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PubMed1982

Small-angle neutron scattering study of Bence-Jones protein Mcg: comparison of structures in solution and in crystal.

Immunoglobulins fragments are composed of globular domains linked by extended polypeptide segments. The molecular flexibility inherent in this arrangement allows for significant potential differences between structures observed in the crystalline state and those attained in solution. Small-angle neutron scattering measurements in dilute solution were performed on the Mcg Bence-Jones protein dimer, for which performed on the Mcg Bence-Jones protein dimer, for which accurate atomic coordinates have been determined by crystallographic methods [Edmundson, A. B., Ely, K. R., Abola, E. E., Schiffer, M., & Panagiotopoulos, N. (1975) Biochemistry 14, 3953-3961; Schiffer, M. (1980) Biophys. J. 32, 230-232]. The measured radius of gyration (Rg) in H2O buffer is 24.0 +/- 0.4 A and in D2O buffer is 23.3 +/- 0.1 A; the calculated value of Rv (Rg in vacuo) is 24.0 A. The above values compare well with the calculated Rg value of 23.6 A when refined coordinates of the trigonal crystal form of the Mcg Bence-Jones protein are used. On the basis of a match point of 44.2% D2O concentration, the experimental partial specific volume is 0.74 cm3/g. The experimentally derived molecular weight of 47 000 is in very good agreement with that (45 500) calculated from the amino acid composition. For comparison with different Fab's (antigen binding fragments) exhibiting various "elbow bends" due to the flexibility of the switch peptide between variable and constant domains of the immunoglobulin chains, calculation of the Rg value of the Mcg dimer was performed as a function of the elbow bend. The Rg varied from 22.8 to 26.0 A as the elbow bend was opened from 100 degrees to 180 degrees; the maximum radius of gyration of the particle was 26.5 A with the switch peptide stretched by separating the variable and constant domains by an additional 1.5 A at an elbow bend of 180 degrees.

Bence Jones Protein↗

PubMed2004

Interactions of poly(amidoamine) dendrimers with the surfactants SDS, DTAB, and C12EO6: an equilibrium and structural study using a SDS selective electrode, isothermal titration calorimetry, and small angle neutron scattering.

Interactions in aqueous solutions of different generations of poly(amidoamine) (PAMAM) dendrimers containing amine, hydroxyl, or delta-glucolactone functional groups at the periphery with the anionic surfactant sodium dodecyl sulfate (SDS) were investigated. We used a SDS-specific electrode (EMF) for SDS monomer concentration monitoring, isothermal titration calorimetry (ITC) for binding information, and small angle neutron scattering (SANS) for structural studies. ITC experiments monitoring the interaction of the dendrimers with cationic dodecyltrimethylammonium bromide (DTAB) and nonionic hexaethylene glycol mono-n-dodecyl ether (C12EO6) showed no significant binding effects. In contrast, SDS binds to all of the above dendrimers. EMF and ITC data demonstrated a regular trend for both the onset of binding and binding saturation as the generation in each family of dendrimers increased. In addition, generation G6 exhibited a noncooperative binding process at very low SDS concentrations. Furthermore, the onset of cooperative binding in the EMF experiments started at lower concentrations as the weight % (w/v), the size, and the numbers of the internal or surface groups increased. On the other hand, the binding capacity of the dendrimers showed only a small dependence on the above parameters. At SDS concentrations approaching the binding limit and also at selective concentrations within the binding range, SANS measurements indicated that in all cases the bound surfactant is in the micellar form. From the electromotive force (EMF) measurements, ITC data, and SANS data, the stoichiometry of the supramolecular complexes was determined.

Calorimetry↗

PubMed2000

Small-angle neutron scattering study of the n-decane effect on the bilayer thickness in extruded unilamellar dioleoylphosphatidylcholine liposomes.

