Biomedical subjects

M Marraud

Publications and source records attributed to M Marraud.

At least 19 recordsLinked to original sources

PubMed2001

Structure of epitopes recognized by the antibodies to alpha(181-192) peptides of neuronal nicotinic acetylcholine receptors: extrapolation to the structure of acetylcholine-binding domain.

Using the alpha(181-192) peptides of neuronal nicotinic acetylcholine receptor (nAChR) and Ala-substituted peptide analogues, amino acid residues critical for specific monoclonal antibody (mAb) binding were identified. By means of 2D nuclear magnetic resonance (2D-NMR) analysis followed by molecular modeling, it was found that mAb binding resulted in stabilization of the free alpha3(181-192) peptide flexible conformation yielding an extended structure with residues 6-11 of the peptide being in direct contact with the Ab. Since the Ab binds the native AChR as well, it is suggested that the corresponding fragment of AChR alpha3 subunit is exposed to solution and also appears in extended conformation.

Alanine↗

PubMed2001

Unexpected stability of the urea cis-trans isomer in urea-containing model pseudopeptides.

In contrast to the situation observed in the crystal state, the urea moiety in N-Boc-N'-carbamoyl-gem-diaminoalkyl derivatives (single-residue ureidopeptides) 1-4 exclusively assumes a cis-trans conformation in solution. When R(3) = H, the resulting structure can be further stabilized by an intramolecular hydrogen bond that closes an eight-membered pseudocycle. The root-mean-square deviation calculated for heavy atoms between a peptide gamma-turn and the folded conformation that we propose to call urea turn is 0.60 A. [structure: see text]

Isomerism↗

PubMed2001

Preparative HPLC resolution of the CIS cyclohexane analogs of phenylalanine.

The analytical resolution of different derivatives of cis c(6)Phe (cyclohexane analogs of phenylalanine) was tested by HPLC using the mixed 10-undecenoate/3,5-dimethylphenylcarbamate of amylose bonded on allylsilica gel as the chiral stationary phase. The same chiral support has allowed the enantioseparation of racemate cis-4 on a semipreparative column (150 x 20 mm ID) with a mixture of n-hexane/2-propanol/chloroform as the mobile phase. Some 200-300 mg of the optically pure enantiomers were isolated and transformed into the N-benzyloxycarbonyl amino acids. The X-ray diffraction structure of (1R;2R)-4 is reported.

Chromatography, High Pressure Liquid↗

PubMed2000

The third-dimensional structure of the complex between an Fv antibody fragment and an analogue of the main immunogenic region of the acetylcholine receptor: a combined two-dimensional NMR, homology, and molecular modeling approach.

Binding of autoantibodies to the acetylcholine receptor (AChR) plays a major role in the autoimmune disease Myasthenia gravis (MG). In this paper, we propose a structure model of a putative immunocomplex that gives rise to the reduction of functional AChR molecules during the course of MG. The model complex consists of the [G(70), Nle(76)] decapeptide analogue of the main immunogenic region (MIR), representing the major antigenic epitope of AChR, and the single chain Fv fragment of monoclonal antibody 198, a potent MG autoantibody. The structure of the complexed decapeptide antigen [G(70), Nle(76)]MIR was determined using two-dimensional nmr, whereas the antibody structure was derived by means of homology modeling. The final complex was constructed using calculational docking and molecular dynamics. We termed this approach "directed modeling," since the known peptide structure directs the prestructured antibody binding site to its final conformation. The independently derived structures of the peptide antigen and antibody binding site already showed a high degree of surface complementarity after the initial docking calculation, during which the peptide was conformationally restrained. The docking routine was a soft algorithm, applying a combination of Monte Carlo simulation and energy minimization. The observed shape complementarity in the docking process suggested that the structure assessments already led to anti-idiotypic conformations of peptide antigen and antibody fragment. Refinement of the complex by dynamic simulation yielded improved surface adaptation by small rearrangements within antibody and antigen. The complex presented herein was analyzed in terms of antibody-antigen interactions, properties of contacting surfaces, and segmental mobility. The structural requirements for AChR complexation by autoantibodies were explored and compared with experimental data from alanine scans of the MIR peptides. The analysis revealed that the N-terminal loop of the peptide structure, which is indispensable for antibody recognition, aligns three hydrophobic groups in a favorable arrangement leading to the burial of 40% of the peptide surface in the binding cleft upon complexation. These data should be valuable in the rational design of an Fv mutant with much improved affinity for the MIR and AChR to be used in therapeutic approaches in MG.

