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Light scattering and small-angle neutron scattering experiments were performed on comicelles of several combinations of oppositely charged (block co)polymers in aqueous solutions. Fundamental differences between the internal structure of this novel type of micelle --termed complex coacervate core micelle (C3Ms), polyion complex (PIC) micelle, block ionomer complex (BIC), or interpolyelectrolyte complex (IPEC)-- and its traditional counterpart, i.e., a micelle formed via self-assembly of polymeric amphiphiles, give rise to differences in scaling behaviour. Indeed, the observed dependencies of micellar size and aggregation number on corona block length, N (corona) , are inconsistent with scaling predictions developed for polymeric micelles in the star-like and crew-cut regime. Generic C3M characteristics, such as the relatively high core solvent fraction, the low core-corona interfacial tension, and the high solubility of the coronal chains, are causing the deviations. A recently proposed scaling theory for the cross-over regime, as well as a primitive first-order self-consistent field (SCF) theory for obligatory co-assembly, follow our data more closely.

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Using small-angle neutron scattering combined with a containerless aerodynamic levitation technique for high temperatures, we have measured the temperature dependence of the correlation length ξ of near-critical magnetic fluctuations in the solid phase of the completely miscible fcc alloy Co80Pd20. A fit to our data yields a critical exponent ν = 0.76 ± 0.05 for the divergence of ξ(T) above the ferromagnetic transition temperature Tc. This value of ν is consistent with the prediction of the three-dimensional Heisenberg model for magnetic critical scattering.

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We present a combined 1H-NMR and small angle neutron scattering in situ study of the anionic polymerization of butadiene using t-butyllithium as the initiator. Both initiation and propagation phases were explored. This combined approach allows the structural and kinetic characteristics to be accessed and cross compared. The use of the D22 instrument (ILL Grenoble) permits the attainment of Q approximately equal to 2 x 10(-3) A. This, in turn, led to the identification of coexisting large-scale and smaller aggregates during all stages of the polymerization. The smaller aggregates contain most of the reacted monomers. Their structure changes from high functionality wormlike chains at early stages of the reaction to starlike aggregates where the crossover occurs at a degree of polymerization of approximately equal to 40. The initiation event involved these small, high functionality (approximately equal to 120) aggregates that apparently consisted of cross-associated t-butyllithium with the newly formed allylic-lithium head groups. As the initiation event progressed the initiation rate increased while the functionality of these small aggregates decreased and their size increased. Propagation, in the absence of initiation, was found to have a rate constant that was molecular weight dependent. At approximately 11 kg/mol the measured polymerization rate was found to increase while no further structural changes were seen.

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Protein folding by chaperonins is powered by ATP binding and hydrolysis. ATPase activity drives the folding machine through a series of conformational rearrangements, extensively described for the group I chaperonin GroEL from Escherichia coli but still poorly understood for the group II chaperonins. The latter--archaeal thermosome and eukaryotic TRiC/CCT--function independently of a GroES-like cochaperonin and are proposed to rely on protrusions of their own apical domains for opening and closure in an ATP-controlled fashion. Here we use small-angle neutron scattering to analyze structural changes of the recombinant alpha-only and the native alphabeta-thermosome from Thermoplasma acidophilum upon their ATPase cycling in solution. We show that specific high-salt conditions, but not the presence of MgATP alone, induce formation of higher order thermosome aggregates. The mechanism of the open-closed transition of the thermosome is strongly temperature-dependent. ATP binding to the chaperonin appears to be a two-step process: at lower temperatures an open state of the ATP-thermosome is predominant, whereas heating to physiological temperatures induces its switching to a closed state. Our data reveal an analogy between the ATPase cycles of the two groups of chaperonins and enable us to put forward a model of thermosome action.

