AI (apo AI), we have developed an approach related to the solubilization of this protein in 30% n-propanol. We have previously reported the promotion of a native-like structure for apo AI solubilized in n-propanol, as depicted by circular dichroism, fluorescence, and limited proteolytic digestion as compared to the lipid associated form of apo AI. In the present study, we labeled the Lys residues of apo AI with 13C by reductive methylation and used 13C NMR to confirm the formation of a native-like structure of apo AI in this environment. Furthermore, by the above criteria (circular dichroism and 13C NMR) and by using urea and temperature as denaturing agents, we show that the denaturation of the native-like structure of apo AI in n-propanol is a biphasic process. These studies show that in 30% n-propanol, apo AI contains two independently folded structural domains, of markedly different stabilities that might correspond to the amino-terminal and the carboxy-terminal halves of the molecule.

1-Propanol↗
PubMed1995

NMR sequential assignments and solution structure of chlorotoxin, a small scorpion toxin that blocks chloride channels.

The solution structure of chlorotoxin, a small toxin purified from the venom of the Leiurus quinquestriatus scorpion, has been determined using 2D 1H NMR spectroscopy. Analysis of the NMR data shows that the structure consists of a small three-stranded antiparallel beta-sheet packed against an alpha-helix, thereby adopting the same fold as charybdotoxin and other members of the short scorpion toxin family [Arseniev et al. (1984) FEBS Lett. 165, 57-62; Martins et al. (1990) FEBS Lett. 260, 249-253; Bontems et al. (1991) Science 254, 1521-1523]. Three disulfide bonds of chlorotoxin (Cys5-Cys28, Cys16-Cys33, and Cys20-Cys35), cross-linking the alpha-helix to the beta-sheet, follow the common pattern found in the other short scorpion toxins. The fourth disulfide bridge (Cys2-Cys19) links the small N-terminal beta strand to the rest of the molecule, in contrast to charybdotoxin where this disulfide bridge is absent and the first strand interacts with the rest of the molecule by several contacts between hydrophobic residues. Another structural difference between chlorotoxin and charybdotoxin is observed at the level of the alpha-beta turn. This difference is accompanied by a change in the electrostatic potential surface, which is largely positive at the level of this turn in chlorotoxin, whereas no such positive potential surface can be found at the same position in charybdotoxin. In the latter protein, the positive surface is formed by different charged residues situated on the solvent-exposed site of the C-terminal beta-sheet.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Homo- and heteronuclear two-dimensional NMR studies of the globular domain of histone H1: full assignment, tertiary structure, and comparison with the globular domain of histone H5.

The globular domain of chicken histone H1 (GH1) has been studied by 1H homonuclear and 1H-15N heteronuclear 2D NMR spectroscopy. After the full assignment of the proton and 15N resonances, the tertiary structure of GH1 was determined by an iterative procedure using distance geometry and restrained simulated annealing. The secondary structure elements of GH1, three helices (S5-A16, S24-A34, N42-K56) followed by a beta-hairpin (L59-L73), are folded in a manner very similar to the corresponding parts of the globular domain of chicken histone H5 (GH5) [Clore et al. (1987) EMBO J. 6, 1833-1842; Ramakrishnan et al. (1993) Nature 362, 219-223]. However, subtle differences are detected between the two structures and between the electrostatic potentials surrounding the molecules. The most important differences are located in the loop between the second and third helices, a region that could be responsible for the different affinity for DNA. The most positively charged regions are not found in exactly the same position in GH1 and GH5. Nevertheless, their location seems to agree with the model where nucleosome binding takes place through contact points located at one DNA terminus and close to the dyad axis of the nucleosome [Schwabe & Travers (1993) Curr. Biol. 3, 628-630].

Amino Acid Sequence↗
PubMed1993

Homo- and heteronuclear two-dimensional NMR studies of the globular domain of histone H1: sequential assignment and secondary structure.