Amino Acid Sequence↗

PubMed2000

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.

Amino Acid Sequence↗

PubMed2000

Piv-Propsi

The pseudodipeptide, (S)-N-isopropyl [N-(pivaloyl)pyrrolidin-2-yl]methylaminooxyacetamide, C(15)H(29)N(3)O(3), adopts a global extended conformation with the hydroxylamine group in the g(+)/g(-) structure. The C-terminal amide NH interacts intramolecularly with the hydroxylamine O atom. Both NH bonds of each molecule are hydrogen bonded to the C-terminal amide carbonyl of a neighbouring molecule.

Journal Article↗

PubMed2000

A silaproline-containing dipeptide.

The silaproline-containing dipeptide N-(3, 3-dimethyl-1-pivaloyl-1-aza-3-sila-5-cyclopentylcarbonyl)-L- alanine isopropylamide, C(17)H(33)N(3)O(3)Si, has two independent molecules in the asymmetric unit and each adopts a beta-II folded conformation, where the amide on the terminal C interacts intramolecularly with the pivaloyl O atom. The five-membered silaproline ring is C(beta)-puckered, an infrequent conformation for the homologous proline ring.

Crystallography, X-Ray↗

PubMed1999

Engineered glycolytic glyceraldehyde-3-phosphate dehydrogenase binds the anti conformation of NAD+ nicotinamide but does not experience A-specific hydride transfer.

Glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a NAD-dependent oxidoreductase which catalyzes the oxidative phosphorylation of d-glyceraldehyde-3-phosphate (G3P) to form 1, 3-diphosphoglycerate. The currently accepted mechanism involves an oxidoreduction step followed by a phosphorylation. GAPDH is classified as a B-specific oxidoreductase. The inspection of several crystal structures of GAPDHs indicates that the efficient hydride transfer from the hemithioacetal intermediate to the C4 position of the pyridinium si face requires optimal nicotinamidium-protein contacts for a suitable pyridinium-ring orientation. In previous studies carried out on Escherichia coli GAPDH (C. Corbier, A. Mougin, Y. Mely, H. W. Adolph, M. Zeppezauer, D. Gerard, A. Wonacott, and G. Branlant, Biochimie 72, 545-554, 1990; J. Eyschen, C. Corbier, B. Vitoux, G. Branlant, and M. T. Cung, Protein Pept. Lett. 1, 19-24, 1994), the role of the invariant Asn 313 residue, as an anchor which favors the syn orientation of the nicotinamide ring, was examined. Here, we report further investigations on the molecular factors responsible for the cofactor stereospecificity. Two single [Gly317] and [Ala317] GAPDH mutants and one double [Thr313-Gly317] GAPDH mutant were constructed on the basis of a molecular modelling study from the crystal structure of holo GAPDH from E. coli (E. Duée, L. Olivier-Deyris, E. Fanchon, C. Corbier, G. Branlant, and O. Dideberg, J. Mol. Biol. 257, 814-838, 1996). The Kd constants of [Ala317], [Gly317], and [Thr313-Gly317] GAPDH mutants for NAD are 5, 13, and 300 times higher than that of wild-type GAPDH. Transferred nuclear Overhauser effect spectroscopy demonstrates that the wild-type syn orientation of bound nicotinamide remains unchanged in the [Gly317] and [Ala317] mutants, whereas a conformational equilibrium between the syn and anti forms occurs in the [Thr313-Gly317] double mutant with a preference for the anti conformer. Although the double mutant preferably binds the nicotinamide ring in an anti conformation, it still exhibits B hydride transfer stereospecificity. Yet, the catalytic efficiency is much less than that of the wild type. This indicates that the holo GAPDH mutant fraction with an anti orientation of bound NAD is not capable of forming the ternary complex with G3P which would be required for an efficient A-specific catalytic process. The reasons of this catalytic inefficiency are discussed in relation with the historical and functional models which were advanced to explain the stereospecificity of NAD(P)-dependent dehydrogenases.