Dioleoylphosphatidylcholine (DOPC) and n-decane were mixed and hydrated afterwards in an excess of heavy water at 1 wt.% of DOPC. From this dispersion, unilamellar liposomes were prepared by extrusion through polycarbonate filter with 500-A pores. Small-angle neutron scattering (SANS) was conducted on these liposomes. From the Kratky-Porod plot ln[I(Q)Q2] vs. Q2 of SANS intensity I(Q) in the range of scattering vectors Q corresponding to the interval 0.001 A(-2) < or = Q2 < or = 0.006 A(-2), the liposome bilayer radius of gyration Rg and the bilayer thickness parameter d(g) = 12(0.5)Rg were obtained. The values of d(g) indicated that the bilayer thickness is within the experimental error constant up to n-decane/DOPC approximately 0.5 molar ratio, and then increases by 2.4 +/- 1.3 A up to n-decane/DOPC = 1.2 molar ratio.

Alkanes↗

PubMed1996

Solution structures of GroEL and its complex with rhodanese from small-angle neutron scattering.

BACKGROUND: Molecular chaperonins 60 are cylindrical oligomeric complexes which bind to unfolded proteins and assist in their folding. Studies to identify the location of the protein substrate have produced contradictory results: some suggest that the substrate-binding site is buried within the interior of the complex, whereas others indicate an external (polar) location. RESULTS: Small-angle neutron scattering (SANS) measurements were made on GroEL chaperonin and on a complex of GroEL with rhodanese. The radius of gyration and the molecular weight determined from SANS measurements of GroEL agree well with those from its crystal structure. The positions of residues which were unresolved in the crystal structure have been confirmed. In addition, through model fitting of the SANS data, conformational changes in solution have been assessed and the location of bound rhodanese has been determined. CONCLUSIONS: The overall structure of GroEL in solution is similar to the crystal structure. In GroEL the N-terminal and C-terminal residues are organized compactly near the equator of the cylinder and the apical domains are flared by about 5 degrees. The best fit of SANS data suggests the existence of an equilibrium between the complex and single rings and monomers. SANS data for the GroEL-rhodanese complex are consistent with a model wherein one rhodanese molecule binds across the opening to the chaperonin cavity, rather than within it.

Binding Sites↗

PubMed1998

Small-angle neutron scattering and dynamic light scattering studies of N- and C-terminal fragments of ovotransferrin.

In order to rationalize the physicochemical heterogeneities between the N- and C-lobes of ovotransferrin (OTf), we have analyzed the structural characteristics of the isolated fragments corresponding to the N- and C-terminal halves of OTf (OTf/2N and OTf/2C) with and without iron by means of small-angle neutron scattering (SANS) using the contrast variation method with solvents of various D2O/H2O mixtures, and dynamic light scattering (DLS) measurements. The analyses of the internal structural characteristics from SANS data revealed that the radius of gyration (Rg) for both fragments decreased to the same extent with iron binding, and the structural distortion of OTf/2C was smaller than that of OTf/2N, decreasing with iron uptake. The DLS studies showed that the change in the diffusion coefficient induced by iron binding to OTf/2C was greater than that to OTf/2N. It was inferred that the OTf/2C molecule tends to become more compact on the whole by iron binding as compared to the OTf/2N molecule.

Animals↗

PubMed2003

Small-angle neutron scattering study of the lipid bilayer thickness in unilamellar dioleoylphosphatidylcholine vesicles prepared by the cholate dilution method: n-decane effect.

Previous X-ray diffraction studies on fully hydrated fluid lamellar egg phosphatidylcholine phases indicated a approximately 10 A increase of bilayer thickness in the presence of excess n-decane [Biochim. Biophys. Acta 597 (1980) 455], while the small-angle neutron scattering (SANS) on unilamellar extruded dioleoylphosphatidylcholine (DOPC) vesicles detected substantially smaller 2.4+/-1.3 A bilayer thickness increase at n-decane/DOPC molar ratio of 1.2 [Biophys. Chem. 88 (2000) 165]. The purpose of the present study is to investigate the n-decane effect on the bilayer thickness in unilamellar DOPC vesicles prepared by the sodium cholate (NaChol) dilution method. Mixed DOPC+NaChol micelles at DOPC and NaChol concentrations of 0.1 mol/l were prepared in 2H(2)O containing 0.135 mol/l NaCl. This micellar solution was diluted in 0.135 mol/l NaCl in 2H(2)O to reach the final DOPC and NaChol concentrations of 0.008 mol/l. Thirty microliters of n-decane solution in methanol was added to 1 ml of this dispersion. After methanol evaporation, SANS was conducted on the dispersions. From the Kratky-Porod plot ln[I(Q)Q(2)] vs. Q(2) of SANS intensity I(Q) in the range of scattering vector values Q corresponding to interval 0.001 A(-2)<or=Q(2)<or=0.006 A(-2), the bilayer radius of gyration R(g) and the bilayer thickness parameter d(g)=12(0.5)R(g) were obtained. The values of d(g) indicate that the bilayer thickness increases by 2.4+/-0.9 A up to n-decane/DOPC molar ratio of 1.6. This result confirms the previous SANS findings obtained with extruded vesicles.

