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G Zaccai

Publications and source records attributed to G Zaccai.

At least 73 records · Page 4Linked to original sources
PubMed1986

Denaturation of a halophilic enzyme monitored by small-angle neutron scattering.

Malate dehydrogenase from Halobacterium maris mortui exists in 4 M-NaCl as a stable protein dimer, with which are associated unusually large amounts of salt and water. In 1 M-NaCl at 25 degrees C, it denatures with a time-constant of about 0.5 h-1. Small-angle neutron scattering data from the protein under these conditions were monitored regularly over more than 12 hours during denaturation. They are quantitatively consistent with a model in which the protein dimer loses its exceptional salt and water-binding properties, and dissociates into monomers that partially unfold and have the interactions with solvent expected from their relatively charged amino acid composition. The exceptional salt and water-binding by the native enzyme, therefore, is associated with the native structure of the dimer.

Halobacterium↗
PubMed1986

Solution structure of halophilic malate dehydrogenase from small-angle neutron and X-ray scattering and ultracentrifugation.

Data from small-angle X-ray and neutron scattering and ultracentrifugation experiments on solutions of malate dehydrogenase from Halobacterium maris mortui are analysed together to yield a model for the enzyme particle formed by the protein and its interactions with water and salt in the solvent. The halophilic enzyme is stable only in high concentrations of salt and the model has structural features that are absent from non-halophilic malate dehydrogenase. The complementarity of the information derived from the three experimental methods is discussed extensively and quantitatively. It derives from the fact that mass density (ultracentrifugation), electron density (X-rays) and neutron scattering density are independent of each other. Each method gives a different "view" of the same particle, and an analysis of the combined data provided thermodynamic and structural parameters with, apart from the chemical composition of the solutions, only one other assumption: a constant partial specific volume for water equal to 1.00 cm3 g-1. Both the insights gained by this novel approach and its limitations are carefully pointed out. In solvents between 1 M and 5 M-NaCl, the enzyme forms a particle of invariant volume, consisting of a protein dimer (87,000 g mol-1) with which are associated 0.87 g of water and 0.35 g of salt per gram of protein. The partial specific volume of the protein calculated from the combined experimental data is 0.753(+/- 0.030) cm3 g-1, in good agreement with the value calculated from the amino acid composition. The particle has a radius of gyration of 32 A and an equivalent Stokes radius of 43 A. By combining the data from the X-ray and neutron scattering studies, the radii of gyration of the protein moiety alone and of the associated water and salt distribution were calculated. They are 28 A and about 40 A, respectively. The large-angle scattering curves show that the shapes of the particle and of the protein moiety alone are similar. At very low resolution they can be approximated by an ellipsoid of axial ratio 1:1:0.6 (or 1:1:1.5). At higher resolution, it becomes apparent that the particle has a significantly larger interface with solvent than an homogeneous ellipsoid or globular protein. The model has a globular protein core similar to non-halophilic malate dehydrogenase, with about 20% of the protein extending loosely out of the core, forming the large interface with solvent. The main interactions with water and salt take place on this outer part.

Halobacterium↗
PubMed1986

Small-angle neutron scattering study of the ternary complex formed between bacterial elongation factor Tu, guanosine 5'-triphosphate, and valyl-tRNAVal.

The formation of the ternary complex between bacterial elongation factor Tu, GTP, and valyl-tRNAVal has been studied by small-angle neutron scattering. Titrations of the protein with amino-acyl-tRNA solutions in both H2O and 70% D2O confirm the expected stoichiometry. The molecular weight obtained for the protein alone is significantly higher than expected and can be explained by postulating a monomer-dimer equilibrium. The titration data are then internally consistent with a dissociation of the dimer on ternary complex formation. The radius of gyration for the ternary complex and the calculation of the separation of the centers of mass of the protein and tRNA components suggest a compact model for the ternary complex.

Escherichia coli↗
PubMed1985

Methionyl-tRNA synthetase from E. coli: direct evidence for exchange of protomers in the dimeric enzyme by using deuteration and small-angle neutron scattering.

Direct demonstration of the reversible dissociation of native dimeric methionyl-tRNA synthetase from E. coli has been obtained using small angle neutron scattering and deuterated enzyme. Structural parameters of the fully deuterated dimer are very similar to the hydrogenated one. Analysis of the variations of the intensity and of the radius of gyration of a stoichiometric mixture of the two types of dimer (hydrogenated and deuterated), as a function of D2O content in the solvent, enabled us to characterize an hybrid dimer, having both hydrogenated and deuterated protomers. By separating the contribution of each protomer to the scattering, the radius of gyration of the protomer in situ and the distance between the centers of mass of each protomer in the dimer are determined.

Amino Acyl-tRNA Synthetases↗
PubMed1984

Neutron small-angle scattering studies of ribonuclease in mixed aqueous solutions and determination of the preferentially bound water.

Neutron small-angle measurements of ribonuclease A in mixed solutions of ethanol-water and glycerol-water have been used to estimate the region near the molecule that only contains water. When glycerol is used as probe, the region corresponds to an interaction parameter xi of 0.23 +/- 0.05 g of water/g of protein. For ethanol xi is -0.07 +/- 0.05, corresponding to the macromolecule being equally accessible to ethanol and water. The observations of the radius of gyration in the mixed solutions are used to show that the volume excluding glycerol is found on the exterior of the protein.

Ethanol↗
PubMed1983

Optical properties and small-angle neutron scattering of bovine heart mitochondrial oligomycin sensitivity conferring protein.