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Mapping of protein positions in the ribosomal subunits was first achieved for the 30S subunit by means of neutron scattering about 15 years ago. Since the 50S subunit is almost twice as large as the 30S subunit and consists of more proteins, it was difficult to apply classical contrast variation techniques for the localisation of the proteins. Polarisation dependent neutron scattering (spin-contrast variation) helped to overcome this restriction. Here a map of 14 proteins within the 50S subunit from Escherichia coli ribosomes is presented including the proteins L17 and L20 that are not present in archeal ribosomes. The results are compared with the recent crystallographic map of the 50S subunit from the archea Haloarcula marismortui.

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Using small-angle neutron scattering (SANS), we have measured the salt-dependent static structure factor of di- and trinucleosomes from chicken erythrocytes and from COS-7 cells. We also determined the sedimentation coefficients of these dinucleosomes and dinucleosomes reconstituted on a 416-bp DNA containing two nucleosome positioning sequences of the 5S rDNA of Lytechinus variegatus at low and high salt concentrations. The internucleosomal distance d was calculated by simulation as well as Fourier back-transformation of the SANS curves and by hydrodynamic simulation of sedimentation coefficients. Nucleosome dimers from chicken erythrocyte chromatin show a decrease in d from approximately 220 A at 5 mM NaCl to 150 A at 100 mM NaCl. For dinucleosomes from COS-7 chromatin, d decreases from 180 A at 5 mM to 140 A at 100 mM NaCl concentration. Our measurements on trinucleosomes are compatible with a compaction through two different mechanisms, depending on the salt concentration. Between 0 and 20 mM NaCl, the internucleosomal distance between adjacent nucleosomes remains constant, whereas the angle of the DNA strands entering and leaving the central nucleosome decreases. Above 20 mM NaCl, the adjacent nucleosomes approach each other, similar to the compaction of dinucleosomes. The internucleosomal distance of 140-150 A at 100 mM NaCl is in agreement with distances measured by scanning force microscopy and electron microscopy on long chromatin filaments.

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Chaperonins are double-ring protein assemblies with a central cavity that provides a sequestered environment for in vivo protein folding. Their reaction cycle is thought to consist of a nucleotide-regulated alternation between an open substrate-acceptor state and a closed folding-active state. The cavity of ATP-charged group I chaperonins, typified by Escherichia coli GroEL [1], is sealed off by a co-chaperonin, whereas group II chaperonins--the archaeal thermosome and eukaryotic TRiC/CCT [2]--possess a built-in lid [3-5]. The mechanism of the lid's rearrangements requires clarification, as even in the absence of nucleotides, thermosomes of Thermoplama acidophilum appear open in vitrified ice [6] and closed in crystals [4]. Here we analyze the conformation of the thermosome at each step of the ATPase cycle by small-angle neutron scattering. The apo-chaperonin is open in solution, and ATP binding induces its further expansion. Closure seems to occur during ATP hydrolysis and before phosphate release, and represents the rate-limiting step of the cycle. The same closure can be triggered by the crystallization buffer. Thus, the allosteric regulation of group II chaperonins appears different from that of their group I counterparts.

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We applied neutron scattering in conjunction with deuterium (D-) labeling in order to obtain information about the domain structure of GroEL and GroES isolated and in the complex. Each subunit of the heptameric GroES consists of two domains, a core domain (Met1 to Lys15 and Lys34 to Ala97) and an intervening loop region (Glu16 to Ala33). Neutron scattering shows that both regions change their conformation upon GroEL/GroES complex formation. The interdomain angle between the core regions of the heptameric GroES increases from 120 to 140 degrees, leading to a less dome-like shape of GroES, and the loop regions turn inwards by 75 degrees. The 23 C-terminal amino acids of the 14 GroEL subunits (Lys526 to Met548), which are unresolved in the crystal structure, are located either at the bottom of the cavity formed by the seven-membered GroEL ring or at the inner wall of the cavity. Upon complex formation the apical domains of GroEL move outwards, which facilitates binding of GroES at a Gro-EL-GroES center-to-center distance of (87 +/- 8) A. These structural changes may be important for the dissociation of the unfolded protein bound to the central cavity upon GroES binding. The overall structure determined by neutron scattering in solution tallies with the crystallographic model published after completion of this study. Differences in the conformation of GroES observed in the complex by the two methods support the view that the chaperonin complex is a flexible molecule which might switch in solution between different conformations.