Alkanes↗

PubMed2003

Determination of the B820 subunit size of a bacterial core light-harvesting complex by small-angle neutron scattering.

The B820 subunit is an integral pigment-membrane protein complex and can be obtained by both dissociation of the core light-harvesting complex (LH1) in photosynthetic bacteria and reconstitution from its component parts in the presence of n-octyl beta-D-glucopyranoside (OG). Intrinsic size of the B820 subunit from Rhodospirillum rubrum LH1 complex was measured by small-angle neutron scattering in perdeuterated OG solution and evaluated by Guinier analysis. Both the B820 subunits prepared by dissociation of LH1 and reconstitution from apopolypeptides and pigments were shown to have a molecular weight of 11,400 +/- 500 and radius of gyration of 11.0 +/- 1.0 A, corresponding to a heterodimer consisting of one pair of alphabeta-polypeptides and two bacteriochlorophyll a molecules. Molecular weights of micelles formed by OG alone in solutions were determined in a range from 30,000 to 50,000 over concentrations of 1-5% (w/v), and thus are much larger than that of the B820 subunit. Similar measurement on the pigment-depleted apopolypeptides revealed highly heterogeneous behavior in the OG solutions, indicating that aggregates with various sizes were formed. The result provides evidence that bacteriochlorophyll a molecules play a crucial role in stabilizing and maintaining the B820 subunits in the dimeric state in solution. Further measurements on individual alpha- and beta-polypeptides exhibited a marked difference in aggregation property between the two polypeptides. The alpha-polypeptides appear to be uniformly dissolved in OG solution in a monomeric form, whereas the beta-polypeptides favor a self-associated form and tend to form large aggregates even in the presence of detergent. The difference in aggregation tendency was discussed in relation to the different behavior between alpha- and beta-polypeptides in reconstitution with bacteriochlorophyll a molecules.

Apoproteins↗

PubMed2001

Small-angle neutron scattering reveals an oxygen-dependent conformational change of the immunogen keyhole limpet hemocyanin type 1 (KLH1).

The respiratory protein of the keyhole limpet, Megathura crenulata, the hemocyanin (KLH), commonly used as an immunogen, binds oxygen cooperatively, which implies the existence of different conformations. For the first time, two different conformations of KLH1 were detected upon oxygenation, a deoxy and an oxy state, using small-angle neutron scattering. Rearrangements in the quaternary structure of KLH1 were predicted from the different radii of gyration and the shifts of the minima and maxima in the scattering curves. Upon oxygenation, KLH1 becomes smaller and more compact. Model reconstruction of KLH1 indicates a hollow cylinder with two rings located close to both ends, which move slightly together upon oxygenation.

Animals↗

PubMed2005

Small-angle neutron scattering study of adsorbed pluronic tri-block copolymers on laponite.

The adsorption of selected poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) tri-block copolymers on synthetic clay particles (laponite) has been investigated. The adsorbed amount and distribution of polymer was determined as a function of relative block composition and size, using the technique of contrast variation small-angle neutron scattering. The pluronic molecules appear to adsorb via a preferential segregation of hydrophobic PPO segments at the surface, with hydrophilic PEO segments dangling into solution. The effect of the PPO segments is substantial with large increases in adsorbed amount and layer thickness as the anchor fraction decreases/PEO chain length increases. This is in direct contrast to the behavior observed for PEO homopolymer adsorption (of much higher molecular weights) where the adsorbed amount and layer thickness are smaller and change little with molecular weight.

