Biomedical subjects

Y Guillou

Publications and source records attributed to Y Guillou.

At least 19 recordsLinked to original sources

PubMed1999

Pseudo-native motifs in the noncovalent heme-apocytochrome c complex. Evidence from antibody binding studies by enzyme-linked immunosorbent assay and microcalorimetry.

When beef heart apocytochrome c is unfolded, it folds upon noncovalent heme binding (Dumont, M. E., Corin, A. F., and Campbell, G.A. (1994) Biochemistry, 33, 7368-7378). Here, the conformation of the heme-apocytochrome noncovalent complex is compared with that of holocytochrome c. A purification method was designed for obtaining in large amounts apocytochrome c that was shown by amino acid analysis and mass spectroscopy to be chemically intact. The apoprotein and its noncovalent complex were characterized by absorption, fluorescence, circular dichroism, and sedimentation velocity, confirming previous reports. Sedimentation-diffusion equilibrium showed that the apoprotein and its noncovalent complex with heme were monomeric. Surprisingly, whereas apocytochrome c was quite soluble, the noncovalent complex slowly formed heavy aggregates, thus precluding experiments at the concentrations needed for structural studies. Two monoclonal antibodies that bind strongly to distinct antigenic sites on native holocytochrome were used to probe the noncovalent complex conformation. For both antibodies, the affinity for the noncovalent complex was only about 5-10-fold smaller than that for native holocytochrome c, and about 50-100-fold larger than that for apocytochrome c. These results indicate that the noncovalent complex, although not entirely native, carries some pseudo-native structural motifs.

Animals↗

PubMed1997

Lack of coupling between secondary structure formation and collapse in a model polypeptide that mimics early folding intermediates, the F2 fragment of the Escherichia coli tryptophan-synthase beta chain.

The isolated, 101-residue long C-terminal (so called F2) fragment of the beta chain from Escherichia coli tryptophan synthase was shown previously to fold into an ensemble of conformations that are condensed, to contain large amounts of highly dynamic secondary structures, and to behave as a good model of structured intermediates that form at the very early stages of protein folding. Here, solvent perturbations were used to investigate the forces that are involved in stabilizing the secondary structure (monitored by far-UV CD) and the condensation of the polypeptide chain (monitored by dynamic light scattering) in isolated F2. It was observed that neither the ionic strength, nor the pH (between 7 and 10), nor salts of the Hofmeister series affected the global secondary structure contents of F2, whereas some of these salts affected the collapse slightly. Addition of trifluoroethanol resulted in a large increase in both the amount of secondary structure and the Stokes radius of F2. Conversely, F2 became more condensed upon raising the temperature from 4 to 60 degrees C, whereas in this temperature range, the secondary structure undergoes significant melting. These observations lead to the conclusion that, in isolated F2, there is no coupling between the hydrophobic collapse and the secondary structure. This finding will be discussed in terms of early events in protein folding.

Chemical Phenomena↗

PubMed1997

The "pre-molten globule," a new intermediate in protein folding.

In vitro folding studies of several proteins revealed the formation, within 2-4 msec, of transient intermediates with a large far-UV ellipticity but no amide proton protection. To solve the contradiction between the secondary structure contents estimated by these two methods, we characterized the isolated C-terminal fragment F2 of the tryptophan synthase beta 2 subunit. In beta 2, F2 forms its tertiary interactions with the F1 N-terminal region. Hence, in the absence of F1, isolated F2 should remain at an early folding stage with no long-range interactions. We shall show that isolated F2 folds into, and remains in, a "state" called the pre-molten globule, that indeed corresponds to a 2- to 4-msec intermediate. This condensed, but not compact, "state" corresponds to an array of conformations in rapid equilibrium comprising native as well as nonnative secondary structures. It fits the "new view" on the folding process.

Circular Dichroism↗

PubMed1995

Importance of residues 2-9 in the immunoreactivity, subunit interactions, and activity of the beta 2 subunit of Escherichia coli tryptophan synthase.