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The function of proliferating cell nuclear antigen (PCNA) in DNA replication and repair is to form a sliding clamp with replication factor C (RF-C) tethering DNA polymerase delta or epsilon to DNA. In addition, PCNA has been found to interact directly with various proteins involved in cell cycle regulation. The crystal structure of yeast PCNA shows that the protein forms a homotrimeric ring lining a hole through which double-stranded DNA can thread, thus forming a moving platform for DNA synthesis. Human and yeast PCNA are highly conserved at a structural and functional level. We determined the solution structure of functionally active human PCNA by small-angle neutron scattering. Our measurements strongly support a trimeric ring-like structure of functionally active PCNA in solution, and the data are in good agreement with model calculations based on the crystal structure from yeast PCNA. The human PCNA used in the small-angle neutron scattering experiments was active before and after the measurements in a RF-C independent and a RF-C dependent assay suggesting that the trimeric structure is the in vivo functional form.

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Earlier neutron small-angle scattering experiments had revealed the low resolution structure of the complex between sodium dodecyl sulfate (SDS) and the single polypeptide (452 amino acid residues) of a water-soluble enzyme. The saturated complex consists of three globular micelles which are connected by short flexible polypeptide segments. New experiments, described here, were performed at subsaturating concentrations of free SDS in equilibrium with the complex. The data show a decrease in stoichiometry from one bound dodecyl sulfate (DS) anion per two amino acid residues near the critical micelle concentration (CMC) to one per four residues at half the CMC. At 0.3 CMC, a two-micelle complex is formed by the recombination of the small amino-terminal micelle with the middle one; and the center-to-center distance between the carboxyl-terminal micelle and the middle one decreases from 7.5 to 6.2 nm. These structural data allow us to better understand earlier results obtained with high-performance agarose gel chromatography of the same SDS-protein complexes.

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Following the 'strategy of the glassy ribosome' single protonated ribosomal proteins (r-proteins) were reconstituted into deuterated 50S subunits of Escherichia coli. The deuteration of both rRNA and r-proteins were individually adjusted to such a degree that the ribosomal matrix appeared nearly homogeneous with respect to coherent neutron scattering and had a scattering density equivalent to a D2O solution of about 90%. Neutron scattering of ribosomal subunits was recorded in reconstitution buffer containing three different concentrations of D2O around 90% D2O (contrast variation). The signal-to-noise ratio achieved allowed us to make a direct determination of the radii of gyration of r-proteins within the 50S subunit and thus provides the first information relating to the shape of these proteins in situ. We present the radii of gyration of 11 r-proteins incorporated into 50S subunits and of 9 isolated r-proteins in solution. In addition, the data concerning the overall dimensions of the r-proteins we report on indicate that conformational changes of at least two individual r-proteins occur during the assembly process of the ribosome.

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Selected pairs of protonated ribosomal proteins were reconstituted into deuterated 50S subunits from Escherichia coli ribosomes. The rRNA of the deuterated ribosomal matrix was derived from cells grown in 76% D2O, the deuterated protein moiety from cells grown in 84% D2O. This procedure warrants that the coherent neutron scattering of deuterated proteins and rRNA is nearly the same and equals that of a D2O solution of approximately 90%. The neutron scattering is recorded in a reconstitution buffer containing approximately 90% D2O. The result is a significant improvement of the coherent signal:noise ratio over traditional methods; due to this dilute solutions can be used, thus preventing unfavorable inter-particle effects. From the diffraction pattern the distance between the mass centers of gravity of the two protonated proteins can be deduced. In this way, 50 distances between proteins within the large subunit have been determined which provide a basis for future models of the large ribosomal subunit describing the spatial distribution of the ribosomal proteins. A model containing seven ribosomal proteins is presented.