The mitochondrial oligomycin sensitivity conferring protein (OSCP) was isolated from beef heart mitochondria according to Senior (1979) [Senior, A.E. (1979) Methods Enzymol. 55,391-397] with a supplementary step of Sephadex chromatography for more extensive purification. The ultraviolet and fluorescence spectra of OSCP were consistent with the presence of tyrosyl residues and the absence of tryptophanyl residues. From the circular dichroism spectrum of OSCP, 43% alpha-helical structure was calculated; the dichroism spectra of OSCP in H2O and D2O were identical. A molecular weight (Mr) of 22 000 for OSCP was determined by sodium dodecyl sulfate gel electrophoresis at different concentrations of the polyacrylamide gel. The radius of gyration (Rg) and the shape of OSCP in H2O and D2O were studied by small-angle neutron scattering. The experimentally determined Rg value of OSCP in H2O was 24 +/- 1 A, and its Mr was 25 000 +/- 3000. Comparison of the experimental Rg value with that expected for a compact globular protein of the same molecular weight (17 A) led to the conclusion that OSCP is a considerably elongated molecule protein with an axial ratio higher than 3. In D2O buffer, the Rg value was higher than that in H2O, a situation in contrast with that observed for most globular hydrophilic proteins; this might be due to a preferential location of the positively charged lysine residues.

Adenosine Triphosphatases↗
PubMed1983

Neutron diffraction analysis of cytochrome b5 reconstituted in deuterated lipid multilayers.

Cytochrome b5 was reconstituted with a highly deuterated phospholipid to form ordered multilayers consisting of repeated centrosymmetric pairs of asymmetric lipid-protein bilayers. Lamellar neutron diffraction data were collected to approximately 29 A resolution, and have been interpreted using models for the interaction of the membrane-binding domain of cytochrome b5 with the lipid bilayer. A range of different models was examined, and those in which the protein penetrates well into the bilayer, possibly spanning it, are favored.

Animals↗
PubMed1983

Assignment of segments of the bacteriorhodopsin sequence to positions in the structural map.

Specific amino acid sequence segments have been assigned to locations in the structural map of bacteriorhodopsin using two-dimensional neutron diffraction data and a model building analysis. Models are constructed computationally by building specific regions of the amino acid sequence as alpha helices and then positioning the helices on axes indicated by the density map of Henderson and Unwin (Nature [Lond.]. 1975, 257:28-32). Neutron diffraction data were collected from samples of stacked, oriented "native" purple membranes as well as purple membranes containing different kinds of deuterated amino acids. Models differing in the assignments of helices to specific axes and in rotations of the helices about those axes were tested against the neutron data using a weighted residual factor to rank the models. This residual factor was calculated between observed and predicted intensity differences for pairs of data sets. Using this approach, a small set of related models has been found that predicts the observed intensity changes between five independent data sets. These models are inconsistent with the proposed locations of the retinal chromophore and the carboxyl terminus and with any of the previously proposed models for bacteriorhodopsin.

Amino Acid Sequence↗
PubMed1982

Spontaneous aggregation of the mitochondrial natural ATPase inhibitor in salt solutions as demonstrated by gel filtration and neutron scattering. Application to the concomitant purification of the ATPase inhibitor and F1-ATPase.

(1) The natural ATPase inhibitor (IF1) from beef heart mitochondria has a tendency to form aggregates in aqueous solutions. The extent of aggregation and the structure of the aggregates were assessed by gel filtration and small-angle neutron scattering. IF1 polymerization was found to depend on the salt concentrations, pH of the medium and concentration of IF1. The higher the salt concentration, the lower the aggregation state. Aggregation of IF1 was decreased at slightly acidic pH. It increased with the concentration of IF1 as expected from the law of mass action. (2) Neutron scattering showed the aggregation of IF1 in 2 M ammonium sulfate solutions. The predominant species is the dimer which has a somewhat elongated shape. (3) The Sephadex G-50 chromatography that is supposed to deprive beef heart submitochondrial particles of loosely bound IF1 (Racker, E. and Horstman, L.L. (1967) J. Biol. Chem. 242, 2547-2551) was shown to have a limited effectiveness as a trap for IF1. The reason was that IF1 released from the particles formed high molecular weight aggregates that were not separated from the membrane vesicles by Sephadex G-50 chromatography. (4) The above observations provide the basis for a simple method of purification of beef heart IF1 which combines the recovery of the supernatant from submitochondrial particles with the last three steps of the IF1 preparation described by Horstman and Racker (J. Biol. Chem. (1970) 265, 1336-1344). The particles recovered in the sediment were deprived of IF1 and could therefore be used for preparation of F1-ATPase. The advantage of this method is that both IF1 and F1-ATPase can be prepared from the same batch of mitochondria.

Adenosine Triphosphatases↗
PubMed1982

Formation of a catalytically active complex between tRNAAsp and aspartyl-tRNA synthetase from yeast in high concentrations of ammonium sulphate.

The interactions of yeast tRNAAsp with cognate aspartyl-tRNA synthetase have been studied in high concentrations of either sodium chloride or ammonium sulphate by fluorescence titration and small-angle neutron scattering. In solutions containing more than 1M NaCl no complex is formed and enzymatic activity is abolished. In strong contrast, however, the physical measurements showed the formation of a two-to-one tRNA-enzyme complex, with high affinity, in 1.6 M (NH4)2SO4. Aminoacylation assays under the same salt conditions showed the enzymatic fixation of aspartic acid to tRNAAsp to occur at an appreciable rate. The present study emphasizes that the effects of salts on protein-nucleic acid interactions do not depend only on ionic strength but also on the nature of the salt. This study has allowed a rational approach to the crystallisation of a functional tRNAAsp-aspartyl-tRNA synthetase complex (Giegé, Lorber, Ebel, Thierry and Moras (1980) C.R. Acad. Sci. Paris, série D, 291, 393-396).

Amino Acyl-tRNA Synthetases↗