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PubMed1988

Small-angle x-ray scattering investigation of the solution structure of troponin C.

X-ray crystallographic studies of troponin C (Herzberg, O., and James, M.N.G. (1985) Nature 313, 653-659; Sundaralingam, M., Bergstrom, R., Strasburg, G., Rao, S.T., and Roychowdhury, P. (1985a) Science 227, 945-948) have revealed a novel protein structure consisting of two globular domains, each containing two Ca2+-binding sites, connected via a nine-turn alpha-helix, three turns of which are fully exposed to solvent. Since the crystals were grown at pH approximately 5, it is of interest to determine whether this structure is applicable to the protein in solution under physiological conditions. We have used small-angle x-ray scattering to examine the solution structure of troponin C at pH 6.8 and the effect of Ca2+ on the structure. The scattering data are consistent with an elongated structure in solution with a radius of gyration of approximately 23.0 A, which is quite comparable to that computed for the crystal structure. The experimental scattering profile and the scattering profile computed from the crystal structure coordinates do, however, exhibit differences at the 40-A level. A weak Ca2+-facilitated dimerization of troponin C was observed. The data rule out large Ca2+-induced structural changes, indicating rather that the molecule with Ca2+ bound is only slightly more compact than the Ca2+-free molecule.

Algorithms↗

PubMed2005

Ca2+-induced structural changes in phosphorylase kinase detected by small-angle X-ray scattering.

Phosphorylase kinase (PhK), a 1.3-MDa (alphabetagammadelta)(4) hexadecameric complex, is a Ca(2+)-dependent regulatory enzyme in the cascade activation of glycogenolysis. PhK comprises two arched (alphabetagammadelta)(2) octameric lobes that are oriented back-to-back with overall D(2) symmetry and joined by connecting bridges. From chemical cross-linking and electron microscopy, it is known that the binding of Ca(2+) by PhK perturbs the structure of all its subunits and promotes redistribution of density throughout both its lobes and bridges; however, little is known concerning the interrelationship of these effects. To measure structural changes induced by Ca(2+) in the PhK complex in solution, small-angle X-ray scattering was performed on nonactivated and Ca(2+)-activated PhK. Although the overall dimensions of the complex were not affected by Ca(2+), the cation did promote a shift in the distribution of the scattering density within the hydrated volume occupied by the PhK molecule, indicating a Ca(2+)-induced conformational change. Computer-generated models, based on elements of the known structure of PhK from electron microscopy, were constructed to aid in the interpretation of the scattering data. Models containing two ellipsoids and four cylinders to represent, respectively, the lobes and bridges of the PhK complex provided theoretical scattering profiles that accurately fit the experimental data. Structural differences between the models representing the nonactivated and Ca(2+)-activated conformers of PhK are consistent with Ca(2+)-induced conformational changes in both the lobes and the interlobal bridges.

Animals↗

PubMed2006

New lamellar structure formed by an adamantyl derivative of cholic acid.

The self-aggregation of the sodium salt of a new adamantyl amide of the 3beta-amino derivative of cholic acid (Na-AdC) in aqueous solution has been investigated by surface tension, dynamic light scattering, fluorescence, small-angle X-ray scattering (SAXS), and transmission electron microscopy (TEM) measurements. These last two techniques suggest that a lamellar phase, consisting of charged bilayers of Na-AdC separated by solvent and periodically stacked, is formed in aqueous solution. The structure of the bilayer is inferred from the resolution of the crystal of the compound in its acid form. The adamantyl moieties, which are mutually interlocked, reside in the central region of the bilayer, and the carboxylic groups are directed toward the hydrophilic region. The structure is open enough to allow water molecules to interact with a fluorescence probe located at the central hydrophobic region.

Adamantane↗

PubMed1994

Abnormal bone mineralization after fluoride treatment in osteoporosis: a small-angle x-ray-scattering study.

