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E Pebay-Peyroula

Publications and source records attributed to E Pebay-Peyroula.

32 records · Page 2Linked to original sources
PubMed1997

X-ray structure of bacteriorhodopsin at 2.5 angstroms from microcrystals grown in lipidic cubic phases.

Lipidic cubic phases provide a continuous three-dimensional bilayer matrix that facilitates nucleation and growth of bacteriorhodopsin microcrystals. The crystals diffract x-rays isotropically to 2.0 angstroms. The structure of this light-driven proton pump was solved at a resolution of 2.5 angstroms by molecular replacement, using previous results from electron crystallographic studies as a model. The earlier structure was generally confirmed, but several differences were found, including loop conformations and side chain residues. Eight water molecules are now identified experimentally in the proton pathway. These findings reveal the constituents of the proton translocation pathway in the ground state.

Bacteriorhodopsins↗
PubMed1997

The crystal structure of plant acetohydroxy acid isomeroreductase complexed with NADPH, two magnesium ions and a herbicidal transition state analog determined at 1.65 A resolution.

Acetohydroxy acid isomeroreductase catalyzes the conversion of acetohydroxy acids into dihydroxy valerates. This reaction is the second in the synthetic pathway of the essential branched side chain amino acids valine and isoleucine. Because this pathway is absent from animals, the enzymes involved in it are good targets for a systematic search for herbicides. The crystal structure of acetohydroxy acid isomeroreductase complexed with cofactor NADPH, Mg2+ ions and a competitive inhibitor with herbicidal activity, N-hydroxy-N-isopropyloxamate, was solved to 1.65 A resolution and refined to an R factor of 18.7% and an R free of 22.9%. The asymmetric unit shows two functional dimers related by non-crystallographic symmetry. The active site, nested at the interface between the NADPH-binding domain and the all-helical C-terminus domain, shows a situation analogous to the transition state. It contains two Mg2+ ions interacting with the inhibitor molecule and bridged by the carboxylate moiety of an aspartate residue. The inhibitor-binding site is well adjusted to it, with a hydrophobic pocket and a polar region. Only 24 amino acids are conserved among known acetohydroxy acid isomeroreductase sequences and all of these are located around the active site. Finally, a 140 amino acid region, present in plants but absent from other species, was found to make up most of the dimerization domain.

Alcohol Oxidoreductases↗
PubMed1996

Protein-detergent interactions in single crystals of membrane proteins studied by neutron crystallography.

The detergent micelles surrounding membrane protein molecules in single crystals can be investigated using neutron crystallography combined with H2O/D2O contrast variation. If the protein structure is known then the contrast variation method allows phases to be determined at a contrast where the detergent dominates the scattering. The application of various constraints allows the resulting scattering length density map to be realistically modeled. The method has been applied to two different forms of the membrane protein porin. In one case both hydrogenated and partially deuterated protein were used, allowing the head group and tail to be distinguished.

Crystallography↗
PubMed1995

Detergent structure in tetragonal crystals of OmpF porin.

BACKGROUND: The high-resolution structures of five porins have been solved by X-ray crystallography including the trigonal crystal form of the trimeric OmpF porin from Escherichia coli. In an accompanying article, the structure of the tetragonal form of OmpF porin is presented. In contrast to the trigonal crystal form, the protein surfaces normally in contact with lipids in the membrane are exposed and interact with amphiphiles in the tetragonal crystal. Thus, the tetragonal form can be used to investigate protein-detergent interactions. RESULTS: Using single-crystal neutron diffraction studies and two different detergents (one of them deuterated in its hydrophobic moiety), details of the amphiphile-protein interactions are revealed. Detergent molecules bind to the so-called hydrophobic zone that surrounds the OmpF porin trimer and which is exposed to lipid in the native environment. The aromatic rings on both sides of the hydrophobic zone coincide with the boundary between non-polar and polar moieties of the detergents. CONCLUSIONS: In the tetragonal crystal form of OmpF porin, the membrane-exposed area is accessible from the aqueous solution. It is coated by a film of detergent molecules, which presumably mimics the interactions of the protein with lipids in the biological membrane. In the trigonal form, protein-protein interactions predominate in the hydrophobic zone. These may reflect the tight interactions between trimers that are observed in the biological membrane.

Bacterial Outer Membrane Proteins↗
PubMed1994

Crystallization and preliminary crystallographic data for acetohydroxy acid isomeroreductase from Spinacia oleracea.

