PubMed Health⌕ Search

Biomedical subjects

J F Lefèvre

Publications and source records attributed to J F Lefèvre.

At least 19 recordsLinked to original sources
PubMed1999

Solution structure of a conformationally constrained Arg-Gly-Asp-like motif inserted into the alpha/beta scaffold of leiurotoxin I.

A monoclonal antibody, AC7, directed against the RGD-binding site of the GPIIIa subunit of the platelet fibrinogen receptor, interacts with activated platelet. The H3 region (H3, RQMIRGYFDV sequence) of the complementarity-determining region 3 heavy chain of AC7 inhibits platelet aggregation and fibrinogen binding to platelet. H3 contains the arginine, glycine and aspartate residues, but in an unusual order. The solution structure of the decapeptide has been studied by proton NMR. The NMR data suggested a helical equilibrium. To test whether the helical structure of H3 was biologically relevant, a conformationally constrained peptide with the RGD-like motif was designed. The sequence of a scorpion toxin (leiurotoxin I) has been modified in order to constrain the H3 sequence in a rigid helical conformation. The structure of leiurotoxin I consists of a beta-sheet and an alpha-helix, linked by three disulfide bridges. The structural feature of the chimeric peptide (H3-leiurotoxin) has been determined by standard two-dimensional NMR techniques. H3-Leiurotoxin structure closely resembles that of leiurotoxin I.

Amino Acid Sequence↗
PubMed1998

Solution conformation of the synthetic bovine proenkephalin-A209-237 by 1H NMR spectroscopy.

Proenkephalin-A has been described to generate enkephalins, opoid peptides, and several derived peptides, which display various biological effects, including antinociception and immunological enhancement. Recently, we have isolated from bovine chromaffin granules a new antibacterial peptide, named enkelytin, which corresponds to the bisphosphorylated form of PEAP209-237 (Goumon, Y., Strub, J. M., Moniatte, M., Nullans, G., Poteur, L., Hubert, P., Van Dorsselaer, A., Aunis, D., and Metz-Boutigue, M. H. (1996) Eur. J. Biochem. 235, 516-525). In this paper, the three-dimensional solution structure of synthetic PEAP209-237 was investigated by NMR. These studies indicate that this peptide, which is unstructured in water, folds into an alpha-helical structure in trifluoroethanol/water (1/1). NMR data revealed two possible three-dimensional models of PEAP209-237. In both models, the proline residue Pro-227 induces a 90 degrees hinge between two alpha-helical segments (Ser-215 to Ser-221 and Glu-228 to Arg-232) leading to an overall L-shaped structure for the molecule. The negative charge of PEAP209-237 and the low amphipathy of the two alpha-helical segments imply new mechanisms to explain the antibacterial activity of enkelytin.

Amino Acid Sequence↗
PubMed1998

The assembly of immunoglobulin-like modules in titin: implications for muscle elasticity.

Titin, a giant muscle protein, forms filaments that span half of the sarcomere and cover, along their length, quite diversified functions. The region of titin located in the sarcomere I-band is believed to play a major rôle in extensibility and passive elasticity of muscle. In the I-band, the titin sequence contains tandem immunoglobulin-like (Ig) modules intercalated by a potentially non-globular region. By a combined approach making use of small angle X-ray scattering and nuclear magnetic resonance techniques, we have addressed the questions of what are the average mutual orientation of poly-Igs and the degree of flexibility around the domain interfaces. Various recombinant fragments containing one, two and four titin I-band tandem domains were analysed. The small-angle scattering data provide a picture of the domains in a mostly extended configuration with their long axes aligned head-to-tail. There is a small degree of bending and twisting of the modules with respect to each other that results in an overall shortening in their maximum linear dimension compared with that expected for the fully extended, linear configurations. This shortening is greatest for the four module construct ( approximately 15%). 15N NMR relaxation studies of one and two-domain constructs show that the motions around the interdomain connecting regions are restricted, suggesting that titin behaves as a row of beads connected by rigid hinges. The length of the residues in the interface seems to be the major determinant of the degree of flexibility. Possible implications of our results for the structure and function of titin in muscles are discussed.

Connectin↗
PubMed1998

Bacterial iron transport: 1H NMR determination of the three-dimensional structure of the gallium complex of pyoverdin G4R, the peptidic siderophore of Pseudomonas putida G4R.