The epitope recognized by a monoclonal antibody (mAb19) directed against the beta 2 subunit of Escherichia coli tryptophan synthase was found to be carried by residues 2-9 of the beta chain. The affinities of mAb19 for peptides of different lengths containing the 2-9 sequence were close to 0.6 x 10(9) M-1, the affinity of mAb19 for native beta 2. In view of these results, a model is proposed to account for the kinetics of appearance of the epitope during in vitro renaturation of beta 2 (Murry-Brelier, A., and Goldberg, M.E. (1988) Biochemistry 27, 7633-7640). A mutant producing beta chains lacking residues 1-9 (beta delta 1-9) was prepared. The beta delta 1-9 protein was able to fold into a heat stable homodimer resembling wild type beta 2. Isolated beta delta 1-9 had no detectable enzymatic activity. It could bind alpha chains extremely weakly and be slightly activated. In the presence of the 1-9 peptide, the beta delta 1-9 protein could bind alpha chains much more strongly and generate a 50% active enzyme. Thus, although having little role in the overall folding and stability of the protein, the 1-9 sequence of the beta chain appears strongly involved in the alpha-beta interactions and in the enzymatic activity.

Antibodies, Monoclonal↗

PubMed1994

Native disulfide bonds greatly accelerate secondary structure formation in the folding of lysozyme.

To assess the respective roles of local and long-range interactions during protein folding, the influence of the native disulfide bonds on the early formation of secondary structure was investigated using continuous-flow circular dichroism. Within the first 4 ms of folding, lysozyme with intact disulfide bonds already had a far-UV CD spectrum reflecting large amounts of secondary structure. Conversely, reduced lysozyme remained essentially unfolded at this early folding time. Thus, native disulfide bonds not only stabilize the cfinal conformation of lysozyme but also provide, in early folding intermediates, the necessary stabilization that favors the formation of secondary structure.

Circular Dichroism↗

PubMed1992

Kinetic resolution of peptide bond and side chain far-UV circular dichroism during the folding of hen egg white lysozyme.

The kinetics of regain of the native ellipticity in the far- and near-UV spectra have been investigated during the refolding at pH 7.8 and 20 degrees C of guanidine-unfolded, nonreduced hen egg white lysozyme. Stopped-flow studies showed that the ellipticities at 260 and 289.5 nm exhibit biphasic kinetics with rate constants of about 50 s-1 and 2.5 s-1 for the rapid and slow phase, respectively. The ellipticity in the far-UV obeyed triphasic kinetics. In addition to a rapid and a slow phase with rate constants similar to those observed in the near-UV, a "burst" of ellipticity was shown to occur in the dead time of the experiments. The effects of low pH and of concentrations of guanidine ranging from 0.075 to 1.5 M on the rapid and slow rate constants were studied. Under all conditions investigated, the rate constants observed in the far- and near-UV for a given phase were the same, thus suggesting that the molecular events observed in the two regions of the UV spectrum are either identical or strongly coupled. Continuous-flow experiments at different wavelengths between 214 and 240 nm under conditions where the dead time for the observation was only 4 ms, followed by a detailed analysis of the kinetics of ellipticity change at each wavelength, provided the spectrum of the molecular species formed at the end of the burst phase. This spectrum was found to closely fit that predicted from the secondary structure of native lysozyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

A possible initial folding intermediate: the C-terminal proteolytic domain of tryptophan synthase beta chains folds in less than 4 milliseconds into a condensed state with non-native-like secondary structure.