Journal Article↗

PubMedDate not supplied

A small angle light scattering device for planar connective tissue microstructural analysis.

The planar fibrous connective tissues of the body are composed of a dense extracellular network of collagen and elastin fibers embedded in a ground matrix, and thus can be thought of as biocomposites. Thus, the quantification of fiber architecture is an important step in developing an understanding of the mechanics of planar tissues in health and disease. We have used small angle light scattering (SALS) to map the gross fiber orientation of several soft membrane connective tissues. However, the device and analysis methods used in these studies required extensive manual intervention and were unsuitable for large-scale fiber architectural mapping studies. We have developed an improved SALS device that allows for rapid data acquisition, automated high spatial resolution specimen positioning, and new analysis methods suitable for large-scale mapping studies. Extensive validation experiments revealed that the SALS device can accurately measure fiber orientation for up to a tissue thickness of at least 500 microns to an angular resolution of approximately 1 degree and a spatial resolution of +/-254 microns. To demonstrate the new device's capabilities, structural measurements from porcine aortic valve leaflets are presented. Results indicate that the new SALS device provides an accurate method for rapid quantification of the gross fiber structure of planar connective tissues.

Animals↗

PubMed2001

Small-angle neutron scattering study of N-dodecyl-N,N-dimethylamine N-oxide induced solubilization of dioleoylphosphatidylcholine bilayers in liposomes.

Mixtures of N-dodecyl-N,N-dimethylamine N-oxide (DDAO) and 1,2-dioleoylphosphatidyl choline (DOPC) in chloroform/methanol were evaporated, dried and hydrated in excess 2H2O. Aqueous dispersions thus prepared were extruded through polycarbonate filter with pores of diameter 500A. These samples were studied using small-angle neutron scattering. DDAO destabilizes the bilayer in unilamellar liposomes and solubilizes it into mixed micelles whose shape changes with the DDAO : DOPC molar ratio. Bilayers or/and bilayer fragments have been observed up to DDAO : DOPC = 1.5, rod-like particles (tubular, cylindric micelles) at 2.5 < DDAO : DOPC < 3.5, and transition to globular particles (spheroid micelles) at DDAO: DOPC > 4. In bilayers or/and bilayer fragments, DDAO modulates the thickness of the bilayer.

Detergents↗

PubMed2006

Modular structure of solubilized human apolipoprotein B-100. Low resolution model revealed by small angle neutron scattering.

Being intimately involved in cholesterol transport and lipid metabolism human low density lipoprotein (LDL) plays a prominent role in atherogenesis and cardiovascular diseases. The receptor-mediated cellular uptake of LDL is triggered by apolipoprotein B-100 (apoB-100), which represents the single protein moiety of LDL. Due to the size and hydrophobic nature of apoB-100, its structure is not well characterized. Here we present a low resolution structure of solubilized apoB-100. We have used small angle neutron scattering in combination with advanced shape reconstruction algorithms to generate a three-dimensional model of lipid-free apoB-100. Our model clearly reveals that apoB-100 is composed of distinct domains connected by flexible regions. The apoB-100 molecule adopts a curved shape with a central cavity. In comparison to LDL-associated apoB-100, the lipid-free protein is expanded, whereas according to spectroscopic data the secondary structure is widely preserved. Finally, the low resolution model was used as a template to reconstruct a hypothetical domain organization of apoB-100 on LDL, including information derived from a secondary structure prediction.

Algorithms↗

PubMed1970

The small angle light scattering of biological cells. Theoretical considerations.

The light scattered by living biological cells (assumed homogeneous spheres with a relative refractive index, m = 1.05) at small angles has been calculated by the Hodkinson approximation and the more rigorous Mie theory. Both methods predict that relative volume distributions may be estimated from low angle scattering measurements on each cell in a population. Under conditions of short wavelength illumination or strong absorption, absolute volume information may also be obtained.

Cell Biology↗

PubMed1990

Small-angle neutron scattering from the reconstituted TF1 of H(+)-ATPase from thermophilic bacterium PS3 with deuterated subunits.

