All hierarchical levels are involved in conformational transitions of the 4 x 6-meric tarantula hemocyanin upon oxygenation

The respiratory protein of the tarantula Eurypelma californicum is a 4 x 6-meric hemocyanin that binds oxygen with high cooperativity. This requires the existence of different conformations which have been confirmed by small angle X-ray scattering (SAXS). Here we present reconstructed 3D-models of the oxy- and deoxy-forms of tarantula hemocyanins, as obtained by fitting small angle X-rays scattering curves on the basis of known X-ray structures and electron microscopy of related hemocyanins. For the first time, the involvement of movements at all levels of the quaternary structure was confirmed for an arthropod hemocyanin upon oxygenation. The two identical 2 x 6-meric half-molecules of the native 4 x 6-mer were shifted in the oxy-state along each other compared with the deoxy-state by about 14 A. In addition, the angle between the two 2 x 6-meric half-molecules increased by 13 degrees. Within these 2 x 6-mers the two hexamers were rotated against each other by about 26 degrees with respect to the deoxy-state. In addition, the distance between the two trimers of each hexamer increased upon oxygenation by about 2.5 A. These strongly coupled movements are based on the particular hierarchical structure of the 4 x 6-mer. It also shows a concept of allosteric interaction in hierarchically assembled proteins to guarantee the involvement of all subunits of a native oligomer to establish very high Hill coefficients.     

Small angle x-ray scattering of the hemoglobin from Biomphalaria glabrata

The hemoglobin from Biomphalaria glabrata is an extracellular respiratory protein of high molecular mass composed by subunits of 360 kDa, each one containing two 180 kDa chains linked by disulfide bridges. In this work, small angle x-ray scattering (SAXS) measurements were performed with the hemoglobin at pH 5.0 and 7.5. Radii of gyration of 98.6 +/- 0.5 and 101.8 +/- 0.2 A and maximum diameters of 300 +/- 10 and 305 +/- 10 A, respectively, were obtained from Guinier plot extrapolation and analytical curve fitting. The pair distance distribution functions p(r) corresponded to globular particles with a somewhat anisotropic shape for both preparations. Computer analysis of the low angle part of the scattering curve led to the determination of the low resolution envelope of the protein, revealing a P(222) symmetry. Shape reconstruction from ab initio calculations using the complete scattering curve furnished a compact prolate three-dimensional (3D) bead model for the protein. Hydrodynamic parameters were obtained from experiments and theoretical calculations using the 3D model. The results of the structural and biochemical studies reported herein indicate that the multisubunit structure of this hemoglobin is compatible with a tetrameric arrangement.     

Structural and conformational analysis of scorpion (Buthus sindicus) hemocyanin using low resolution techniques

Blue oxygen binding protein hemocyanin from scorpion Buthus sindicus has been investigated using low resolution techniques. The native protein is a polymer of eight different types of subunits arranged in four cubic hexameric form (4x6-mers) as previously annotated using a combination of various types of chromatographic and electrophoretic techniques. In addition, both "top face" as well as the "side view" of the native assembly has also been identified from the negatively stained specimens using transmission electron microscopy confirming the overall structural features of arthropodan hemocyanins. These results are also supported from data obtained from another low resolution technique i.e. Small Angle X-ray scattering (SAXS). SAXS results under oxygenated and deoxygenated states represent a validation case for this technique with key conformational changes of Rg 88.0 --> 86.0 A; +/- 1% (Dmax 280.0 --> 290.0 A; +/- 2%), respectively suggesting that the oxygenated hemocyanin is longer then the deoxygenated hemocyanin by almost 2 A;. Likewise, active conformations of the purified structural and functional subunit Bsin1 under oxygenated and deoxygenated states also determined by SAXS measurements revealed a Rg value of 25.2 --> 25.7 A; +/- 1% (Dmax 75.0 --> 75.5 A; +/- 2%), respectively suggesting very little or no contribution of the individual subunit in the overall conformational change in the native assembly during molecular breathing. Preliminary molecular shapes for the oxy-molecules, calculated directly from the scattering profile-alone in a model-independent procedure, superimpose well on other closely related known three-dimensional structures of the same size. Structural and functional aspects of the native as well as purified subunit and the application of these low resolution techniques like transmission electron microscopy and Small Angle X-ray scattering have been discussed.     

