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G Deléage

Publications and source records attributed to G Deléage.

15 recordsLinked to original sources
PubMed1995

Dimerization kinetics of HIV-1 and HIV-2 reverse transcriptase: a two step process.

The dimerization processes of the human immunodeficiency virus (HIV) types 1 and 2 reverse transcriptase (RTs) from their subunits have been investigated using a number of complementary approaches (fluorescence spectroscopy, size exclusion-HPLC and polymerase activity assay). The formation of the native heterodimeric form of HIV-1 and HIV-2 RT occurs in a two step process. The first step is a concentration-dependent association of the two subunits (p66 and p51) to give a heterodimeric intermediate, which slowly isomerizes to the "mature" heterodimeric form of the enzyme. For both RTs, the first step behaves as a second order reaction with similar association rate constants (in the range of 2 x 10(4) to 4 x 10(4) M-1 s-1). This initial dimerization results in a 25% quenching of the intrinsic fluorescence and a 30% decrease in the accessibility of the tryptophan hydrophobic cluster to solvent as revealed by iodide quenching experiments and by monitoring the binding of 1-anilino-8-naphthalenesulphonate. The formation of the intermediate-RT form appears to involve hydrophobic regions of the subunits containing tryptophan residues. This intermediate form is devoid of polymerase activity, but is able to bind primer/template with high affinity. The final stage of the mature RT-heterodimer formation occurs in a slow first order reaction, which is 12-fold faster for HIV-2 (1.2 h-1) than HIV-1 RT (0.1 h-1). At micromolar concentrations, this slow isomerization constitutes the rate limiting step of the RT maturation and the structural change involved appears to be partly associated with the catalytic site, as shown using fluorescent labelled primer/template. On the basis of both the presently available X-ray structure of the HIV-1 RT and the predicted structure of HIV-2 RT, the thumb subdomain of the p51 subunit seems to be involved in this maturation step, which is probably the interaction of this domain with the RNAse H domain of the large subunit. The placement of the fingers subdomain of p51 in the palm subdomain of the p66 subunit may also be associated with formation of mature heterodimeric RTs.

Anilino Naphthalenesulfonates↗
PubMed1995

ANTHEPROT 2.0: a three-dimensional module fully coupled with protein sequence analysis methods.

ANTHEPROT is a fully interactive graphics program devoted to the analysis of the sequences and structures of proteins. This program, originally developed to facilitate the protein sequence analysis coupled with multiple alignments and predicted secondary structures of proteins, now comprises a powerful 3D module to display and handle macromolecular structures. All the methods that were previously integrated into ANTHEPROT are now directly coupled with a 3D window that provides the user all the classic features of a molecular modeling package. Indeed, it allows real-time rotation and translation of 3D structures with many kinds of models in depth-cueing mode (space filling, backbone, wire models, main chain, and ribbons), selections (atom type, residue type, segments, and chain), color-coding systems (amino acid properties, predicted or observed secondary structures, temperature B factor, and subunits), geometric calculations (Ramachandran plot, distances, and angles), and fitting molecules. Stereo views are possible as well as HPGL standard files. A module specifically devoted to the determination of 3D structures using nuclear magnetic resonance is also available. This major release of our program for IBM rs6000 workstations is available by anonymous ftp to ibcp.fr for academic institutions.

Antigens↗
PubMed1991

Intrinsic tryptophan fluorescence of Schizosaccharomyces pombe mitochondrial F1-ATPase. A powerful probe for phosphate and nucleotide interactions.

Mitochondrial F1 from the yeast Schizosaccharomyces pombe, in contrast to the mammalian enzyme, exhibits a characteristic intrinsic tryptophan fluorescence with a maximal excitation at 291 nm and a maximal emission at 332 nm. Low values of Stern-Volmer quenching constants, 4.0 M-1 or 1.8 M-1, respectively, in the presence of either acrylamide or iodide, indicate that tryptophans are mainly buried inside the native enzyme. Upon subunit dissociation and unfolding by 6 M guanidine hydrochloride (Gdn.HCl), the maximal emission is shifted to 354 nm, a value very similar to that obtained with N-acetyltryptophanamide, a solute-tryptophan model compound. The tryptophan content of each isolated subunit has been estimated by fluorescence titration in the presence of Gdn.HCl with free tryptophan as a standard. Two tryptophans and one tryptophan are found respectively in the alpha and epsilon subunits, whereas none is detected in the beta, gamma, and delta subunits. These subunit contents are consistent with the total of seven tryptophans estimated for native F1 with alpha 3 beta 3 gamma 1 delta 1 epsilon 1 stoichiometry. The maximal emission of the isolated epsilon subunit is markedly blue-shifted to 310-312 nm by interaction with the isolated delta subunit, which suggests that the epsilon subunit tryptophan might be a very minor contributor to the native F1 fluorescence measured at 332 nm. This fluorescence is very sensitive to phosphate, which produces a marked blue shift indicative of tryptophans in a more hydrophobic environment. On the other hand, ADP and ATP quench the maximal emission at 332 nm, lower tryptophan accessibility to acrylamide, and reveal tryptophan heterogeneity.