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Small-angle neutron scattering (SANS) measurements on dilute and concentrated dispersions of kappa-casein micelles in a buffer at pH = 6.7 were made using the D11 diffractometer in Grenoble. Results indicate that the micelles have a dense core with a fluffy outer layer. This outer layer appears to give rise to a steeply repulsive interaction on contact. In fact, the hard-sphere model best fits the measured scattering intensities. Adding chymosin to the dispersion initiated a fractal flocculation of the micelles and consecutively a coalescence of the micelles. This unexpected second process resembled that of spinodal demixing. The dispersion phase thus separates into a water and a protein phase on a time scale of hours. The observed phenomona contribute to the understanding of the cheese-making process.

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By means of neutron solution scattering we determined the position and orientation of core enzyme and sigma-factor within the Escherichia coli RNA polymerase holoenzyme with the aim of improving existing models. The individual components, core enzyme (E) and sigma-factor (sigma), were highlighted by deuterium labeling and their center-to-center distances determined in the monomeric and the dimeric holoenzyme. The following distance parameters were obtained: dE1-sigma 1 = 8.6(+/- 1) nm, dE1-E2 = 11.5(+/- 1) nm, d sigma 1-sigma 2 = 12.0(+/- 0.7) nm, dE1-sigma 2 = 9(+/- 3) nm. Using a triangulation procedure the position of the sigma-factors, sigma 1 and sigma 2, were determined with respect to the mass center of the core enzyme molecules, E1 and E2, assuming a symmetrical arrangement of the holoenzyme molecules in the dimer (C2 symmetry). In addition, the orientation of the sigma-factor with respect to core enzyme was estimated by means of model calculations. The obtained model of holoenzyme depicts the sigma-factor as buried in a groove of core enzyme, probably between the large subunits beta' and beta.

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The structure of the complex between sodium dodecyl sulfate (SDS) and a deuterated bifunctional enzyme, N-5'-phosphoribosylanthranilate isomerase/indole-3-glycerol-phosphate synthase (Mr 49,484), has been studied in dilute solution by small-angle neutron scattering. The complex nearly acquired its final size, as shown by molecular-sieve chromatography, at the chosen SDS concentration of 1.6 mM, which is slightly below the critical micelle concentration of 1.8 mM (at the ionic strength of 0.1 M). The 452 amino-acid residues of the bifunctional enzyme were combined with 216 detergent molecules. The complex was found to be composed of three protein-decorated SDS micelles of unequal size, connected by short flexible polypeptide segments. The largest of the three micelles was the middle one. The SDS-protein complex contained the dodecyl hydrocarbon moieties in three globular cores. Each core was surrounded by a hydrophilic shell, formed by the hydrophilic and amphiphilic stretches of the polypeptide chain, and by the sulfate head groups of the detergent. The average thickness of these shells was 0.7-0.8 nm. The three-micelle complex was cleaved with trypsin at a single site, possibly in a micelle-connecting segment, into a single-micelle fragment at the carboxyl-terminal which comprised 73 SDS molecules and 163 amino-acid residues, and a dual-micelle fragment. One of the micelles within this larger fragment contained 42 SDS molecules and about 90 amino-acid residues; the other micelle contained 101 SDS molecules and about 190 amino-acid residues. The individual micelle sizes seemed to be determined by the amino-acid sequence.

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The phase structure of isolated bacterial lipid A, the lipid anchor of the lipopolysaccharides of the outer membrane of Gram-negative bacteria, has been investigated by neutron small-angle scattering. The shape of the scattering curves obtained at different H2O/2H2O ratios revealed a lamellar organisation of the lipid A at neutral pH both above and below its main phase temperature (approximately 40-45 degrees C). Analysis of the scattering curves and interpretation of the corresponding thickness distance distribution functions of the lamellar aggregates led to a model in which the lipid A molecules form a bilayer of about 5 nm in thickness. This value for the thickness of the bilayer, as well as the neutron-scattering density profile across the bilayer, can be explained by a molecular model which shows interdigitation of the fatty acid chains of the lipid A.