Small angle X-ray scattering studies and modeling of Eudistylia vancouverii chlorocruorin and Macrobdella decora hemoglobin

Annelids possess giant extracellular oxygen carriers that exhibit a hexagonal bilayer appearance and have molecular masses of approximately 3.5 MDa. By small angle x-ray scattering (SAXS), Eudistylia vancouverii chlorocruorin and Macrobdella decora hemoglobin were investigated in solution. On the basis of the experimental SAXS data, three-dimensional models were established in a two-step approach (trial and error and averaging). The main differences between the complexes concern the structure of their central part and the subunit architecture. Usage of our SAXS models as templates for automated model generation (program DAMMIN) led to refined models that fit perfectly the experimental data. Special attention was paid to the inhomogeneous density distribution observed within the complexes. DAMMIN models without a priori information could not reproducibly locate low-density areas. The usage of templates, however, improved the results considerably, in particular by applying electron microscopy-based templates. Biologically relevant information on the presence of low-density areas and hints for their presumable location could be drawn from SAXS and sophisticated modeling approaches. Provided that different models are analyzed carefully, this obviously opens a way to gain additional biologically relevant structural information from SAXS data.     

Small angle X-ray scattering study of meso-tetrakis (4-sulfonatophenyl) porphyrin in aqueous solution: a self-aggregation model

The aggregate morphology of meso-tetrakis(4-sulfonatophenyl) porphyrin (TPPS(4)) in aqueous solution is investigated by using small angle x-ray scattering (SAXS) technique. Measurements were performed at pH 4.0 and 9.0 to monitor the pH influence on the structural parameters of the aggregates. Radii of gyration were obtained from distance distribution functions p(r) analysis. The experimental data of TPPS(4) at pH 4.0 showed well-defined oscillations characteristic of large aggregates in contrast to the SAXS curve of 5 mM TPPS(4) at pH 9.0, where both a significant decrease in the intensity and the disappearance of the oscillation peaks suggest the dissociation of the aggregate. A 340-A long "hollow" cylinder with shell thickness of 20 A, compatible to the porphyrin molecule dimension, represents well the scattering curve of the aggregates at pH 4.0. According to the fitting parameters, 26 porphyrin molecules self-associate into a ringlike configuration in the plane of the cylinder cross-section. The total number of porphyrin molecules in the whole aggregate was also estimated as approximately 3000. The model compatible to SAXS data of a hollow cylinder with J-aggregation in the cross-section and H-aggregation (columnar stacking) between the cylinder layers is consistent with optical absorption spectroscopic data both in the literature and obtained in this work.     

Synchrotron SAXS studies on the structural stability of Carcinus aestuarii hemocyanin in solution

The effect of GuHCl and of NaCl on the structural properties of the hemocyanin (Hc) from Carcinus aestuarii has been studied by small angle x-ray scattering (SAXS) using synchrotron radiation. SAXS data collected as a function of perturbant concentration have been used to analyze conformational states of hexameric holo and apoHc as well as the holo and apoforms of the monomeric subunit CaeSS2. In the case of the holoprotein in GuHCl, two concentration domains were identified: at lower concentration, the perturbant induces aggregation of Hc molecules, whereas at higher concentration the aggregates dissociate with concomitant denaturation of the protein. In contrast, with apoHc the denaturation occurs at rather low GuHCl, pointing to an important effect of the active site bound copper for the stabilization of Hc tertiary structure. The effects of NaCl are similar to those of GuHCl as far as CaeSS2 is concerned, namely oligomerization precedes denaturation, whereas in the case of the hexameric form no aggregation occurs. To improve data analysis, on the basis of the current models for Hc monomers and oligomers, the fraction of each aggregation state and/or unfolded protein has been determined by fitting experimental SAXS curves with form factors calculated from Monte Carlo methods. In addition, a global analysis has been carried out on the basis of a thermodynamic model involving an equilibrium between a monomer in a nativelike and denatured form as well as a class of equilibria among the monomer and other aggregates.     

Direct comparison of the crystal and solution structure of glucose/xylose isomerase from Streptomyces rubiginosus

Glucose isomerase (D-xylose ketol-isomerase, EC 5.3.1.5.) catalyses the isomerization reaction of glucose and xylose. The small angle X-ray scattering (SAXS) data of glucose/xylose isomerase from Streptomyces rubiginosus were recorded for protein solution using synchrotron radiation. The experimental data were compared with theoretical scattering calculated on the basis of the known crystal structure (PDB code: 1OAD). The radius of gyration measured by SAXS (R(G)=3.30 nm) was almost identical and the maximum dimension in the distance distribution function was by about 2.5 % lower than the corresponding values calculated on the basis of the crystal structure.     

