Biomedical subjects

G Zaccaï

Publications and source records attributed to G Zaccaï.

14 recordsLinked to original sources

PubMed1994

Stability against denaturation mechanisms in halophilic malate dehydrogenase "adapt" to solvent conditions.

Malate dehydrogenase from Haloarcula marisomortui (hMDH) is active, soluble and mildly unstable in an unusually wide range of salt conditions and temperatures, making it a particularly interesting model for the study of solvent effects on protein stability. Its denaturation (loss of activity due to concomitant dissociation and unfolding) kinetics was studied as a function of temperature and concentration of NaCl, potassium phosphate or ammonium sulphate in H2O or 2H2O. A transition-state-theory analysis was applied to the data. In all cases, stability (resistance to denaturation) increased with increasing salt concentration, and when 2H2O replaced H2O. Each salt condition was associated with a particular energy regime that dominated stability. In NaCl/H2O, a positive enthalpy term, delta H not equal to 0, always dominated the activation free energy of denaturation, delta G not equal to 0. In potassium phosphate/H2O and ammonium sulphate/H2O, on the other hand, stability was dominated by a negative activation entropy, delta S not equal to 0. and delta H not equal to 0 changed sign between 10 degrees C and 20 degrees C, consistent with a strong hydrophobic effect contribution, in these salting-out solvents. Decreasing stability at low temperatures, favouring cold denaturation, was observed. Replacing H2O by 2H2O strengthened the hydrophobic effect in all conditions. As a consequence, conditions were found in which hMDH was not halophilic; below 10 degrees C, it was stable in approximately 0.1 M NaCl/2H2O. The solution structure and preferential solvent interactions of hMDH in H2O or 2H2O solvents containing NaCl were studied by densimetry and neutron scattering. Despite the different stability of the protein in H2O or 2H2O, an experimentally identical invariant solution particle was formed in both solvents. It had a total volume of 1.165 cm3 g-1 and bound about 0.4 g of H2O (0.44 g of 2H2O) and about 0.08 g NaCl g protein. The impact of these results on a stabilisation model for hMDH, involving ion binding, is discussed.

Ammonium Sulfate↗

PubMed1994

The triple isotopic substitution method in small angle neutron scattering. Application to the study of the ternary complex EF-Tu.GTP.aminoacyl-tRNA.

The TIS (triple isotopic substitution) method in small angle neutron scattering was applied to determine the radius of gyration of polypeptide elongation factor Tu (EF-Tu) from E. coli associated with GDP and within the ternary complex EF-Tu.GTP.aminoacyl-tRNA. The results showed that, within errors of about 1 A, there is no change in the radius of gyration of the EF-Tu moiety upon ternary complex formation. Experiments were performed in H2O buffer, in which complex formation could be followed on an absolute scale because of the relatively large contrast of both protein and tRNA. The TIS method is based on the analysis of a scattering curve that is the difference between the scattering of two solutions containing appropriately deuterium labelled particles. A necessary condition for the application of the method is that the two solutions are identical in all respects except for the extent of deuterium label. The main properties of TIS that make it very useful for the study of complex particles in solution were confirmed by this study. These are the elimination of interparticle effects in the difference curve, the 'invisibility' of unlabelled parts of the particles and the independence of the difference scattering curve on the buffer 2H2O-H2O content. The last property is of particular interest for the study of interactions that may be influenced by 2H2O, since, contrary to classical contrast variation methods, TIS experiments can be performed in H2O buffer alone.

Guanosine Triphosphate↗

PubMed1990

Neutron scattering study of the (gamma-B) catalytic domains of complement proteases activated C1r and C1s.

The catalytic domains of activated C1r and C1s, comprising the C-terminal region of the A chain (gamma), disulphide-linked to the B chain, were obtained by limited proteolysis of the native proteases with chymotrypsin and plasmin, respectively, and studied by small angle neutron scattering. For activated C1s (gamma-B), a molar mass of 45,000 +/- 5000 g/mol, and a relatively large radius of gyration (Rg) of 28 +/- 1 A were determined, excluding a single globular domain. The corresponding values for activated C1r (gamma-B)2 (90,000 g/mol, Rg = 34 +/- 1 A) are consistent with a dimer involving the loose packing of two (gamma-B) subunits. Various models of the dimer are discussed in the light of neutron scattering and other data.

Chymotrypsin↗

PubMed1989

Tertiary structure of bacteriorhodopsin. Positions and orientations of helices A and B in the structural map determined by neutron diffraction.