Sodium fluoride treatment of osteoporosis is known to stimulate bone formation and to increase bone mass, but recent clinical trials failed to prove its antifracture effectiveness. The formation of bone with abnormal structure and, therefore, increased fragility is discussed as a possible explanation. Until now, however, exact information on the mineral structure of osteoporotic bone after fluoride treatment has been lacking. Bone biopsies were taken from three patients with postmenopausal osteoporosis before and after fluoride treatment (60 mg NaF/day for 1-2 years), from one patient with iatrogenic fluorosis, as well as from three normal controls. The mineral in these samples was investigated by a combination of backscattered electron imaging and small-angle x-ray scattering. Depending on the total dose of fluoride, an increasing amount of new bone is laid down on the surface of preexisting trabeculae. Its mineral structure is identical to that of heavy fluorosis and is characterized by the presence of additional large crystals, presumably located outside the collagen fibrils. These large crystals, which are not present in the controls or in osteoporotic bone before fluoride treatment, contribute to increase the mineral density without significantly improving the biomechanical properties of the bone. The possible success of fluoride treatment depends not only on the amount of newly formed bone but also on the rate of bone turnover. Indeed, as soon as significant amounts of fluoride are present, bone turnover leads to the replacement of old (normal) bone by new (pathologically mineralized) bone.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed2001

Structural characterization of the pH-denatured states of ferricytochrome-c by synchrotron small angle X-ray scattering.

The ferricytochrome-c (cyt-c) shows a complex unfolding pathway characterized by a series of stable partially folded states. When titrated with HCl at low ionic strength, two transitions are detected. At pH 2, cyt-c assumes the U1 unfolded state, whereas the successive addition of Cl(-) ion from either HCl or NaCl induces the recompaction to a molten globule conformation (A1 and A2 states, respectively). A second unfolded state (U2) is also observed at pH 12. Recent data evidence different features for the local structure of the heme in the different states. To derive relationships between local and overall conformations, we analyzed the structural characteristics of the different states by synchrotron small angle X-ray scattering. The results show that in the acidic-unfolded U1 form the protein assumes a worm-like conformation, whereas in the alkaline-unfolded U2 state, the cyt-c is globular. Moreover, the molten globule states induced by adding HCl or NaCl to U1 appear structurally different: in the A1 state cyt-c is dimeric and less compact, whereas in the A2 form the protein reverts to a globular-like conformation. According to the local heme structure, a molecular model for the different forms is derived.

Animals↗

PubMed2006

Small angle x-ray scattering study of local structure and collapse transition of (1,3)-beta-D-glucan-chitosan gels.

Scleroglucan is a (1,3)-beta-D-glucan polysaccharide produced by the fungus Sclerotium. Dissolved in water at room temperature it adopts a linear, rigid, triple helical structure. Gelation of scleroglucan can be obtained by Schiff-base formation between partly periodate oxidized scleroglucan and the primary amine groups of chitosan. The scleraldehyde-chitosan gels exhibit a collapse transition when exposed to volume fractions of isopropanol, Wp, larger than 65%. The aim of the present study is to provide structural information concerning the local polymer distribution and the collapse transition in (1,3)-beta-D-glucan-chitosan gels. Small angle x-ray scattering was used to investigate solutions and gels of scleroglucan in water, as well as in an aqueous mixture containing 65% isopropanol. The results reveal that in aqueous solution, the polysaccharide scleroglucan chains have an approximately cylindrical cross section of external diameter close to 17 A. The gels display the same local structure, but form clusters on a longer distance scale. For the collapsed gels in the water-isopropanol mixture, partial phase separation occurs in which ordered domains of approximate size of 110 A develop. This study indicates that local ordering in liquid-crystalline-type domains is a possible molecular mechanism contributing to the collapse of gels composed of semiflexible polymers. The triple helical structure of the molecule appears not to be conserved in the majority phase in this solvent, but it is conserved in the liquid crystalline domains.

2-Propanol↗

PubMed2006

Investigation of humate-cetyltrimethylammonium complexes by small-angle X-ray scattering.