Low resolution structure determination shows procollagen C-proteinase enhancer to be an elongated multidomain glycoprotein

Procollagen C-proteinase enhancer (PCPE) is an extracellular matrix glycoprotein that can stimulate the action of tolloid metalloproteinases, such as bone morphogenetic protein-1, on a procollagen substrate, by up to 20-fold. The PCPE molecule consists of two CUB domains followed by a C-terminal NTR (netrin-like) domain. In order to obtain structural insights into the function of PCPE, the recombinant protein was characterized by a range of biophysical techniques, including analytical ultracentrifugation, transmission electron microscopy, and small angle x-ray scattering. All three approaches showed PCPE to be a rod-like molecule, with a length of approximately 150 A. Homology modeling of both CUB domains and the NTR domain was consistent with the low-resolution structure of PCPE deduced from the small angle x-ray scattering data. Comparison with the low-resolution structure of the procollagen C-terminal region supports a recently proposed model (Ricard-Blum, S., Bernocco, S., Font, B., Moali, C., Eichenberger, D., Farjanel, J., Burchardt, E. R., van der Rest, M., Kessler, E., and Hulmes, D. J. S. (2002) J. Biol. Chem. 277, 33864-33869) for the mechanism of action of PCPE.     

Low-resolution molecular structures of isolated functional units from arthropodan and molluscan hemocyanin

Synchrotron x-ray scattering measurements were performed on dilute solutions of the purified hemocyanin subunit (Bsin1) from scorpion (Buthus sindicus) and the N-terminal functional unit (Rta) from a marine snail (Rapana thomasiana). The model-independent approach based on spherical harmonics was applied to calculate the molecular envelopes directly from the scattering profiles. Their molecular shapes in solution could be restored at 2-nm resolution. We show that these units represent stable, globular building blocks of the two hemocyanin families and emphasize their conformational differences on a subunit level. Because no crystallographic or electron microscopy data are available for isolated functional units, this study provides for the first time structural information for isolated, monomeric functional subunits from both hemocyanin families. This has been made possible through the use of low protein concentrations (< or = 1 mg/ml). The observed structural differences may offer advantages in building very different overall molecular architectures of hemocyanin by the two phyla.     

Microfibrillar structure of PGG-glucan in aqueous solution as triple-helix aggregates by small angle x-ray scattering.

The conformation of polysaccharide PGG-Glucan, isolated from yeast cell walls, in aqueous solution was investigated by small angle x-ray scattering (SAXS) and multidetector gel permeation chromatography coupled with postcolumn delivery (GPC/PCD) techniques in comparison with scleroglucan. It was shown that both polysaccharides exhibit a rigid rod-like conformation in aqueous solution by SAXS experiments. The mass per unit length (M/L) and radius (R) of rod cross section of PGG-Glucan were measured to be 6300 daltons/nm and 1.89 nm, while those of scleroglucan are 2300 and 0.83, respectively. Utilizing a GPC/light scattering technique, the average aggregation number of PGG-Glucan is 9, while that of scleroglucan is around 3. From the comparison of the M/L and R of the respective rod cross sections as well as their aggregation number data, it is concluded that PGG-Glucan is composed of triple helices, which tend to aggregate as triplets in solution, whereas scleroglucan is composed of a single triple helix. The aggregation number distribution of PGG-Glucan was found to range from 1 to about 25 determined by GPC/PCD. From the observation of a Debye-Scherrer ring type of peak in the macroscopic scattering cross section of PGG-Glucan by SAXS, the existence of a small amount of ordered clusters of PGG-Glucan can be deduced. The "lattice parameter" of these ordered fasces-like clusters is consistent with the radius of the individual triple-helical rods forming a microfibrillar superstructure. These results indicate that higher aggregated forms of PGG-Glucan containing up to 8 triple helices behave as ordered fasces-like clusters. We conclude that PGG-Glucan is triple-helix aggregates formed by rigid rods stacking together side by side. We propose a molecular structural model for PGG-Glucan conformations.     

Salt-dependent DNA superhelix diameter studied by small angle neutron scattering measurements and Monte Carlo simulations.