A recombinant 75 amino acid polypeptide corresponding to the globular domain of the chicken histone H1 (GH1) has been studied by 1H homonuclear and 1H-15N heteronuclear 2D NMR spectroscopy. Sequential assignment of the backbone and beta-proton resonances has enabled us to determine the secondary structure of GH1. It was found to consist of three helical regions (T7-S17, L25-Y37, E40-K56) and probably a beta-hairpin (L59-L73). This structure is similar to the structure of the globular domain of histone H5 (GH5) obtained both by NMR spectroscopy [Zarbock et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 7628-7632; Clore et al. (1987) EMBO J. 6, 1833-1842] and by X-ray crystallography [Ramakrishnan et al. (1993) Nature 362, 219-223]. The beta-hairpin as suggested for GH1 is also present in the X-ray structure of GH5 but has not been reported for the NMR structure of GH5.

Amino Acid Sequence↗
PubMed1993

Transfer nuclear Overhauser effect study of the conformation of oxytocin bound to bovine neurophysin I.

This study reports the structure of the peptide hormone oxytocin bound to its carrier protein, neurophysin I, obtained by nuclear magnetic resonance techniques. At the pH value of 2.1 in our experiments, the ligand is in fast exchange with its carrier protein, allowing the use of transfer-NOE methods. The number of distance constraints for the peptide being limited, considerable attention has been paid to an accurate distance determination. The resulting accurate distance limits were used as input for a distance geometry calculation followed by a restrained molecular dynamics run. Convergence to a well-defined family of structures for oxytocin in its bound state was reached. Both the backbone and the side-chain conformations differ between the bound form and the crystal structure of free oxytocin [Wood, S. P., et al. (1986) Science 232, 633]. These differences, as well as other structural features of the bound form, are discussed in terms of interactions made with the carrier protein. Transfer-NOE experiments at low peptide protein ratios provide direct experimental evidence for contacts between the oxytocin Tyr2 residue and an aromatic residue of neurophysin. The resonance assignments of the aromatic groups [Whittaker, B. A., et al. (1985) Biochemistry 24, 2782] together with the recently published X-ray structure of the neurophysin II protein complexed with a dipeptide [Chen et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4240] allow us to assign the aromatic signal on the protein to the neurophysin Phe22 residue.

Amino Acid Sequence↗
PubMed1992

Dual anomeric specificity and dual anomerase activity of phosphoglucoisomerase quantified by two-dimensional phase-sensitive 13C EXSY NMR.

The reversible conversion between D-glucose 6-phosphate and D-fructose 6-phosphate catalyzed by yeast phosphoglucoisomerase was studied by phase sensitive two-dimensional 13C-[1H] EXSY NMR spectroscopy at 150.869 and 125.759 MHz, using 13C-enriched substrates in the C2 position of the D-hexose 6-phosphates. The shape of the build-up curves of the cross-peaks associated with the 13C2 resonances of the alpha- and beta-anomers of both D-[2-13C]glucose 6-phosphate and D-[2-13C]fructose 6-phosphate reveals that phosphoglucoisomerase selectively catalyzes the reversible conversion between alpha-D-[2-13C]glucose 6-phosphate and beta-D-[2-13C]fructose 6-phosphate. Quantitative analysis of the build-up curves by three different methods allowed us to conclude that phosphoglucoisomerase not only selectively channels the latter isomerization but also considerably accelerates the anomerization of both D-hexose 6-phosphates. The rate constants of anomerization were indeed much higher in the presence than in the absence of enzyme. The major finding in the present study consists in the anomeric specificity of phosphoglucoisomerase toward the beta-anomer of D-fructose 6-phosphate both as a substrate and a product, contrary to previous proposals. This finding supports recent evidence suggesting the direct channelling of beta-D-fructose 6-phosphate from phosphoglucoisomerase to phosphofructokinase.

Catalysis↗
PubMedDate not supplied

Monitoring of solid-phase organic synthesis on macroscopic supports by high-resolution magic angle spinning NMR.

In this paper we demonstrate the efficiency of high-resolution magic angle spinning NMR to monitor solid-phase organic chemistry on macroscopic systems such as Synphase lanterns. The use of the LED sequence eliminates the peaks due to the use of protonated solvents and was also sufficient to decrease the signals due to the matrix. As a direct result, we established that reaction kinetics on the lantern proved to be significantly more rapid than on an equivalent polystyrene resin. More generally, the macroscopic nature of the support facilitates both sample preparation and spectral recording and hence opens up the perspective of an automated on-line analysis in combinatorial chemistry.

Journal Article↗