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Escherichia coli phenylalanyl-tRNA synthetase is a tetrameric protein composed of two types of protomers. In order to resolve the subunit organization, neutron small-angle scattering experiments have been performed in different contrasts with all types of isotope hybrids that could be obtained by reconstituting the alpha 2 beta 2 enzyme from the protonated and deuterated forms of the alpha and beta subunits. Experiments have been also made with the isolated alpha promoter. A model for the alpha 2 beta 2 tetramer is deduced where the two alpha promoters are elongated ellipsoids (45 x 45 x 160 A3) lying side by side with an angle of about 40 degrees between their long axes and where the two beta subunits are also elongated ellipsoids (31 x 31 x 130 A3) with an angle of 30 degrees between their axes. This model was obtained by assuming that the two pairs of subunits are in contact in an orthogonal manner and by taking advantage of the measured distance between the centers of mass of the alpha 2 and beta 2 pairs (d = 23 +/- 2 A).

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The spatial arrangement of Tet repressor dimer, both free and in complex with an 80 bp DNA fragment spanning the wild-type Tn10-encoded tet transcriptional control sequence containing a tandem repeat of two operators, has been determined by neutron small-angle scattering. The active, free Tet repressor dimer is an elongated and flat molecule with a maximum dimension of 11 +/- 1.5 mm which can be approximated by an ellipsoid with the half-axes 6 nm, 2.5 nm and 1 nm. The overall conformation undergoes no detectable change when the repressor dimer is bound to a DNA fragment containing a single tet operator. The normal distance between the centre of gravity of the protein and the DNA axis is 3.0 +/- 0.1 nm, indicating that the repressor dimer is mainly located on one side of the DNA. When bound to the wild type tet control DNA, the two repressor dimers have a centre-to-centre distance of 11.0 +/- 0.5 nm. Their minimal distance is 5 +/- 2 nm. Protein-protein contacts via loop formation of the DNA by repressor binding is excluded. The repressors are well separated and have no direct contact. A model is proposed where the two repressor dimers are located on opposite sides of the DNA and the DNA is not strongly bent in the complex.

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In this paper we demonstrate that neutron small angle scattering is a suitable method to study the spatial arrangement of large specific protein-DNA complexes. We studied the complex of DNA-dependent RNA polymerase of Escherichia coli and a 130 base-pair DNA fragment containing the strong promoter A1 of bacteriophage T7. Contrast variation of the complex with deuterium allowed us to "visualize" either RNA polymerase, or DNA, or both components in situ. From the corresponding scattering curves information was derived about: (1) Conformational changes of RNA polymerase and DNA by complex formation: comparison of the scattering profiles of the isolated and complexed components showed that by specific complex formation the cross-section of RNA polymerase decreases, while the DNA fragment does not undergo a gross conformational change. (2) The spatial arrangement of RNA polymerase and DNA in the specific complex from the cross-sectional radii of gyration of the complex the normal distance dn between the centre of gravity of the RNA polymerase and the axis of the DNA fragment was derived as 5.0 (+/- 0.3) nm. On the basis of these and footprinting data a low resolution model of the RNA polymerase-promoter complex is proposed. The main feature of this model is the positioning of RNA polymerase to only one side of the DNA.

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Translating ribosomes of Escherichia coli were prepared either in the pre-translocation or in the post-translocation states by a special technique based on the use of poly(U)-Sepharose columns where the template was coupled to the matrix through splittable -S-S- bridges. Elongation factors were absent from the final preparations. A neutron scattering study of the translating ribosomes in the two functional states was performed at different contrasts (various 1H2O/2H2O mixtures). Under conditions of a high contrast for the protein constituent the radius of gyration of the post-translocation-state ribosomes was found to be slightly greater than that of the pre-translocation-state ribosomes. Using the results of this study the conclusion can be drawn that translocation is accompanied by a spatial displacement of some parts of the ribosome with a magnitude of several ångström units.

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