Acrylamide↗
PubMed1991

ANTHEPROT: an interactive graphics software for analyzing protein structures from sequences.

ANTHEPROT is a fully interactive program devoted to the analysis of protein structures using a graphics workstation. It presents four options: The first option can predict secondary structures using five methods, and hydrophobicity, solvent accessibility, flexibility and antigenicity profiles using eighteen scales. The user may introduce his own scales. The results displayed on the screen can be easily analyzed. The second option is for representing results concerning up to eight proteins by one method. To compare these proteins, it is possible to align the profiles or the predicted secondary structure according to various motifs. The secondary structure deduced from crystallographic data may also be introduced. The third option is designed to compare the primary structure of two proteins and to visualize on the screen regions that exhibit similarity. Six different comparison matrices may be used, but the user can also introduce his own matrices. The last option is for studying the proteolytic peptides resulting from a chemical or enzymatic digestion of a given protein. It is possible to analyze the protein cleavage using eleven chemical reagents or enzymes. The results are displayed on the screen as RP-HPLC chromatogram.

Amino Acid Sequence↗
PubMed1990

Interaction between delta and epsilon subunits of F1-ATPase from pig heart mitochondria. Circular dichroism and intrinsic fluorescence of purified and reconstituted delta epsilon complex.

A delta epsilon complex has been purified as a molecular entity from pig heart mitochondrial F1-ATPase. This delta epsilon complex has also been reconstituted from purified delta and epsilon subunits. Both isolated and reconstituted delta epsilon complexes have delta 1 epsilon 1 stoichiometry and are indistinguishable by their chromatographic behavior, their circular dichroism spectra (CD spectra), and their intrinsic fluorescence features. The content of secondary structures deduced from CD spectra of the delta epsilon complex appears to be the sum of the respective contributions of purified delta and epsilon subunits. All intrinsic fluorescence studies carried out on isolated epsilon subunit and delta epsilon complex show that the single tryptophan residue located on epsilon is involved in the interaction between delta and epsilon subunits. Results obtained with F1-ATPase are in favor of the same delta epsilon interaction in the entire enzyme.

Animals↗
PubMed1990

Virus-neutralizing monoclonal antibody to a conserved epitope on the duck hepatitis B virus pre-S protein.

In this study we used duck hepatitis B virus (DHBV)-infected Pekin ducks and heron hepatitis B virus (HHBV)-infected heron tissue to search for epitopes responsible for virus neutralization on pre-S proteins. Monoclonal antibodies were produced by immunizing mice with purified DHBV particles. Of 10 anti-DHBV specific hybridomas obtained, 1 was selected for this study. This monoclonal antibody recognized in both DHBV-infected livers and viremic sera a major (36-kilodalton) protein and several minor pre-S proteins in all seven virus strains used. In contrast, pre-S proteins of HHBV-infected tissue or viremic sera did not react. Thus, the monoclonal antibody recognizes a highly conserved DHBV pre-S epitope. For mapping of the epitope, polypeptides from different regions of the DHBV pre-S/S gene were expressed in Escherichia coli and used as the substrate for immunoblotting. The epitope was delimited to a sequence of approximately 23 amino acids within the pre-S region, which is highly conserved in four cloned DHBV isolates and coincides with the main antigenic domain as predicted by computer algorithms. In in vitro neutralization assays performed with primary duck hepatocyte cultures, the antibody reduced DHBV infectivity by approximately 75%. These data demonstrate a conserved epitope of the DHBV pre-S protein which is located on the surface of the viral envelope and is recognized by virus-neutralizing antibodies.

Amino Acid Sequence↗
PubMed1990

Do synapsin I and microtubule-associated proteins bind to a common site on polymerized tubulin?

Synapsin I plays an important role in the regulation of neurotransmitter release, since it binds to synaptic vesicles and to the cytoskeleton, and it bundles F-actin and microtubules. We have previously shown by tryptic digestion of synapsin I that a 44 kDa fragment contains a binding site for polymerized tubulin. In the present experiments, we test whether synapsin I and microtubule-associated proteins (MAPs) have the same or a different binding site on tubulin molecules. Our results show that heat stable MAPs do not compete with synapsin I for binding to taxol tubulin. In addition, subtilisin digestion of tubulin, which suppresses MAPs binding, does not abolish synapsin I cosedimentation with taxol tubulin. Thus, our results strongly suggest that synapsin I (as reported for kinesin) does not bind to the 4 kDa subtilisin digested C-terminal part of the tubulin molecule.

Animals↗
PubMed1989

Duck hepatitis B virus can tolerate insertion, deletion, and partial frameshift mutation in the distal pre-S region.

In-frame and frameshift mutations were introduced into the pre-S region (1,212 base pairs) of duck hepatitis B virus. The in-frame mutants retained the inserted 12 nucleotides, while the frameshift mutants either reverted to wild type or exhibited a 10-nucleotide compensatory deletion downstream of the original mutation site. Thus, although duck hepatitis B virus has a compact and highly economical genome organization, it can replicate despite alterations of up to 9 amino acid codons in the pre-S and P open reading frames.