Among the fluorescent Pseudomonas species, Pseudomonas putida is a rare case of a nitrogen-fixing bacterium that transforms nitrogen into ammonia. When grown under iron-deficient conditions, it produces two major pyoverdins: pyoverdin G4R and pyoverdin G4RA. Their primary structures have been established using FAB-MS and one- and two-dimensional 15N, 13C, and 1H NMR on both the unlabeled and 15N-labeled compounds [Salah El Din, A. L. M., et al. (1997) Tetrahedron 53, 12539-12552]. The two pyoverdins have a common chromophore derived from 2,3-diamino-6,7-dihydroxyquinoline. The chromophore is bound to the linear heptapeptide L-Asp-L-Orn-D-beta-threo-OHAsp-L-Dab-Gly-L-Ser-L-cyclo-OHOrn . Circular dichroism spectra suggest that the absolute configuration of the metal complex is Delta. The three-dimensional structure in solution of pyoverdin G4R-Ga(III) was determined after interpretation of two-dimensional 1H NMR spectra recorded at 283 and 303 K. The complex is tightly defined with a compact structure with a Delta absolute configuration. The site of complexation of the metal ion is found to be located on the surface of the molecule, showing that the ion can be released without large conformational changes, while the polar groups of the peptide chain, which may be responsible for the recognition of the receptor, are placed on the opposite side of the overall shape. The three-dimensional structure of pyoverdin G4R-Ga(III) is compared with those of other pyoverdins, and the role of the structure in iron uptake is discussed.

Bacterial Proteins↗
PubMed1998

Characterization of antibacterial COOH-terminal proenkephalin-A-derived peptides (PEAP) in infectious fluids. Importance of enkelytin, the antibacterial PEAP209-237 secreted by stimulated chromaffin cells.

Proenkephalin-A (PEA) and its derived peptides (PEAP) have been described in neural, neuroendocrine tissues and immune cells. The processing of PEA has been extensively studied in the adrenal medulla chromaffin cell showing that maturation starts with the removal of the carboxyl-terminal PEAP209-239. In 1995, our laboratory has shown that antibacterial activity is present within the intragranular chromaffin granule matrix and in the extracellular medium following exocytosis. More recently, we have identified an intragranular peptide, named enkelytin, corresponding to the bisphosphorylated PEAP209-237, that inhibits the growth of Micrococcus luteus (Goumon, Y., Strub, J. M., Moniatte, M., Nullans, G., Poteur, L., Hubert, P., Van Dorsselaer, A., Aunis, D., and Metz-Boutigue, M. H. (1996) Eur. J. Biochem. 235, 516-525). As a continuation of this previous study, in order to characterize the biological function of antibacterial PEAP, we have here examined whether this COOH-terminal fragment is released from stimulated chromaffin cells and whether it could be detected in wound fluids and in polymorphonuclear secretions following cell stimulation. The antibacterial spectrum shows that enkelytin is active against several Gram-positive bacteria including Staphylococcus aureus, but it is unable to inhibit the Gram-negative bacteria growth. In order to relate the antibacterial activity of enkelytin with structural features, various synthetic enkelytin-derived peptides were tested. We also propose a computer model of synthetic PEAP209-237 deduced from 1H NMR analysis, in order to relate the antibacterial activity of enkelytin with the three-dimensional structure. Finally, we report the high phylogenetic conservation of the COOH-terminal PEAP, which implies some important biological function and we discuss the putative importance of enkelytin in the defensive processes.

Adrenal Glands↗
PubMed1998

Alpha-helix mimicry of a beta-turn.

It is shown here that the three-dimensional arrangement of the amino acids in an RGDF beta-turn (sequence involved in cell adhesion) resembles that of an alpha-helix with a shuffled RGDF sequence (i.e. RGXFD). A miniprotein was designed and constructed which arranges the RGXFD sequence into a well defined helical conformation. The designed protein is bioactive and folds into the desired structure as assessed by nuclear magnetic resonance spectroscopy. The recognition process mediated by a beta-turn can thus be mimicked by an alpha-helix.

Amino Acid Sequence↗
PubMed1996

Solution structure of PMP-C: a new fold in the group of small serine proteinase inhibitors.

The solution structure and the disulfide pairings of a 36-residue proteinase inhibitor isolated from the insect Locusta migratoria have been determined using NMR spectroscopy and simulated annealing calculations. The peptide, termed PMP-C, was previously shown to inhibit bovine alpha-chymotrypsin as well as human leukocyte elastase, and was also found to block high-voltage-activated Ca2+ currents in rat sensory neurones. PMP-C has a prolate ellipsoid shape and adopts a tertiary fold hitherto unobserved in the large group of small "canonical" proteinase inhibitors. The over-all fold consists mainly of three strands arranged in a right-handed twisted, antiparallel, beta-sheet that demarcates a cavity, together with a linear amino-terminal segment oriented almost perpendicular to the three strands of the beta-sheet. Inside the cavity a phenyl ring constitutes the centre of a hydrophobic core. The proteinase binding loop is located in the carboxy-terminal part of the molecule, between two cysteine residues involved in disulfide bridges. Its conformation resembles that found in other small canonical proteinase inhibitors. A comparison of PMP-C structure with the recently published solution structure of the related peptide PMP-D2 shows that the most significant differences are complementary changes involved in the stabilization of similar folds. This comparison led us to review the structure of PMP-D2 and to identify two salt bridges in PMP-D2.