Subunits alpha, beta and gamma of adenosine triphosphatase (H(+)-ATPase) from the thermophilic bacterium PS3 (TF1) have been over-expressed in Escherichia coli. alpha and beta subunits deuterated to the level of 90% were obtained by culturing E. coli in 2H2O medium. Both the subunits and the reconstituted alpha beta gamma complex, TF1, which contain the deuterated components in various combinations, were studied in solution by small-angle neutron scattering. The individual shapes of the subunits and their organization in the alpha beta gamma-TF1 complex were examined using the techniques of selective deuteration and contrast variation. The alpha and beta subunits are well approximated as ellipsoids of revolution having minor semi-axes of 20.4(+/- 0.4) and 20.0(+/- 0.2) A, and major semi-axes of 53.0(+/- 1.4) and 55.8(+/- 0.9) A, respectively. In the TF1 complex, three beta subunits are aligned to form an equilateral triangle, with their major axes tilted by 35 degrees with respect to the 3-fold axis of the complex. The beta-beta distance is about 53 A. Three alpha subunits are similarly arranged, positioned between the beta subunits, and with their direction of tilt opposite to that of the beta subunits. The centers of the alpha and beta subunits lie in the same plane, forming a hexagon. Adjacent subunits overlap in this model, suggesting that they are not simple ellipsoids of revolution.

Bacteria↗

PubMed2001

Structure determination of micelle-like intermediates in amyloid beta -protein fibril assembly by using small angle neutron scattering.

Increasing evidence supports the hypothesis that amyloid beta-protein (Abeta) assembly is a key pathogenic feature of Alzheimer's disease. Thus, understanding the assembly process offers opportunities for the development of strategies for treating this devastating disease. In prior studies, Abeta was found to form micelle-like aggregates under acidic conditions. These structures exhibited an average observed hydrodynamic radius of 7 nm. They were found to be in rapid equilibrium with Abeta monomers or low molecular weight oligomers, and were centers of fibril nucleation. Here the technique of small angle neutron scattering has been used to determine the structure of these Abeta micelles. The data reveal that the micellar assemblies comprise 30-50 Abeta monomers and have elongated geometries. The best fit of the data to a uniform spherocylinder yields a radius approximately 2.4 nm and cylinder length approximately 11 nm. These structure parameters remain constant over more than a decade in concentration range. The concentration independence of the length of the cylindrical aggregate indicates the presence of an internal nonrepetitive structure that spans the entire length of the Abeta assembly.

Amyloid beta-Peptides↗

PubMed2001

Morphology of fast-tumbling bicelles: a small angle neutron scattering and NMR study.

Bilayered micelles, or bicelles, which consist of a mixture of long- and short-chain phospholipids, are a popular model membrane system. Depending on composition, concentration, and temperature, bicelle mixtures may adopt an isotropic phase or form an aligned phase in magnetic fields. Well-resolved (1)H NMR spectra are observed in the isotropic or so-called fast-tumbling bicelle phase, over the range of temperatures investigated (10-40 degrees C), for molar ratios of long-chain lipid to short-chain lipid between 0.20 and 1.0. Small angle neutron scattering data of this phase are consistent with the model in which bicelles were proposed to be disk-shaped. The experimentally determined dimensions are roughly consistent with the predictions of R.R. Vold and R.S. Prosser (J. Magn. Reson. B 113 (1996)). Differential paramagnetic shifts of head group resonances of dimyristoylphosphatidylcholine (DMPC) and dihexanoylphosphatidylcholine (DHPC), induced by the addition of Eu(3+), are also consistent with the bicelle model in which DHPC is believed to be primarily sequestered to bicelle rims. Selective irradiation of the DHPC aliphatic methyl resonances results in no detectable magnetization transfer to the corresponding DMPC methyl resonances (and vice versa) in bicelles, which also suggests that DHPC and DMPC are largely sequestered in the bicelle. Finally, (1)H spectra of the antibacterial peptide indolicidin (ILPWKWPWWPWRR-NH(2)) are compared, in a DPC micellar phase and the above fast-tumbling bicellar phases for a variety of compositions. The spectra exhibit adequate resolution and improved dispersion of amide and aromatic resonances in certain bicelle mixtures.