Glyceraldehyde-3-Phosphate Dehydrogenases↗

PubMed1999

Solution structure of a retro-inverso peptide analogue mimicking the foot-and-mouth disease virus major antigenic site. Structural basis for its antigenic cross-reactivity with the parent peptide.

The antigenic activity of a 19-mer peptide corresponding to the major antigenic region of foot-and-mouth disease virus and its retro-enantiomeric analogue was found to be completely abolished when they were tested in a biosensor system in trifluoroethanol. This suggests that the folding pattern, which is alpha-helix in trifluoroethanol (confirmed by CD measurement), does not correspond to the biologically relevant conformation(s) recognized by antibodies. The NMR structures of both peptides were thus determined in aqueous solution. These studies showed that the two peptides exhibit similar folding features, particularly in their C termini. This may explain in part the cross-reactive properties of the two peptides in aqueous solution. However, the retro-inverso analogue appears to be more rigid than the parent peptide and contains five atypical beta-turns. This feature may explain why retro-inverso foot-and-mouth disease virus peptides are often better recognized than the parent peptide by anti-virion antibodies.

Amino Acid Sequence↗

PubMed1999

Efficient access to all four stereoisomers of phenylalanine cyclopropane analogues by chiral HPLC

Bonded polysaccharide-derived chiral stationary phases were found to be useful for the preparation of the four stereoisomers of the cyclopropane analogue of phenylalanine (c(3)Phe) as well as for the direct determination of the enantiomeric purity of c(3)Phe derivatives by HPLC. Three chiral stationary phases, consisting of cellulose and amylose derivatives chemically bonded on allylsilica gel, were tested. The mixed 10-undecenoate/3, 5-dimethylphenylcarbamate of cellulose, 10-undecenoate/3, 5-dimethylphenylcarbamate of amylose and 10-undecenoate/p-methylbenzoate of cellulose were the starting polysaccharide derivatives for CSP-1, CSP-2, and CSP-3, respectively. Using mixtures of n-hexane/chloroform/2-propanol as mobile phase on a semi-preparative column (150 mm x 20 mm ID) containing CSP-2, we separated about 1.7 g of racemic cis-methyl 1-tert-butoxycarbonylamino-2-phenylcyclopropanecarboxylate (cis-6) and 1.2 g of racemic trans-methyl-1-tert-butoxycarbonylamino-2-phenylcycloprop-anecarboxyl ate (trans-6) by successive injections. Copyright 1999 Wiley-Liss, Inc.

Journal Article↗

PubMed1998

Solution structures of the fibronectin-like Leishmania gp63 SRYD-containing sequence in the free and antibody-bound states--transferred NOE and molecular dynamics studies.

The anti-SRYD monoclonal antibody (mAbSRYD) raised against the IASRYDQL synthetic octapeptide, the 250-257 sequence of the Leishmania major surface glycoprotein gp63 recognizes both SRYD-containing peptides and the whole cognate major surface protein on intact parasites. Two SRYD-containing peptides, which antigenically and functionally mimic the RGDS sequence of fibronectin and efficiently inhibit parasite attachment to the macrophage receptors, were studied by two-dimensional transferred nuclear Overhauser effect experiments in the presence of mAbSRYD. The antibody-bound IASRYDQL octapeptide solution conformation was determined on the basis of 55 interproton-distance restraints, derived from NMR measurements. Eighteen structures which were first generated using an approach combining distance geometry and molecular dynamics, converge by energy minimization toward a folded structure with an average rmsd from the experimental data of less than 0.05 nm for the overall backbone and 0.025 nm for the SRYD motif. A distorted gamma-turn was found, stabilized by the backbone-backbone D255-NH to R253-CO hydrogen bond, while the R253 and D255 side chains are pointing in opposite directions. This latter antibody-bound structure is compared with that of the free octapeptide in dimethylsulfoxide solution, and with the crystal structure of the RYD fragment in OPG2 Fab, an antireceptor antibody that mimics the RGD cell adhesion site. On this basis, a mechanism for IASRYDQL-receptor interaction is discussed.