The structural examination of the complexes formed between humic acid and cationic surfactants has environmental implications. A humic acid (HA) dissolved in 0.1 M NaOH (5 g/L) was reacted with a cationic surfactant (hexadecyltrimethylammonium bromide or CTAB) at initial solution concentrations of 1, 5, 10, 20, 30, 40 and 50 mM. The HA precipitated at CTAB concentrations of 20, 30, and 50 mM but the complexes were soluble at 40 mM and below 20 mM. The charge neutralization between humic acid anions and CTAB micelles and the subsequent charge reversal due to hydrophobic interactions explain the behavior of the HA-CTAB complexes. The HA solution (5 g/L), reaction products (supernatants and precipitates), and pure cationic surfactant solutions were studied by the small-angle X-ray scattering (SAXS) technique in order to determine the structure of HA-CTAB complexes. The scattering intensity (I(q)) of various HA-CTAB systems were recorded over a range of scattering vectors (q=0.053-4.0 nm(-1)). HA forms networks in an alkaline solution with a characterization length of 7.8 nm or greater. The HA-CTAB precipitates and the 50-mM CTAB solution gave d(100) and d(110) reflections of a hexagonal structure. The hexagonal array of cylindrical CTAB micelles has a lattice parameter of 5.01 nm in pure solution, and the parameter decreases in the order: 4.96, 4.91, and 4.85 nm for the precipitates of HA-CTAB (50, 30, and 20 mM, respectively), indicating that the structure of CTAB micelles was disturbed by the addition of HA. The molecular properties and behavior of HA in solution were discussed.

Journal Article↗

PubMed2006

Molecular analysis and solution structure from small-angle X-ray scattering of the human natural killer inhibitory receptor IRp60 (CD300a).

Natural killer (NK) cells are lymphocytes of the innate immune system specialized in recognition and killing of certain virus-infected and tumor cells. To carry out this task, NK cells are equipped with a complex array of germ line encoded receptors. These receptors deliver either positive or negative signals, and a delicate balance between these signals governs the NK cell cytolytic activity against the target cell. IRp60 (CD300a) is a human NK inhibitory receptor with an immunoglobulin-like fold. In the present study the IRp60 protein was expressed in Escherichia coli as inclusion bodies and refolded by dilution. The refolded protein was purified to homogeneity, biochemical characterized and the solution structure was investigated using small-angle X-ray scattering (SAXS). The SAXS data revealed that IRp60 is monomeric in solution with a molecular shape characteristic of the immunoglobulin-like structures. A homology model of IRp60 was built and validated experimentally against the SAXS data.

Antigens, CD↗

PubMed1988

Differences in the solution structures of oxidized and reduced cytochrome c measured by small-angle X-ray scattering.

While X-ray crystallographic data on cytochrome c show the reduced and oxidized forms to have very similar structures, there is a considerable body of data, mostly from solution studies, that indicates the reduced form is more stable and that the interior of the protein is less accessible to solvent in this state. These observations have led to the hypothesis that while the time-averaged structure is preserved between the two forms, the dynamics of the two forms are different. The oxidized form has been proposed to undergo more large-amplitude, low-frequency motions than the reduced form. The crystal structure data were derived from crystals grown in high salt concentrations, but the solution studies were done at relatively low ionic strength. Small-angle X-ray scattering has been used to examine the effects of the ionic strength and oxidation state on the solution structure of cytochrome c. We find that the radius of gyration and the maximum linear dimension of oxidized cytochrome c are significantly larger than those for reduced cytochrome c, in 5 mM phosphate buffer at pH 7.3, and further that this difference is suppressed by addition of 200 mM sodium chloride. We conclude that there is a real structural difference between the two forms at low ionic strength in solution and that this difference is likely to contribute to the observed differences in accessibility and compressibility.

Cytochrome c Group↗

PubMed1994

Conformation of (2-->1)-beta-D-fructan in aqueous solution.