The isolated F2-V8 peptide corresponding to the 101 C-terminal residues of Escherichia coli tryptophan synthase beta chains folds into a heat-stable, yet fluctuating, condensed state that contains a lot of secondary structure. However, this state has non-native-like secondary and supersecondary structures [Chaffotte, A., Guillou, Y., Delepierre, M., Hinz, H.-J., & Goldberg, M. E. (1991) Biochemistry 30, 8067-8074]. To characterize the rate of appearance of this state, stopped-flow studies on the far-ultraviolet circular dichroism (CD) and on the binding of 1-anilino-8-naphthalenesulfonate (ANS) have been conducted during the folding of guanidine-unfolded F2-V8. It was shown that both the CD signal at 222 nm and the ANS binding properties of folded isolated F2-V8 were regained, at 20 degrees C, within the dead time of the stopped-flow apparatus, which was 4 ms. At 12 degrees C, the binding of ANS was also completed within this dead time, but the ellipticity showed some minor later changes. After a rapid overshoot of the CD signal that occurred during the 4-ms dead time, a small readjustment of the ellipticity to the final value occurred more slowly and was completed after about 25 ms. Thus, even at 12 degrees C, the hydrophobic core and most of the secondary structure of folded F2-V8 were formed in less than 4 ms. These observations strongly suggest that the previously described condensed non-native-like state of F2-V8 results from a very rapid, nonspecific, hydrophobic collapse. It is proposed that such a state may be a general early intermediate in protein folding.

Circular Dichroism↗

PubMed1992

Inclusion bodies of the thermophilic endoglucanase D from Clostridium thermocellum are made of native enzyme that resists 8 M urea.

Endoglucanase D from Clostridium thermocellum was purified from inclusion bodies formed upon its overproduction in Escherichia coli, using 5 M urea as a solubilizing solution. We examined the effects of denaturing agents upon the stability of the pure soluble enzyme as a function of the temperature. At room temperature, guanidinium chloride induces an irreversible denaturation. By comparison, we observed no structural or functional effects at room temperature using high concentrations of urea as denaturing agent. The irreversible denaturation process observed with guanidinium chloride also occurs with urea but only at elevated temperature (greater than or equal to 60 degrees C); in 6 M urea, the activation energy of the denaturation reaction is decreased by a factor of only 1.8. We interpret the high resistance of this protein to urea as reflecting a reduced flexibility of its structure at normal temperatures which should be correlated to the thermophilic origin of this protein.

Cellulase↗

PubMed1991

The isolated C-terminal (F2) fragment of the Escherichia coli tryptophan synthase beta 2-subunit folds into a stable, organized nonnative conformation.

Proteolysis of the beta 2-subunit of Escherichia coli tryptophan synthase by the endoproteinase Glu C from Staphylococcus aureus V8 yields a peptide, F2, corresponding to the C-terminal 101 residues of the beta-chain. The conformation and stability of isolated F2 in phosphate buffer at pH 7.8 (where native beta 2 is stable) have been investigated. Circular dichroism spectra in the far-UV showed the presence of large amounts of secondary structure (19% alpha-helices, 34% extended beta-structures). Circular dichroism spectra in the near-UV and sedimentation velocity studies indicated an open globular structure with the aromatic side chains in a symmetric (or disordered) environment. NMR spectra and rates of amide proton exchange showed that F2 fluctuates rapidly between several conformations. The thermal denaturation of F2 observed by the loss of far-UV circular dichroism with increasing temperature appeared noncooperative, and indicates a high thermal stability (Tm = 70 degrees C). Differential scanning microcalorimetry confirmed the absence of cooperativity and indicated a very low value for the calorimetric enthalpy of denaturation (delta H = 17 kJ/mol). All these properties were compatible with a molten globule. However, the low sedimentation coefficient of F2 suggested a very hydrated and/or expanded structure, and the secondary structure content of isolated F2 (see above) differed widely from that reported in the literature for F2 within the context of native beta 2 (49% alpha-helices and 13% extended beta-structures). Thus, neither the secondary nor the tertiary structure of isolated F2 resembled those of native F2. In this respect, isolated F2 is not a "molten globule".(ABSTRACT TRUNCATED AT 250 WORDS)

Circular Dichroism↗

PubMed1989

Pressure-induced changes in the secondary structure of the Escherichia coli methionine repressor protein.