Amino Acid Sequence↗

PubMed1998

Azaproline as a beta-turn-inducer residue opposed to proline.

Azaproline (AzPro) is an analogue of proline containing a nitrogen atom in place of the C(alpha)H group. AzPro has been introduced in various model peptides, and especially in the Boc-Ala-AzPro-Ala-NHiPr tripeptide. The structural consequence of that modification has been investigated in solution by using IR and 1H NMR, with reference to the cognate proline-containing peptide. Contrary to proline, which induces beta-folding of the Pro-Ala sequence, azaproline apparently favors betaVI-folding of the Ala-AzPro one with high occurrence. Opening of the AzPro pyrazolidine ring to get N-methylazaalanine fundamentally does not change the structural properties of the azatripeptide, but allows the existence of open conformers to an extent depending on the solvent.

Aza Compounds↗

PubMed1998

Anatomy of the antigenic structure of a large membrane autoantigen, the muscle-type nicotinic acetylcholine receptor.

The neuromuscular junction nicotinic acetylcholine receptor (AChR), a pentameric membrane glycoprotein, is the autoantigen involved in the autoimmune disease myasthenia gravis (MG). In animals immunized with intact AChR and in human MG, the anti-AChR antibody response is polyclonal. However, a small extracellular region of the AChR alpha-subunit, the main immunogenic region (MIR), seems to be a major target for anti-AChR antibodies. A major loop containing overlapping epitopes for several anti-MIR monoclonal antibodies (mAbs) lies within residues alpha 67-76 at the extreme synaptic end of each alpha-subunit: however, anti-MIR mAbs are functionally and structurally quite heterogeneous. Anti-MIR mAbs do not affect channel gating, but are very effective in the passive transfer of MG to animals; in contrast, their Fab or Fv fragments protect the AChR from the pathogenic effects of the intact antibodies. Antibodies against the cytoplasmic region of the AChR can be elicited by immunization with denatured AChR and the precise epitopes of many such mAbs have been identified; however, it is unlikely that such antibodies are present in significant amounts in human MG. Antibodies to other extracellular epitopes on all AChR subunits are present in both experimental and human MG; these include antibodies to the acetylcholine-binding site which affect AChR function in various ways and also induce acute experimental MG. Finally, anti-AChR antibodies cross-reactive with non-AChR antigens exist, suggesting that MG may result from molecular mimicry. Despite extensive studies, many gaps remain in our understanding of the antigenic structure of the AChR; especially in relation to human MG. A thorough understanding of the antigenic structure of the AChR is required for an in-depth understanding, and for possible specific immunotherapy, of MG.

Amino Acid Sequence↗

PubMed1997

Two-dimensional 1H-NMR and CD structural analysis in a micellar medium of a bovine alphaS1-casein fragment having benzodiazepine-like properties.

The conformation of the benzodiazepine-like decapeptide, YLGYLEQLLR, corresponding to residues 91-100 of bovine alphaS1-casein, has been examined in SDS micelles using CD, two-dimensional 1H-NMR and restrained molecular-dynamics simulation. Evidence is presented that the decapeptide adopts a rigid structure in water/SDS micellar medium, but not in water or dimethylsulfoxide. The three-dimensional structure, consistent with the proton-proton distances obtained from the quantitative analysis of the two-dimensional NOEs, was generated by restrained energy minimization and molecular-dynamics simulation. In water/SDS micellar medium, YLGYLEQLLR adopts an amphipathic helicoid structure with distinct hydrophobic and hydrophilic faces. The relative disposition of the tyrosine aromatic rings was compared with that of the aromatic rings in the benzodiazepines.

Animals↗

PubMed1997

Aza-peptides. II. X-ray structures of aza-alanine and aza-asparagine-containing peptides.