Animals↗
PubMed1988

ANTHEPROT: a package for protein sequence analysis using a microcomputer.

A simple microcomputer package is described to make the theoretical analysis of protein sequences. Several methods designed to compare two sequences, to model proteolytic reactions and to predict the secondary structure, the hydrophobic/hydrophilic regions and the potential antigenic sites of proteins have been included in an Apple II microcomputer software. The package comprises 21 programs as well as the secondary structure database of Kabsch and Sander (1983).

Algorithms↗
PubMed1986

Efficient reconstitution of mitochondrial energy-transfer reactions from depleted membranes and F1-ATPase as a function of the amount of bound oligomycin sensitivity-conferring protein (OSCP).

Pig heart mitochondrial membranes depleted of F1 and OSCP by various treatments were analyzed for their content in alpha and beta subunits of F1 and in OSCP using monoclonal antibodies. Membrane treatments and conditions of rebinding of F1 and OSCP were optimized to reconstitute efficient NADH- and ATP-dependent proton fluxes, ATP synthesis and oligomycin-sensitive ATPase activity. F1 and OSCP can be rebound independently to depleted membranes but to avoid unspecific binding of F1 to depleted membranes (ASUA) which is not efficient for ATP synthesis, F1 must be rebound before the addition of OSCP. The rebinding of OSCP to depleted membranes reconstituted with F1 inhibits the ATPase activity of rebound F1, while it restores the ATP-driven proton flux measured by the quenching of ACMA fluorescence. The rebinding of OSCP also renders the ATPase activity of bound F1 sensitive to uncouplers. The rebinding of OSCP alone or F1 alone, does not modify the NADH-dependent proton flux, while the rebinding of both F1 and OSCP controls this flux, inducing an inhibition of the rate of NADH oxidation. Similarly, oligomycin, which seals the F0 channel even in the absence of F1 and OSCP, inhibits the rate of NADH oxidation. OSCP is required to adjust the fitting of F1 to F0 for a correct channelling of protons efficient for ATP synthesis. All reconstituted energy-transfer reactions reach their optimal value for the same amount of OSCP. This amount is consistent with a stoichiometry of two OSCP per F1 in the F0-F1 complex.

Adenosine Triphosphatases↗
PubMed1986

Structural aspects and orientation mechanism of mitochondrial F1 adenosinetriphosphatase. Evidence for a negative electric birefringence due to a permanent moment perpendicular to the long axes of the particle.

The electric birefringence technique was used to investigate the steady-state birefringence, the orientational relaxation time, and the orientation mechanism of pig heart mitochondrial F1 adenosine-5'-triphosphatase (F1-ATPase). The electrooptical properties of this enzyme in solution were studied as functions of pH, protein concentration, and applied electric field. The F1-ATPase exhibits a surprising negative electric birefringence with a specific Kerr constant of -1.5 X 10(-3) esu cgs. The field-independent relaxation time was found to be 0.65 +/- 0.05 microseconds, corresponding to a rotational diffusion constant of 2.55 X 10(5) s-1. The overall size and shape of F1-ATPase have been calculated from both translational and rotational diffusion constants. The enzyme may be assumed to be an oblate ellipsoid of revolution with dimensions of about 170 X 170 X 70 A. The orientation mechanism of F1-ATPase was analyzed by fitting experimental birefringence rising curves with theoretical rising functions. The ratio of the permanent to induced dipole moment is found to be very high; therefore, the birefringence of F1-ATPase is due to a strong permanent dipole moment in a direction perpendicular to the long axes of the particle. These particular electric properties can be explained by the oligomeric structure of the protein and seem likely to play a role in its mechanism of functioning.

Animals↗
PubMed1983

Correlations between ATP hydrolysis, ATP synthesis, generation and utilization of delta pH in mitochondrial ATPase-ATP synthase.

Using fluorescence quenching of 9-amino-6-chloro-2- methoxyacridine induced either by ATP hydrolysis in the ATPase-ATP synthase complex or by succinate oxidation in inverted submitochondrial particles, correlation have been established between ATP hydrolysis, ATP synthesis and the generation and utilization of delta pH. The results obtained are best explained in terms of local circuits of protons.

ATP Synthetase Complexes↗
PubMedDate not supplied

An algorithm for protein secondary structure prediction based on class prediction.

An algorithm has been developed to improve the success rate in the prediction of the secondary structure of proteins by taking into account the predicted class of the proteins. This method has been called the 'double prediction method' and consists of a first prediction of the secondary structure from a new algorithm which uses parameters of the type described by Chou and Fasman, and the prediction of the class of the proteins from their amino acid composition. These two independent predictions allow one to optimize the parameters calculated over the secondary structure database to provide the final prediction of secondary structure. This method has been tested on 59 proteins in the database (i.e. 10,322 residues) and yields 72% success in class prediction, 61.3% of residues correctly predicted for three states (helix, sheet and coil) and a good agreement between observed and predicted contents in secondary structure.

Algorithms↗