Amino Acid Sequence↗
PubMed1996

NMR data show that the carcinogen N-2-acetylaminofluorene stabilises an intermediate of -2 frameshift mutagenesis in a region of high mutation frequency.

The heteroduplex, D(ACCGGCGCCACA) . d(TGTGG-CCGGT), containing two bulged bases, a cytosine and the guanine G7, either unmodified or modified with the carcinogen N-2-acetylaminofluorene, have been studied by NMR as models of slipped-mutagenic intermediates (SMI). The melting temperature of the modified heteroduplex is strongly increased compared with that of the unmodified heteroduplex. NMR studies have shown that all the bases of the unmodified heteroduplex are stacked within the helix, without any disruption of the sequential connectivities. The two strands are in a B-like conformation. Nevertheless, exchangeable-proton studies have revealed that base pairing is very weak, or even lacking, over two base pairs apart from the bulge. Concerning the modified heteroduplex, no B-like connectivity is observed in the G5-C9 segment. Moreover, the cytosine C8 is rejected outside the helix, whereas the N-2-acetylaminofluorene moiety is inserted within the helix. The G5.C18, C6.G17 and C9.G16 bases are remarkably stable when the temperature is increased, in agreement with the high melting temperature. Some small unassigned peaks reveal the presence of the minor conformation in equilibrium. The strong stabilisation of the N-2-acetylaminofluorene-modified heteroduplex compared with the unmodified duplex is in agreement with the high N-2-acetylaminofluorene-induced mutation frequency compared with the spontaneous frequency and with the hypothesis of mutagenesis occurring during replication.

2-Acetylaminofluorene↗
PubMed1996

Stabilization of proteins by glycosylation examined by NMR analysis of a fucosylated proteinase inhibitor.

Here we investigate the effects of the naturally occurring threonine-linked L-fucose moiety on the structure, dynamics and stability of the proteinase inhibitor PMP-C (Pars intercerebralis major peptide C). The three-dimensional structure of PMP-C fucosylated on Thr 9 has been determined by NMR spectroscopy and simulated annealing. The fucose ring is very well ordered, held in place by hydrophobic and hydrogen bond interactions with Thr 16 and Arg 18. Comparing the NMR data and the structure of the fucosylated inhibitor with those of the nonfucosylated form shows that conformational changes only occur in the vicinity of the fucose moiety. Nevertheless, a comparative analysis of the exchange rates of amide protons indicates that fucosylation is responsible for an overall decrease of the dynamic fluctuations of the molecule. This correlates well with an increase in stability of approximately 1 kcal mol-1 as monitored by thermal denaturation.

Cyclotides↗
PubMed1996

Theory and practice of nuclear spin relaxation in proteins.

NMR relaxation experiments can provide information on overall and internal motions in proteins. This review consists of a concise report on the evolution of the theories for nuclear relaxation followed by an overview of mathematical models for internal motions in proteins. Next, the method of spectral density mapping with recent developments is reviewed. This is followed by a discussion of pulse sequences for relaxation experiments. Finally, we review recent studies correlating relaxation parameters, in particular spectral density functions, with structural features of proteins and with results of molecular dynamics simulations.

Enzymes↗
PubMed1994

Solution structure of PMP-D2, a 35-residue peptide isolated from the insect Locusta migratoria.

The three-dimensional solution structure of PMP-D2, a 35 amino acid peptide isolated from the insect Locusta migratoria, has been determined from two-dimensional 1H NMR spectroscopy data. The structure calculations were performed from 222 NOE-derived interproton distances and 11 dihedral angles calculated from the JHN-H alpha coupling constants, using either a combination of distance geometry and restrained simulated annealing or by restrained simulated annealing alone. PMP-D2 contains three disulfide bridges that have been assigned from NMR data and structure calculations and independently confirmed using chemical and enzymatic methods. The core region of PMP-D2 adopts a compact globular fold, stabilized by hydrophobic interactions, which consists of a short three-stranded antiparallel beta-sheet involving residues 8-11, 15-19, and 25-29. Back-calculation of the NOESY spectra was used to validate the final structures. Analysis of the CD spectra of PMP-D2 under various conditions of ionic strength and in the presence of organic solvents demonstrates the high stability of this molecule. PMP-D2 was recently shown to inhibit Ca2+ currents. This activity is discussed based on the comparison of PMP-D2 three-dimensional structure with the recently established three-dimensional structure of the Ca2+ channel blocker omega-conotoxin GVIA.