The conformation and dilute solution properties of (2-->1)-beta-D-fructan in aqueous solution were studied by gel permeation chromatography, low-angle laser light-scattering photometry, viscometry, small-angle X-ray scattering and electron microscopy. Fractions covering a broad range of weight-average molecular weights (Mw) from 1.49 x 10(4) to 5.29 x 10(6) were obtained from a native sample by ultrasonic degradation and fractional precipitation. For Mw < 4 x 10(4), the intrinsic viscosity [eta] varies with Mw0.71, indicating that the fructan chain behaves as a random coil expanded by an excluded-volume effect in this molecular weight region. For Mw > 10(5), [eta] exhibits an unusually weak dependence on Mw and finally becomes almost independent of molecular weight. This behaviour is interpreted in terms of a globular conformation of the high-molecular-weight fructan molecules. Small-angle X-ray-scattering measurements and electron microscopic observations support this interpretation of the values of [eta] observed.

Aspergillus↗

PubMed2001

Large-scale domain movements and hydration structure changes in the active-site cleft of unligated glutamate dehydrogenase from Thermococcus profundus studied by cryogenic X-ray crystal structure analysis and small-angle X-ray scattering.

Here we describe the large-scale domain movements and hydration structure changes in the active-site cleft of unligated glutamate dehydrogenase. Glutamate dehydrogenase from Thermococcus profundus is composed of six identical subunits of M(r) 46K, each with two distinct domains of roughly equal size separated by a large active-site cleft. The enzyme in the unligated state was crystallized so that one hexamer occupied a crystallographic asymmetric unit, and the crystal structure of the hexamer was solved and refined at a resolution of 2.25 A with a crystallographic R-factor of 0.190. In that structure, the six subunits displayed significant conformational variations with respect to the orientations of the two domains. The variation was most likely explained as a hinge-bending motion caused by small changes in the main chain torsion angle of the residue composing a loop connecting the two domains. Small-angle X-ray scattering profiles both at 293 and 338 K suggested that the apparent molecular size of the hexamer was slightly larger in solution than in the crystalline state. These results led us to the conclusion that (i) the spontaneous domain motion was the property of the enzyme in solution, (ii) the domain motion was trapped in the crystallization process through different modes of crystal contacts, and (iii) the magnitude of the motion in solution was greater than that observed in the crystal structure. The present cryogenic diffraction experiment enabled us to identify 1931 hydration water molecules around the hexamer. The hydration structures around the subunits exhibited significant changes in accord with the degree of the domain movement. In particular, the hydration water molecules in the active-site cleft were rearranged markedly through migrations between specific hydration sites in coupling strongly with the domain movement. We discussed the cooperative dynamics between the domain motion and the hydration structure changes in the active site of the enzyme. The present study provides the first example of a visualized hydration structure varying transiently with the dynamic movements of enzymes and may form a new concept of the dynamics of multidomain enzymes in solution.

Binding Sites↗

PubMed1978

The conformer nature of the multiple forms of beef liver catalase as obtained by biochemical and small-angle X-ray scattering experiments. A model for the quaternary structure of the beef liver catalase molecule.

Two of the five multiple forms of beef liver catalase have been extensively studied using biochemical and biophysical analysis techniques. The two molecules, cat I and cat V, have different isoelectric points (pH 6.55 and 5.6), different surface charges (25.8 and 32.7 elementary charges) and display different numbers of primary amino groups on their surfaces. The numbers of tyrosine residues on the surfaces of the two molecules are also different (16 and 10 at pH 10). Since the two forms of catalase can be interconverted, the described changes may be caused by conformational changes of the four protein subunits within the molecule. This mobility of the polypeptide chains is also demonstrated by the different absorption spectra below 390 nm. Using small-angle X-ray scattering, the radii and the volumes of the two catalase forms were shown to be different (cat 1 is smaller than cat V). All five multiple forms of beef liver catalase are concormers of the molecule. A model for the quaternary sturcture of the beef liver catalase are conformers of the molecule. A model for the quaternary structure of the beef liver catalase molecule is suggested. It consists of a regular configuration of four prolate rotational ellipsoids (semiaxes: a = 52 A, b = c = 21 A) in close contact in which the nearest neighbour subunits are shifted by 37 A parallel to each other. Thus the height of the complete molecule is 141 A and the diameter 94 A.

Animals↗

PubMed1977

On the conformation of serine-specific transfer RNA. Studies by small-angle X-ray scattering and ultraviolet absorption of the molecule in solution.