The effect of hydrostatic pressure on the conformational properties of the E. coli methionine repressor protein in aqueous solution was investigated by infrared spectroscopy. Changes in hydrostatic pressure produce dramatic changes in the spectral region of the conformation-sensitive amide I band. As the pressure is raised up to 18 kbar, the protein undergoes a rearrangement of alpha-helical segments into beta-type structures; after the pressure is released the beta-strands reconvert into less ordered alpha-helical or random segments.

Bacterial Proteins↗

PubMed1988

Interactions of DNA-binding proteins with the 5' region of the human transferrin gene.

We have established by transient expression experiments that the 620 base pairs upstream of the cap site of the human transferrin gene contain the information necessary for efficient expression of the gene in hepatoma cells HepG2 or Hep3B but not in HeLa cells. DNase I footprint analysis reveals that at least five distinct factors present in human or rat liver nuclear extracts interact with different sites of this region. One of these factors, binding to nucleotides -193 to -162, is closely related to or identical with the eukaryotic factor CCAAT-binding transcription factor/nuclear factor I; another one, binding to nucleotides -103 to -83 seems to be related to the CCAAT-binding protein. The binding sites of two other factors, not recognized by HeLa nuclear proteins, each contain an identical 10-nucleotide-long sequence (5' TCTTTGACCT 3') in reverse orientation, separated by 400 base pairs. Results of gel retardation assays, cross-competition experiments, and heat inactivation strongly suggest that the proteins binding to these sites are different. One of these sequences and the binding site of the CCAAT-binding protein related factor are located in the region between nucleotides -119 and -45. We have shown by transient expression experiments with 3' deleted vectors that this region is functionally essential for human transferrin gene expression.

Base Sequence↗

PubMed1988

Crystallization of the met repressor from Escherichia coli.

The met repressor from Escherichia coli has been crystallized in space group P21, with unit cell dimensions a = 35.6 A, b = 62.6 A, c = 44.5 A, beta = 102.4 degrees and one aporepressor dimer per asymmetric unit. Preliminary X-ray diffraction photographs show measurable intensities to beyond 1.5 A resolution, and the crystal form is ideally suited to high-resolution crystallographic analysis (1 A = 0.1 nm).

Amino Acid Sequence↗

PubMed1987

Fourier transform infrared investigation of the Escherichia coli methionine aporepressor.

This study represents the first physicochemical analysis of the recently cloned methionine repressor protein (Met aporepressor) from Escherichia coli. Infrared spectrometry was used to investigate the secondary structure and the hydrogen-deuterium exchange behavior of the E. coli Met aporepressor. The secondary structure of the native bacterial protein was derived by analysis of the amide I mode. The amide I band contour was found to consist of five major component bands (at 1625, 1639, 1653, 1665, and 1676 cm-1) which reflect the presence of various substructures. The relative areas of these component bands are consistent with a high alpha-helical content of the peptide chain secondary structure in solution (43%) and a small amount of beta-sheet structure (7%). The remaining substructure is assigned to turns (10%) and to unordered (or less ordered) structures (40%). The temperature dependence of the infrared spectra of native Met aporepressor in D2O medium over the temperature interval 20-80 degrees C indicates that there are two discrete thermal events: the first thermal event, centered at 42 degrees C, is associated with the hydrogen-deuterium exchange of the hard-to-exchange alpha-helical peptide bonds accompanied by a partial denaturation of the protein, while the second event, centered around 50 degrees C, represents the irreversible thermal denaturation of the protein.

Apoproteins↗

PubMed1987

Operator-constitutive mutations of the Escherichia coli metF gene.

The Escherichia coli metF gene codes for 5,10-methylene-tetrahydrofolate reductase, the enzyme that leads to the formation of N-methyltetrahydrofolate, supplying the methyl group of methionine. Transcription of metF, as well as most of the methionine genes, is repressed by the metJ gene product complexed with S-adenosylmethionine. A metF'-'lacZ gene fusion was used to isolate mutants that have altered expression from the metF promoter. The nucleotide sequences of the metF regulatory region from five such mutants were determined. The mutations were located in the region previously defined as the potential target of the methionine repressor by its similarity to other binding sites. The mutationally defined metF operator thus consists of a 40-base-pair-long region, with five 8-base-pair imperfect palindromes spanning the metF transcription start. The altered operators do not recognize the purified repressor in an in vitro transcription-translation system, although the repressor binds efficiently to the metF wild-type operator.