In order to determine the structural consequences of the N alpha/C alpha H exchange in aza-peptides, we have solved the crystal molecular structures of some derivatives containing the aza-analogue of asparagine [Z-AzAsn(Me)-NMe2 (1), Z-AzAsn(Me)-Pro-NHiPr (2) and Piv-Pro-AzAsn(Me)-NHiPr (5)], aspartic acid [Z-AzAsp(OEt)-Pro-NHiPr (3) and alanine (Boc-AzAla-Pro-NHiPr (4)], by using X-ray diffraction. They reveal that the alpha-nitrogen accommodates a pyramidal (1-4) or planar (5) structure depending on the sequence. When pyramidal, the alpha-nitrogen assumes the R (D-like) chirality. All of the derivatives but 1 adopt either a beta 1-folded (2-4) or beta n-folded (5) structure in which the (AzAsn)N3H bond is intramolecularly hydrogen-bonded to the alpha-nitrogen.

Alanine↗

PubMed1997

Aza-peptides. III. Experimental structural analysis of aza-alanine and aza-asparagine-containing peptides.

To determine the structural perturbations induced by the C alpha H-->N alpha exchange in aza-peptides, we have examined by 1H NMR and IR spectroscopy various derivatives of the aza-analogues of alanine, aspartic acid and asparagine in different organic solvents with increasing polarity. Their general formulas are: R1-AzXaa-NR2R3, R1-Pro-AzXaa-NR2R3 and R1-AzXaa-Pro-NR2R3 (where AzXaa denotes the aza-analogue of the amino acid residue Xaa = Ala, Asp, Asn; R1 = Boc, Z; R2, R3 = H, Me, iPr). The aza-analogue of an amino acid residue appears to be a strong beta-turn-inducing motif, and the AzAsn carboxamide side-chain is capable of interacting, as a proton donor, with the preceding peptide carbonyl group.

Alanine↗

PubMed1997

X-ray structures of aza-proline-containing peptides.

The aza-analogue of proline (AzPro) contains a nitrogen atom in place of the CH alpha of the cognate residue. The resolution of the crystal structures of seven AzPro-containing peptides, presenting a set of ten AzPro motifs, reveals the structural properties of this particular aza-residue. Because of sterical hindrances, both nitrogen atoms are out of planarity, and the reduced electronic conjugation in the two AzPro-adjacent amide groups probably explains the longer amide bond distances and the weak proton-accepting character of the two pyrazolidine nitrogens. The absolute configuration of both AzPro nitrogens depends on the chemical nature of the sequence. In all cases, the AzPro residue assumes the same intrinsic three-dimensional structure and presents folding tendencies opposed to those induced by proline.

Aza Compounds↗

PubMed1996

Phosphorus-31 nuclear magnetic resonance studies on coenzyme binding and specificity in glyceraldehyde-3-phosphate dehydrogenase.

Binding of NAD(P)+ to wild type and a series of mutants of the glycolytic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus designed to alter the cofactor specificity [Clermont, S., Corbier, C., Mely, Y., Gerard, D., Wonacott, A., & Branlant, G. (1993) Biochemistry 21, 10178-10184] has been studied by 31P NMR. In the mutants with the L187A and P188S substitutions, the pyrophosphate signals are split, and the upfield resonance has been assigned to the P(a) phosphate. Titration of the NADP+ 2'-phosphate pKa deduced from its chemical shift shows that the electrostatic environment in the binding site is largely affected by the single point mutations. pKas ranging from 7.7 for the L187A-P188S mutant to < 5.7 for the D32G-L187A-P188S and D32A-L187A-P188S mutants have been observed, thus indicating that the binding of NADP+ is modulated by the ionization state of its 2'-phosphate. In the quintuple mutant L33T-T34G-D35G-L187A-P188S, designed in comparison with the photosynthetic NAD(P)-dependent GAPDH of the chloroplast, the 2'-phosphate has a pKa of 6.8. As further stabilizing interactions like hydrogen bonds or positively charged side chains would lower this pKa, it is suggested that the 2'-phosphate ionization state of bound NADP+ in chloroplastic GAPDH is dianionic. The NADP+ dissociation rate constants (k(off)) of the three mutants D32G, L187A-P188S, and D32G-L187A-P188S, are higher at pH 6.1 than at pH 8.1 and are similar at the same pH, indicating that the difference in binding affinity between these three mutants results from the molecular recognition step or conformational change upon binding.

Coenzymes↗