Towards a structural characterization of charge-driven polymer micelles.

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.

Conformational rearrangements of an archaeal chaperonin upon ATPase cycling.

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.

Mapping proteins of the 50S subunit from Escherichia coli ribosomes.

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.

Spatial arrangement of sigma-factor and core enzyme of Escherichia coli RNA polymerase. A neutron solution scattering study.

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.

Shape and compactness of the isolated ribosomal 16 S RNA and its complexes with ribosomal proteins.

X-ray scattering, neutron scattering and velocity sedimentation techniques were used for studies of ribosomal 16 S RNA in the isolated state and in different complexes with ribosomal proteins. The neutron scattering curve of the ribosomal 30 S subparticle in 42% 2H2O where the protein component is contrast-matched, was taken as a standard of comparison characterizing the dimensions and shape of the 16 S RNA in situ. The following deductions result from the comparisons. The shape of the isolated 16 S RNA at a sufficient Mg2+ concentration (e.g., in the reconstruction buffer) is similar to that of the 16 S RNA in situ, i.e. in the 30 S particle, but it is somewhat less compact. The 16 S RNA in the complex with protein S4 has a shape and compactness similar to those of the isolated 16 S RNA. The 16 S RNA in the complex with four core proteins, namely S4, S7, S8 and S15, has a shape and compactness similar to those of the isolated 16 S RNA. The six ribosomal proteins S4, S7, S8, S15, S16 and S17 are necessary and sufficient for the 16 S RNA to acquire a compactness similar to that within the 30 S particle. The general conclusion is that the overall specific folding of the 16 S RNA is governed and maintained by its own intramolecular interactions, but the additional folding-up (about one-fourth of the linear size of the whole molecule) or the stabilization of the final compactness requires some ribosomal proteins.

Translocation makes the ribosome less compact.

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.

Spatial arrangement of DNA-dependent RNA polymerase of Escherichia coli and DNA in the specific complex. A neutron small angle scattering study.

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.

The solution structure of functionally active human proliferating cell nuclear antigen determined by small-angle neutron scattering.

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.

"Akinetic depression" in schizophrenia.

It is possible that some "postpsychotic depressions" may be a toxic effect of antipsychotic drugs. Out of a total of 94 schizophrenic patients, 28 developed a mild akinesia and 32 never developed extrapyramidal symptoms. Those who developed akinesia became less psychotic, but they also experienced a significant, although modest, increase in depression ratings. Successful treatment of the akinesia resulted in significant improvements in depression, somatic concern, anxiety, emotional withdrawal, blunted affect, and motor retardation on both physicians' and nurses' ratings. A high association between akinesia and both objectively rated and subjectively experienced sedative effect indicates that an 'akinetic depression' is not likely if the patient does not look or feel drowsy. The 32 nonakinetic patients also became less psychotic, but not more depressed.

Direct shape determination of ribosomal proteins in solution and within the ribosome by means of neutron scattering.

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.

Structure of dodecyl sulfate-protein complexes at subsaturating concentrations of free detergent.

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.

Magnetic critical scattering in solid Co80Pd20.

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.

An in situ study of the t-butyllithium initiated polymerization of butadiene in d-heptane via small angle neutron scattering and 1H-NMR.

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.

On the pH-dependence of the reaction of hemoglobin with carbon monoxide.

Flash-photolysis experiments were performed on solutions of carbonmonoxy hemoglobin (human Hb A) as function as pH. The fraction of fast reaction and the amount of photodissociation as produced by a given amount of light quanta has been analyzed in terms of the allosteric model of ligand binding by Monod, Wyman and Changeux. It is shown how the switch-over point of the allosteric transition from the T or R state is controlled by the protons, which act as allosteric effectors.

Kappa-casein micelles: structure, interaction and gelling studied by small-angle neutron scattering.

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.

Low-resolution structure of the tetrameric phenylalanyl-tRNA synthetase from Escherichia coli. A neutron small-angle scattering study of hybrids composed of protonated and deuterated protomers.

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).

Structural elements of the 50 S subunit of E. coli ribosomes.

The large (50 S) subunit from E. coli ribosomes consists of 32 different proteins and two RNA molecules of different length. In an attempt to determine the three-dimensional arrangement of the proteins in the subunit, we are also interested in obtaining direct information on the shape of the proteins within the subunit. This is possible only with ribosomal subunits which, unlike natural protonated subunits, are homogeneous for neutrons. These homogeneous particles are produced by reconstituting 50 S particles from RNA and proteins isolated from bacteria grown at different levels of D2O in the culture medium, 76% D2O for RNA and 84% D2O for proteins. Model calculations and test experiments reveal that the pursued strategy allows direct determination of radii of gyration of 50 S components within the particle with reasonable precision. Data evaluation and interpretation are significantly facilitated by contrast variation of the reconstituted particles. The determination of protein shape parameters is only one aspect of the new strategy. The pair distance measurements are completely independent of its success. Data on radii of gyration of five ribosomal proteins in situ are reported: L1 (26 +/- 2 A), L2 (22 +/- 2 A), L3 (22 +/- 2 A), L4 (20 +/- 2 A), and L23 (13 +/- 2 A).

Inter-protein distances within the large subunit from Escherichia coli ribosomes.

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.

Salt-dependent compaction of di- and trinucleosomes studied by small-angle neutron scattering.

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.