Acetohydroxy acid isomeroreductase (EC 1.1.1.86) is one of the enzymes involved in branched-chain amino acid biosynthesis. The enzyme from spinach (Spinacia oleracea) leaves has been crystallized using the hanging drop vapour diffusion method. The free enzyme crystallized from polymethylene glycol solutions, but these crystals were unsuitable for X-ray diffraction analysis. In the presence of NADPH, Mg(2+) and a reaction intermediate analogue (2-dimethylphosphinoyl-2-hydroxy acetic acid (Hoe 704) or N-hydroxy-N-isopropyloxamate (IpOHA)), much better crystals were obtained. Crystals grown from ammonium sulphate belong to space group P2(1) with cell dimensions a + 193.78(7) A, b = 63.69(2) A, c = 112.84(1) A and beta = 121.22(1) degrees. The molecular mass of the protein, the volume of the unit cell, and crystal density measurements indicated that the asymmetric unit contains two dimers. X-ray diffraction patterns showed measurable reflections to beyond 2.5 A.

Alcohol Oxidoreductases↗
PubMed1994

Detergent organisation in solutions and in crystals of membrane proteins.

The use of neutron scattering in studying the organisation of detergents in pure micelles, in protein/detergent mixed micelles and in crystals of membrane proteins, is reviewed. Small angle scattering has been used to study the size, shape and composition of pure and mixed protein/detergent micelles as well as the effects of adding small amphiphiles. The technique of contrast variation applied to single crystals is described and its application to the determination of the organization of detergent in single crystals of membrane proteins is discussed. A better understanding of protein/detergent interactions should help in producing crystals of membrane proteins more easily as well as clues to the nature of protein/lipid interactions in vivo.

Journal Article↗
PubMed1994

Location of diphenyl-hexatriene and trimethylammonium-diphenyl-hexatriene in dipalmitoylphosphatidylcholine bilayers by neutron diffraction.

Neutron scattering experiments have been performed on oriented dipalmitoylphosphatidylcholine (DPPC) bilayers containing diphenylhexatriene (DPH) or its trimethylammonium analog (TMA-DPH). DPH and TMA-DPH were either protonated or deuterated in one of the phenyl rings which afforded by using proton-deuterium contrast methods the location of these fluorescent probes in the model membrane. Both probes exhibit bimodal distributions in DPPC. The position, population and orientation in the two sites vary depending upon the physical state of the bilayer (gel or fluid) and the presence or absence of the TMA group. In gel (L beta') phase lipids DPH is located close and parallel to the bilayer surface (site I) and near the bilayer center, oriented at approximately 30 degrees with respect to the normal to the surface (site II). On going to the fluid (L alpha) phase, a distribution of orientations around the parallel to the surface is only observed for site II. Orientation of DPH in site I is unchanged. In the gel phase TMA-DPH is found in a position close and parallel to the bilayer surface (site I) and in a position (site II) oriented at an angle of approximately 25 degrees with respect to the bilayer normal, with the trimethylammonium group anchored in the head group domain. On going to the fluid phase there is a change in molecular orientation of each of the sites. In site I the molecule penetrates deeper in the bilayer and adopts a approximately 20 degrees tilt with respect to the surface, with an orientational distribution of +/- 10 degrees. In site II the molecule becomes perpendicular to the membrane surface. Changes in population of sites, both with DPH and TMA-DPH, are observed on going from low to high temperatures. They are however difficult to quantitate due to experimental conditions. The H2O-2H2O exchange experiments afforded an estimate of the water layer thickness as well as the maximum penetration of water into the interior of the bilayer.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1993

Complete amino acid sequence of puroindoline, a new basic and cystine-rich protein with a unique tryptophan-rich domain, isolated from wheat endosperm by Triton X-114 phase partitioning.

A new basic protein has been isolated from wheat endosperm by Triton X-114 phase partitioning. It contains five disulfide bridges and is composed of equal amounts of a polypeptide chain of 115 amino acid residues and of the same chain with a C-terminus dipeptide extension. The most striking sequence feature is the presence of a unique tryptophan-rich domain so that this protein isolated from wheat seeds has been named puroindoline. The similar phase partitioning behavior in Triton X-114 of this basic cystine-rich protein and of purothionins suggests that puroindoline may also be a membranotoxin that might play a role in the defense mechanism of plants against microbial pathogens.

Amino Acid Sequence↗
PubMed1993

Crystal structure of hydrophobic protein from soybean; a member of a new cysteine-rich family.