The scattered X-ray intensities from dilute solutions of tRNASer (yeast) in 0.1 M Soerensen buffer at pH 7.0 were measured at 25 degrees C. The radius of gyration, molecular weight and volume were determined. A model equivalent in scattering is given. The change of the conformation of tRNASer by heating was followed by small-angle X-ray measurements and ultraviolet absorption in a temperature range 20-70 degrees C. The molecule begins to unfold at about 40 degrees C and 70 degrees C has a random coil conformation. Addition of magnesium stabilizes the tRNASer molecule. The reversibility of the melting process was also studied by both methods. An interesting effect was found by ultraviolet absorption: by heating the tRNASer solutions to 55 degrees C and 60 degrees C and subsequently slowly cooling, the melting curves lie at higher absorption values than the corresponding cooling curves. The small-angle data and optical properties of tRNASer are compared with those of tRNAPhe which has already been thoroughly investigated.

Binding Sites↗

PubMed2005

Small angle X-ray scattering (SAXS) and differential scanning calorimetry (DSC) studies of amide phospholipids.

Varying chemically the structure of phospholipids in the region between hydrophobic and hydrophilic segments is expected to have a strong influence on the interaction with water and the phase behavior. This is studied in this work with the motivation to investigate these lipids as potential inhibitors of phospholipase A2. Thus the amide phospholipids L-ether-amide-PC (1-O-hexadecyl-2-N-palmitoyl-2-amino-2-deoxy-sn-glycero-3-phosphocholine), L-ester-amide-PC (1-palmitoyl-2-N-palmitoyl-2-amino-2-deoxy-sn-glycero-3-phosphocholine) and L-ether-amide-PE (1-O-hexadecyl-2-N-palmitoyl-2-deoxy-sn-glycero-3-phosphoethanolamine) have been synthesized and characterized. The phase behavior and thermal transitions in buffer dispersions are examined by a combination of high-sensitivity differential scanning calorimetry (DSC) and small angle X-ray scattering (SAXS) experiments between 10 and 80 degrees C at pH 8.9. The onset temperatures determined from DSC measurements agree well with the starting temperatures of changes in the repeat distance obtained by SAXS measurements. The phases observed are lamellar both below and above the main phase transition. The phase transition temperatures and enthalpies depend strongly on the substitutions in sn-1 position and head group structure. The lamellar repeat distance in gel and liquid-crystalline phases increases with increasing temperature for L-ester-amide-PC and L-ether-amide-PC, whereas the temperature dependence is opposite for the L-ether-amide-PE. The observed behavior is discussed and compared with that of DPPC and DPPE, indicating the strong dependence of hydration and phase behavior on head group structure.

Amides↗

PubMed2005

Structure of strongly interacting polyelectrolyte diblock copolymer micelles.

The structure of spherical micelles of the diblock poly(styrene-block-acrylic acid) [PS-b-PA] copolymer in water was investigated up to concentrations where the polyelectrolyte coronal layers have to shrink and/or interpenetrate in order to accommodate the micelles in the increasingly crowded volume. We obtained the partial structure factors pertaining to the core and corona density correlations with small angle neutron scattering and contrast matching in the water. The counterion structure factor was obtained with small angle x-ray scattering (SAXS) with a synchrotron radiation source. Furthermore, we have measured the flow curves and dynamic visco-elastic moduli. The functionality of the micelles is fixed with a 9 nm diameter PS core and a corona formed by around 100 PA arms. As shown by the SAXS intensities, the counterions are distributed in the coronal layer with the same density profile as the corona forming segments. Irrespective ionic strength and micelle charge, the corona shrinks with increasing packing fraction. At high charge and minimal screening conditions, the polyelectrolyte chains remain almost fully stretched and they interdigitate once the volume fraction exceeds the critical value 0.53+/-0.02. Interpenetration of the polyelectrolyte brushes also controls the fluid rheology: The viscosity increases by three orders of magnitude and the parallel frequency scaling behavior of the dynamic moduli suggests the formation of a physical gel. In excess salt, the coronal layers are less extended and they do not interpenetrate in the present concentration range.