Using small angle neutron scattering we have measured the static form factor of two different superhelical DNAs, p1868 (1868 bp) and pUC18 (2686 bp), in dilute aqueous solution at salt concentrations between 0 and 1.5 M Na+ in 10 mM Tris at 0% and 100% D2O. For both DNA molecules, the theoretical static form factor was also calculated from an ensemble of Monte Carlo configurations generated by a previously described model. Simulated and measured form factors of both DNAs showed the same behavior between 10 and 100 mM salt concentration: An undulation in the scattering curve at a momentum transfer q = 0.5 nm-1 present at lower concentration disappears above 100 mM. The position of the undulation corresponds to a distance of approximately 10-20 nm. This indicated a change in the DNA superhelix diameter, as the undulation is not present in the scattering curve of the relaxed DNA. From the measured scattering curves of superhelical DNA we estimated the superhelix diameter as a function of Na+ concentration by a quantitative comparison with the scattering curve of relaxed DNA. The ratio of the scattering curves of superhelical and relaxed DNA is very similar to the form factor of a pair of point scatterers. We concluded that the distance of this pair corresponds to the interstrand separation in the superhelix. The computed superhelix diameter of 16.0 +/- 0.9 nm at 10 mM decreased to 9.0 +/- 0.7 nm at 100 mM salt concentration. Measured and simulated scattering curves agreed almost quantitatively, therefore we also calculated the superhelix diameter from the simulated conformations. It decreased from 18.0 +/- 1.5 nm at 10 mM to 9.4 +/- 1.5 nm at 100 mM salt concentration. This value did not significantly change to lower values at higher Na+ concentration, in agreement with results obtained by electron microscopy, scanning force microscopy imaging in aqueous solution, and recent MC simulations, but in contrast to the observation of a lateral collapse of the DNA superhelix as indicated by cryo-electron microscopy studies.     

Lumbricus terrestris hemoglobin: A comparison of small-angle X-ray scattering and cryoelectron microscopy data

The quaternary structure of Lumbricus terrestris hemoglobin was investigated by small-angle x-ray scattering (SAXS). Based on the SAXS data from several independent experiments, a three-dimensional (3D) consensus model was established to simulate the solution structure of this complex protein at low resolution (about 3 nm) and to yield the particle dimensions. The model is built up from a large number of small spheres of different weights, a result of the two-step procedure used to calculate the SAXS model. It accounts for the arrangement of 12 subunits in a hexagonal bilayer structure and for an additional central unit of cylinder-like shape. This model provides an excellent fit of the experimental scattering curve of the protein up to h = 1 nm⁻¹ and a nearly perfect fit of the experimental distance distribution function p(r) in the whole range. Scattering curves and p(r) functions were also calculated for low-resolution models based on 3D reconstructions obtained by cryoelectron microscopy (EM). The calculated functions of these models also provide a very good fit of the experimental scattering curve (even at h > 1 nm⁻¹) and p(r) function, if hydration is taken into account and the original model coordinates are slightly rescaled. The comparison of models reveals that both the SAXS-based and the EM-based model lead to a similar simulation of the protein structure and to similar particle dimensions. The essential differences between the models concern the hexagonal bilayer arrangement (eclipsed in the SAXS model, one layer slightly rotated in the EM model), and the mass distribution, mainly on the surface and in the central part of the protein complex. © John Wiley & Sons, Inc. Biopoly 45: 289–298, 1998     

Structural properties of AMP-activated protein kinase: dimerization, molecular shape, and changes upon ligand binding

Heterotrimeric AMP-activated protein kinase (AMPK) is crucial for energy homeostasis of eukaryotic cells and organisms. Here we report on (i) bacterial expression of untagged mammalian AMPK isoform combinations, all containing gamma(1), (ii) an automated four-dimensional purification protocol, and (iii) biophysical characterization of AMPK heterotrimers by small angle x-ray scattering in solution (SAXS), transmission and scanning transmission electron microscopy (TEM, STEM), and mass spectrometry (MS). AMPK in solution at low concentrations (~1 mg/ml) largely consisted of individual heterotrimers in TEM analysis, revealed a precise 1:1:1 stoichiometry of the three subunits in MS, and behaved as an ideal solution in SAXS. At higher AMPK concentrations, SAXS revealed concentration-dependent, reversible dimerization of AMPK heterotrimers and formation of higher oligomers, also confirmed by STEM mass measurements. Single particle reconstruction and averaging by SAXS and TEM, respectively, revealed similar elongated, flat AMPK particles with protrusions and an indentation. In the lower AMPK concentration range, addition of AMP resulted in a significant decrease of the radius of gyration by approximately 5% in SAXS, which indicates a conformational switch in AMPK induced by ligand binding. We propose a structural model involving a ligand-induced relative movement of the kinase domain resulting in a more compact heterotrimer and a conformational change in the kinase domain that protects AMPK from dephosphorylation of Thr(172), thus positively affecting AMPK activity.     