X-ray diffraction methods have been used to determine the structure of the 8.3 kDa hydrophobic protein from soybean and to refine the atomic co-ordinates to a crystallographic R-factor of 18.7% at 1.8 A resolution. The molecule is a four-helix bundle, which together with the connecting loops and a twisted beta-strand form a spiral. The surface contains 70% apolar atoms, and the crystal packing is dominated by hydrophobic interactions, producing a two-dimensional sheet of protein molecules. Most of the 59 water molecules located are involved in hydrophilic contacts and their structural organization does not seem to be affected by the high hydrophobicity of the molecule. From the protein fold it appears that three of the four disulphide bridges are important for keeping the amino and carboxyl-terminal segments in place in the native form, while the central part of the molecule is stabilized by many hydrophobic interactions. Although the protein function is not known, a number of possibilities can be excluded on experimental grounds and by comparison with other members of the family.

Amino Acid Sequence↗
PubMed1992

Crystallographic data for the 9000 dalton wheat non-specific phospholipid transfer protein.

The wheat non-specific phospholipid transfer protein belongs to a family of small proteins sharing a common pattern of four disulphide bridges. Its function in vivo is not known, but it has a high affinity to phospholipids and is involved in phospholipid transfer in vitro. The molecular weight is 9607, and it crystallizes in the space group P2(1) with a = 40.73 A, b = 112.11 A, c = 50.44 A and beta = 106.80 degrees. The crystals diffract to 3 A resolution.

Carrier Proteins↗
PubMed1989

Crystallographic data for soybean hydrophobic protein.

The soybean hydrophobic protein belongs to a family of proteins that contains a number of storage and phospholipid binding proteins. Its function is not known, but its overall hydrophobic nature is typical of many membrane proteins of similar size. The molecular weight is 8.3 x 10(3), and it crystallizes in the space group P2(1)2(1)2(1), with a = 52.01 A, b = 43.50 A and c = 28.80 A. The crystals diffract to 1.8 A resolution, and are thus suitable for X-ray structural studies.

Crystallography↗
PubMedDate not supplied

The interplay between X-ray crystallography, neutron diffraction, image reconstruction, organo-metallic chemistry and biochemistry in structural studies of ribosomes.

Crystals of ribosomes, their complexes with components of protein biosynthesis, their natural, mutated and modified subunits, have been subjected to X-ray and neutron crystallographic analyses. Electron microscopy and 3-dimensional image reconstruction, supported by biochemistry, genetic, functional and organo-metallic studies were employed for facilitating phasing of the crystallographic data. For example, a monofunctional multi heavy-atom cluster (undecagold) was designed for covalent and quantitative binding to ribosomes. The modified particles were crystallized isomorphously with the native ones. Their difference-Patterson maps contain indications for the usefulness of these derivatives for subsequent phasing. Models of the ribosome and its large subunit were reconstructed from tilt series of 2-dimensional sheets. The comparison of the various reconstructed images enabled an initial assessment of the reliability of these models and led to tentative assignments of several functional features. These include the presumed sites for binding mRNA and for codon-anticodon interactions, the path taken by the nascent protein chain and the mode for tRNA binding to ribosomes. These assignments assisted in the design of biologically meaningful crystal systems. The reconstructed models are being used to identify structural features in initial density maps derived from X-ray and neutron diffraction data.

Crystallography↗
PubMedDate not supplied

Detergent binding in trigonal crystals of OmpF porin from Escherichia coli.

The structure of the detergent, ocytyl hydroxyethylsufoxide (C8(HE)SO), bound to the OmpF porin from E coli (in the trigonal crystal form) has been determined by neutron crystallography. Due to a dynamic exchange of detergent molecules with their environment they are not ordered on an atomic scale. The structure reported here is therefore at a resolution of approximately 16 A. The X-ray crystallographically determined structure of the protein provides a starting point for the neutron analysis in which the detergent is visualized primarily thanks to its high contrast against D2O. The structure shows the detergent to be located mainly in two areas. It forms toroidal annuli around each OmpF trimer, these annuli fusing to form a detergent belt surrounding a solvent filled column traversing the crystal. Those areas of the protein to which the detergent binds are formed almost exclusively of hydrophobic residues and form a band about 30 A high around the trimer. Its upper and lower bounds are defined by two bands of aromatic residues, tyrosines pointing away from the detergent belt and interacting with the polar headgroups while phenylalanines point inwards. This strongly suggests that the same areas define, in vivo, the location at which protein interacts with lipid. The hydrophobic moiety of detergent is also found mediating the hydrophobic protein-protein interactions at the interface between two trimers on the crystallographic two-fold axis.

Crystallization↗