Antimicrobial Cationic Peptides↗

PubMed1999

Studies on the structure and mechanism of a bacterial protein toxin by analytical ultracentrifugation and small-angle neutron scattering.

Pneumolysin, an important virulence factor of the human pathogen Streptococcus pneumoniae, is a pore-forming toxin which also possesses the ability to activate the complement system directly. Pneumolysin binds to cholesterol in cell membrane surfaces as a prelude to pore formation, which involves the oligomerization of the protein. Two important aspects of the pore-forming activity of pneumolysin are therefore the effect of the toxin on bilayer membrane structure and the nature of the self-association into oligomers undergone by it. We have used analytical ultracentrifugation (AUC) to investigate oligomerization and small-angle neutron scattering (SANS) to investigate the changes in membrane structure accompanying pore formation. Pneumolysin self-associates in solution to form oligomeric structures apparently similar to those which appear on the membrane coincident with pore formation. It has previously been demonstrated by us using site-specific chemical derivatization of the protein that the self-interaction preceding oligomerization involves its C-terminal domain. The AUC experiments described here involved pneumolysin toxoids harbouring mutations in different domains, and support our previous conclusions that self-interaction via the C-terminal domain leads to oligomerization and that this may be related to the mechanism by which pneumolysin activates the complement system.SANS data at a variety of neutron contrasts were obtained from liposomes used as model cell membranes in the absence of pneumolysin, and following the addition of toxin at a number of concentrations. These experiments were designed to allow visualization of the effect that pneumolysin has on bilayer membrane structure resulting from oligomerization into a pore-forming complex. The structure of the liposomal membrane alone and following addition of pneumolysin was calculated by the fitting of scattering equations directly to the scattering curves. The fitting equations describe scattering from simple three-dimensional scattering volume models for the structures present in the sample, whose dimensions were varied iteratively within the fitting program. The overall trend was a thinning of the liposome surface on toxin attack, which was countered by the formation of localized structures thicker than the liposome bilayer itself, in a manner dependent on pneumolysin concentration. At the neutron contrast match point of the liposomes, pneumolysin oligomers were observed. Inactive toxin appeared to bind to the liposome but not to cause membrane alteration; subsequent activation of pneumolysin in situ brought about changes in liposome structure similar to those seen in the presence of active toxin. We propose that the changes in membrane structure on toxin attack which we have observed are related to the mechanism by which pneumolysin forms pores and provide an important perspective on protein/membrane interactions in general. We discuss these results in the light of published data concerning the interaction of gramicidin with bilayers and the hydrophobic mismatch effect.

Amino Acid Sequence↗

PubMed1998

Small angle neutron scattering analysis of thermal stability of 23S rRNA and the intact 50S subunits of Sulfolobus solfataricus.

The ribosomes of the extremely thermophilic archaebacterium, Sulfolobus solfataricus, are very resistant to thermal denaturation (optimal growth temperature 87 degrees C), remaining essentially intact up to above 90 degrees C. However, the separate ribosomal components (rRNA and r-proteins) are less thermally stable than the ribosome as a whole, indicating that the mode of interaction of all of the components within the ribonucleoprotein particle play an essential role in determining thermal stability. To get some insight into the structural features of the thermophilic ribosome, we performed small angle neutron scattering (SANS) measurements at various temperatures on Sulfolobus solfataricus intact large ribosomal subunits (50S) and deproteinated large ribosomal subunit RNA (23S). Even if the scattering profiles suggest the presence of supramolecular aggregates in all of the samples and at all of the investigated temperatures, the measured form factors indicated for both samples that, at temperatures above 70 degrees C, the suspended particles underwent a structural rearrangement. This finding is likely to reflect single particles' properties, since S. solfataricus ribosomes are known to be biologically activated only above 60 degrees C, and there are indications that such activation requires a conformational rearrangement of the particle. A remarkable superimposition of the percentage variation of the volume from neutron scattering and of the absorbency increment with respect to temperature supports this view.

Neutrons↗