5,10-Methylenetetrahydrofolate Reductase (FADH2)↗

PubMed1986

Interactions of the Escherichia coli methionine repressor with the metF operator and with its corepressor, S-adenosylmethionine.

The metJ gene encoding the methionine aporepressor was placed under the control of a strong and inducible promoter, ptac. Bacterial strains carrying the recombinant plasmid pIP35 overproduced the regulatory protein by a factor of 200 over the wild type strain as determined by the immunoblot technique. The purified metJ gene product negatively controls the expression of the metF gene, in a cell-free system as shown by repression of beta-galactosidase synthesis under the control of the metF promoter. The metJ protein binds to a DNA fragment containing the potential operator of the metF gene with an affinity which is 10 times greater in the presence of S-adenosylmethionine than in its absence. Equilibrium dialysis experiments showed that the met aporepressor binds 2 mol of S-adenosylmethionine per mol of dimer with a dissociation constant of 200 microM.

Escherichia coli↗

PubMed1985

Reversible dissociation of aspartokinase I/homoserine dehydrogenase I from Escherichia coli K 12. The active species is the tetramer.

Dimers of aspartokinase I/homoserine dehydrogenase I from Escherichia coli K 12 have been isolated under very mild conditions. The dimers which cannot be distinguished from the tetramers by their kinetic properties, reassociate in the presence of potassium ions or L-aspartate. The selective sensitivity of aspartokinase I/homoserine dehydrogenase I to mild proteolytic digestion of dimers has been used to probe the reassociation reaction under the conditions of aspartokinase assay. We demonstrate that rapid reassociation occurs and that the protein species present in the assay when dimers are used to test the activity is tetrameric. These results confirm the previously proposed model for the subunit association of aspartokinase I/homoserine dehydrogenase I.

Aspartokinase Homoserine Dehydrogenase↗

PubMed1985

Isolation of the aspartokinase domain of bifunctional aspartokinase I-homoserine dehydrogenase I from E.coli K12.

A proteolytic fragment (Mr approximately 25 000) carrying only the aspartokinase activity has been purified by chromatofocusing after limited proteolysis of aspartokinase I-homoserine dehydrogenase I from E.coli K12. The NH2-terminal sequence shows that it corresponds to the amino terminal peptide of the native enzyme. The results confirm a previous hypothesis about the organization of native aspartokinase I-homoserine dehydrogenase I.

Amino Acid Sequence↗

PubMed1983

A hybrid proteolytic fragment of Escherichia coli aspartokinase I-homoserine dehydrogenase I. Structure, inhibition pattern, dissociation properties, and generation of two homodimers.

A hybrid dimeric fragment of Escherichia coli aspartokinase I-homoserine dehydrogenase I (Fazel, A., Müller, K., Le Bras, G., Garel, J.-R., Véron, M., and Cohen, G. N. (1983) Biochemistry 22, 158-165) has been purified and shown to possess both aspartokinase and homoserine dehydrogenase activities and is rather stable in the presence of L-threonine. Its two activities are still inhibited by threonine, but noncooperatively in contrast to the native protein. The aspartokinase activity is found to be more sensitive to threonine than the dehydrogenase activity. In the absence of threonine, the different chains of the hybrid (Mr = 89,000 + 59,000) dissociate first into monomers, this being followed by the pairing of two homologous chains to form two homodimers. In the presence of L-threonine, the two homodimers do not dissociate to re-form the hybrid fragment. The NH2-terminal analysis of different chains of the hybrid shows that the homodimers correspond, respectively, to the dimer of the native protein (Mr = 2 X 89,000) and to a dimer already described (Véron, M., Falcoz-Kelly, F., and Cohen, G. N. (1972) Eur. J. Biochem. 28, 520-527).

Alcohol Oxidoreductases↗