Positions and rotations of two helices in the tertiary structure of bacteriorhodopsin have been studied by neutron diffraction using reconstituted, hybrid purple membrane samples. Purple membrane was biosynthetically 2H-labeled at non-exchangeable hydrogen positions of leucine and tryptophan residues. Two chymotryptic fragments were purified, encompassing either the first two or the last five of the seven putative transmembrane segments identified in the amino acid sequence of bacteriorhodopsin. The 2H-labeled fragments, diluted to variable extents with the identical, unlabeled fragment, were mixed with their unlabeled counterpart; bacteriorhodopsin was then renatured and reconstituted. The crystalline purple membrane samples thus obtained contained hybrid bacteriorhodopsin molecules in which certain transmembrane segments had been selectively 2H-labeled to various degrees. Neutron diffraction powder patterns were recorded and analyzed both by calculating difference Fourier maps and by model building. The two analyses yielded consistent results. The first and second transmembrane segments in the sequence correspond to helices 1 and 7 of the three-dimensional structure, respectively. Rotational orientations of these two helices were identified using best fits to the observed diffraction intensities. The data also put restrictions on the position of the third transmembrane segment. These observations are discussed in the context of folding models for bacteriorhodopsin, the environment of the retinal Schiff base, and site-directed mutagenesis experiments.

Amino Acid Sequence↗

PubMed1986

Localization of two chymotryptic fragments in the structure of renatured bacteriorhodopsin by neutron diffraction.

The structure of crystalline purple membrane reconstituted from purified bacteriorhodopsin (BR) chymotryptic fragments has been studied by neutron diffraction. In one of the samples studied, the fragment C-2, encompassing the first two predicted transmembrane segments, was prepared from deuterated purple membrane. The diffraction changes when the natural C-2 fragment is substituted by a deuterated one are analysed in terms of a seven-helix model for BR. The assignment of the labelled fragment to one end of the molecule placed new constraints on folding models for the protein.

Bacteriorhodopsins↗

PubMed1984

Retinal location in purple membrane of Halobacterium halobium: a neutron diffraction study of membranes labelled in vivo with deuterated retinal.

Purple membranes were prepared by growing Halobacterium halobium in a medium containing nicotine (which inhibits biosynthesis of retinal) and the oxidation products of fully deuterated beta-carotene. This allowed the in vivo incorporation of deuterated retinal into the membranes. The labelled membranes were crystalline and isomorphous with native membrane as determined by X-ray diffraction, and their optical absorption spectra were very similar. Neutron diffraction data for the two dimensional in-plane lattice from labelled and native membranes were analysed by difference Fourier and direct methods to 8.6 A resolution. The difference Fourier shows the retinal to be located in the centre of the bacteriorhodopsin molecule. The best fit to the data was obtained with the projection of retinal as a 10 A long rod forming an angle of -40 degrees +/- 10 degrees with the x axis centred at x = -0.19 +/- 0.02, y = -0.35 +/- 0.02 in fractional unit cell coordinates. The main peak in the difference Fourier map is at x = -0.17, y = -0.33.

Bacteriorhodopsins↗

PubMed1983

Structure of phenylalanine-accepting transfer ribonucleic acid and of its environment in aqueous solvents with different salts.

Thermodynamic and structural parameters were measured for brewers' yeast tRNAPhe in solution in the range of 0.1-0.9 M monovalent salt (with and without 1 mM MgCl2), pH 7.0, by small-angle neutron scattering. Partial specific volumes and preferential interaction parameters were found to be similar to corresponding values measured by more conventional means in DNA [Eisenberg, H. (1981) Q. Rev. Biophys. 14, 141-172]. There is no evidence of a large conformational change in tRNAPhe in this range, and the molecule has a radius of gyration that is the same as that calculated from the crystal-structure coordinates (23 A). Transfer RNA in solution is made up of polyion tRNA76- and 76 positive monovalent ions (in absence of Mg2+). The data show the polyion to be surrounded by a shell of solvent that is significantly denser than bulk, whose structure depends on salt conditions. In 0.1 M NaCl, it has an excess mass of approximately 85 molecules of water. This would be accounted for, for example, by approximately 850 molecules of water if their density were 10% higher than that for bulk. The radius of gyration of the dense shell is approximately 30 A for NatRNA and approximately 35 A for KtRNA. The present study shows that the solvent around tRNA is a component of its structure that must be taken into account in understanding its function.

Kinetics↗

PubMed1983

Nonspecific binding of lac repressor to DNA. II. A small-angle neutron-scattering study.

Complexes between lac repressor and DNA fragments from mononucleosomes have been studied by small-angle neutron scattering. Both the radius of gyration and the molecular weight of the complexes were measured, and the experimental results were interpreted according to a model with two types of complex (M. Charlier and J.-C. Maurizot, Biophys. Chem. 18 (1983) 303), and a statistical distribution of repressor on the DNA fragments. Good agreement between the model calculations and the experimental results was obtained. We concluded that there was an absence of strong cooperativity and of network formation between the complexes. The second type of binding, which does not induce any spectroscopic change, is marked by an increase in molecular weight of the complexes. Kinetic measurements were also made, which allowed the determination of the lifetime of the nonspecific DNA-repressor complexes.

DNA↗

PubMed1983