Influence of salt on the structure of DMPG studied by SAXS and optical microscopy

Aqueous dispersions of 50 mM dimyristoylphosphatidylglycerol (DMPG) in the presence of increasing salt concentrations (2-500 mM NaCl) were studied by small angle X-ray scattering (SAXS) and optical microscopy between 15 and 35 degrees C. SAXS data show the presence of a broad peak around q approximately 0.12 A(-1) at all temperatures and conditions, arising from the electron density contrasts within the bilayer. Up to 100 mM NaCl, this broad peak is the main feature observed in the gel and fluid phases. At higher ionic strength (250-500 mM NaCl), an incipient lamellar repeat distance around d=90-100 A is detected superimposed to the bilayer form factor. The data with high salt were fit and showed that the emergent Bragg peak is due to loose multilamellar structures, with the local order vanishing after approximately 4d. Optical microscopy revealed that up to 20 mM NaCl, DMPG is arranged in submicroscopic vesicles. Giant (loose) multilamellar vesicles (MLVs) start to appear with 50 mM NaCl, although most lipids are arranged in small vesicles. As the ionic strength increases, more and denser MLVs are seen, up to 500 mM NaCl, when MLVs are the prevailing structure. The DLVO theory could account for the experimentally found interbilayer distances.     

Direct comparison of the crystal and solution structure of xylanase from Trichoderma longibrachiatum

The small angle X-ray scattering (SAXS) data of xylanase XYNII (endo-1,4-beta-xylan xylanohydrolase EC 3.2.1.8) from Trichoderma longibrachiatum, an enzyme catalysing the reaction of accidental hydrolysis of beta-1,4-D-xylosidic linkages of xylan, were recorded for protein solution using synchrotron radiation. The experimental data were compared with those of theoretical scattering calculated on the basis of the known crystal structure. The radius of gyration measured by SAXS (RG = 1.7 nm) was about 3.5% larger and the maximum dimension in the distance distribution function about 5 % larger than the corresponding values calculated on the basis of the crystal structure.     

Small angle x-ray study on the structure of active and inactive ribulose bisphosphate carboxylase from Alcaligenes eutrophus. Evidence for a configurational change

Two small angle x-ray scattering curves have been obtained from active and inactive ribulose 1,5-bisphosphate carboxylase from Alcaligenes eutrophus. The radius of gyration was calculated to be R = 47.8 +/- 0.1 nm for the active enzyme and R = 49.2 +/- 0.1 nm for the inactive enzyme. The maximum particle dimension amounts to 13.5 +/- 0.5 nm for the active and 15.7 +/- 0.5 nm for the inactive enzyme. A model of the active carboxylase is presented. It is in good agreement with models derived from electron microscopical data. Model calculations for the inactive enzyme show some evidence for a configurational change.     

Structural parameters of amylopectin clusters and semi-crystalline growth rings in wheat starches with different amylose content

Small-angle X-ray scattering (SAXS) and scanning electron microscopy (SEM) were used to investigate the internal structure of wheat starch granules with different amylose content. Different approaches were used for treatment (interpretation) of SAXS data to assess the values of structural parameters of amylopectin clusters and the size of crystalline and amorphous lamella in different wheat starches. The average values of the semi-crystalline growth rings thickness in starches have been determined and the relationship between structural characteristics and thermodynamic melting parameters is discussed.     

Raman microscopy and X-ray diffraction,a combined study of fibrillin-rich microfibrillar elasticity

Fibrillin-rich microfibrils are essential elastic structures contained within the extracellular matrix of a wide variety of connective tissues. Microfibrils are characterized as beaded filamentous structures with a variable axial periodicity (average 56 nm in the untensioned state); however, the basis of their elasticity remains unknown. This study used a combination of small angle x-ray scattering and Raman microscopy to investigate further the packing of microfibrils within the intact tissue and to determine the role of molecular reorganization in the elasticity of these microfibrils. The application of relatively small strains produced no overall change in either molecular or macromolecular microfibrillar structure. In contrast, the application of larger tissue extensions (up to 150%) resulted in a markedly different structure, as observed by both Raman microscopy and small angle x-ray scattering. These changes occurred at different levels of architecture and are interpreted as ranging from alterations in peptide bond conformation to domain rearrangement. This study demonstrates the importance of molecular elasticity in the mechanical properties of fibrillin-rich microfibrils in the intact tissue.