Journal Article↗

PubMed2003

3D structure of Sulfolobus solfataricus carboxypeptidase developed by molecular modeling is confirmed by site-directed mutagenesis and small angle X-ray scattering.

Sulfolobus solfataricus carboxypeptidase (CPSso) is a thermostable zinc-metalloenzyme with a M(r) of 43,000. Taking into account the experimentally determined zinc content of one ion per subunit, we developed two alternative 3D models, starting from the available structures of Thermoactinomyces vulgaris carboxypeptidase (Model A) and Pseudomonas carboxypeptidase G2 (Model B). The former enzyme is monomeric and has one metal ion in the active site, while the latter is dimeric and has two bound zinc ions. The two models were computed by exploiting the structural alignment of the one zinc- with the two zinc-containing active sites of the two templates, and with a threading procedure. Both computed structures resembled the respective template, with only one bound zinc with tetrahedric coordination in the active site. With these models, two different quaternary structures can be modeled: one using Model A with a hexameric symmetry, the other from Model B with a tetrameric symmetry. Mutagenesis experiments directed toward the residues putatively involved in metal chelation in either of the models disproved Model A and supported Model B, in which the metal-binding site comprises His(108), Asp(109), and His(168). We also identified Glu(142) as the acidic residue interacting with the water molecule occupying the fourth chelation site. Furthermore, the overall fold and the oligomeric structure of the molecule was validated by small angle x-ray scattering (SAXS). An ab initio original approach was used to reconstruct the shape of the CPSso in solution from the experimental curves. The results clearly support a tetrameric structure. The Monte Carlo method was then used to compare the crystallographic coordinates of the possible quaternary structures for CPSso with the SAXS profiles. The fitting procedure showed that only the model built using the Pseudomonas carboxypeptidase G2 structure as a template fitted the experimental data.

Amino Acid Sequence↗

PubMed2005

The onset of amelogenin nanosphere aggregation studied by small-angle X-ray scattering and dynamic light scattering.

Proteins with predominantly hydrophobic character called amelogenins play a key role in the formation of the highly organized enamel tissue by forming nanospheres that interact with hydroxyapatite crystals. In the present investigation, we have studied the temperature and pH-dependent self-assembly of two recombinant mouse amelogenins, rM179 and rM166, the latter being an engineered version of the protein that lacks a 13 amino acid hydrophilic C-terminus. It has been postulated that this hydrophilic domain plays an important role in controlling the self-assembly behavior of rM179. By small-angle X-ray and neutron scattering, as well as by dynamic light scattering, we observed the onset of an aggregation of the rM179 protein nanospheres at pH 8. This behavior of the full-length recombinant protein is best explained by a core-shell model for the nanospheres, where hydrophilic and negatively charged side chains prevent the agglomeration of hydrophobic cores of the protein nanospheres at lower temperatures, while clusters consisting of several nanospheres start to form at elevated temperatures. In contrast, while capable of forming nanospheres, rM166 shows a very different aggregation behavior resulting in the formation of larger precipitates just above room temperature. These results, together with recent observations that rM179, unlike rM166, can regulate mineral organization in vitro, suggest that the aggregation of nanospheres of the full-length amelogenin rM179 is an important step in the self-assembly of the enamel matrix.

Amelogenin↗

PubMed1982

Small-angle X-ray scattering study of the interaction between human alpha 2-macroglobulin and trypsin.

The interaction between human alpha 2-macroglobulin and trypsin yields a complex of two trypsin molecules and one alpha 2-macroglobulin molecule as the main product. This is indicated from small-angle X-ray titration measurements in 0.02 M Tris/HCl buffer of pH 7.40 containing 0.2 M NaCl and 5 mM EDTA. The radius of gyration for the complex was determined to be 7.1 nm. The complex formation coincides with a decrease in the first side maximum of the scattering curve for alpha 2-macroglobulin. A comparison of the experimental data with those calculated from various models indicates that the data are consistent with the scattering from a hollow cylinder containing two sites, each of which is partly filled with one trypsin molecule.

Humans↗