Amino Acid Sequence↗
PubMed1994

NMR evidence of the stabilisation by the carcinogen N-2-acetylaminofluorene of a frameshift mutagenesis intermediate.

Two heteroduplexes d(C1A2C3T4C5G6C7A8C9A10C11)-d (G12T13G14T15G16G17A18G19T20G21) containing a bulged guanine either unmodified or modified with the carcinogen N-2-acetylaminofluorene (AAF) have been studied by nuclear magnetic resonance (NMR) as models of slipped mutagenic intermediates (SMI). Conformational equilibria are observed in both the unmodified and the AAF-modified heteroduplexes. The major conformation of the unmodified duplex is one where the extra guanine is stacked in the helix and the major conformation of the AAF-modified heteroduplex is one where the AAF is external to the helix. Unusual sugar proton chemical shifts of C5- and G6-AAF indicate that the AAF ring is pointing out in the 5' direction. A strong increase in the modified heteroduplex melting temperature (+15 degrees C) is observed. Moreover, in contrast to the unmodified heteroduplex, which shows extensive melting in the vicinity of the bulged guanine, the base pairs around the bulge in the AAF-modified heteroduplex remain paired at temperatures up to 30 degrees C. This exceptional stability of the site around the bulged modified guanine is suggested to be responsible for the high rate of -1 mutation induced by AAF at repetitive sequences.

2-Acetylaminofluorene↗
PubMed1994

Solution structure of pyoverdin GM-II.

The three-dimensional structure in solution of ferri-pyoverdin GM-II isolated from the culture medium of Pseudomonas fluorescens was determined by application of NMR methods to the Ga3+ analogue. Distance geometry calculations were performed with FILMAN using interproton distances and coupling constants as constraints. Further conformational analysis was carried out by energy minimization with MM2 and AMBER. Back-calculation of the NOESY spectra shows that the resulting structures are in agreement with the experimental data.

Amino Acid Sequence↗
PubMed1994

Protein dynamics.

Modern NMR has revitalized the study of protein dynamics. Multidimensional spectra and the heteronuclear spectroscopy allow a substantial gain in resolution. Dynamics can be analyzed at individual sites and data on segmental and sequence-dependent flexibility are accumulating. This review summarizes the wide variety of NMR approaches for observing internal motions, including the folding processes, and the attempts to correlate dynamics to the biological activity of proteins. The implications of mobility on structure determination by NMR is also discussed.

Magnetic Resonance Spectroscopy↗
PubMed1994

Nuclear magnetic resonance measurements of slow conformational dynamics in macromolecules.

The combined use of rotating-frame relaxation methods, temperature-dependent measurements of line shapes and magnetization transfer experiments allows in favorable cases the examination in some detail of exchange processes that occur on the millisecond time scale. It is possible to determine not only the rate constants, but also the activation parameters and chemical shifts even for events that are in fast exchange on the chemical shift time scale. Such measurements complement the information obtainable from heteronuclear relaxation methods that probe mainly the fast librational motions in macromolecules and may provide information important for functional studies of biological macromolecules.

Amino Acid Sequence↗
PubMed1994

Structural studies of two antiaggregant RGDW peptides by 1H and 13C NMR.

The structural features of Arg-Gly-Asp-related sequences have been investigated by 1H and 13C NMR. Two linear peptides which inhibit platelet aggregation with a high efficiency have been studied: D-Arg-Gly-Asp-Trp and L-Arg-Gly-Asp-Trp. Analysis of pH titration effects, amide proton exchange rates and inter-proton distances obtained from ROESY spectra suggest that these small fragments predominantly adopt a type II' beta-turn structure in solution. Folding features of a non-active cyclic peptide based on the same sequence (cyclo-[Arg-Gly-Asp-Trp]2) have also been investigated. The biological relevance of these structures is discussed.

Amino Acid Sequence↗
PubMed1993

Structural and dynamic studies of two antigenic loops from haemagglutinin: a relaxation matrix approach.

We have investigated the dynamics and structural behaviour of two antigenic peptides using 1H NMR. The two cyclic peptides mimic the antigenic site A of influenza haemagglutinin protein; they only differ in the way they were cyclized and in the size of their respective linkers. Homonuclear relaxation parameters extracted from a complete NOE matrix were interpreted in terms of local dynamics. A set of distance constraints was deduced from these parameters which allowed 3D models to be constructed using distance geometry. NOE back-calculation was used to check the validity of the final models. Strong variations of internal motion amplitude have been found in both peptides along their backbone. Motions with high amplitudes have been localized in the Gly-Pro-Gly sequence which forms a beta-turn in both structures.

Amino Acid Sequence↗