Small-angle X-ray study on the quaternary structure of erythrocruorin from Helisoma trivolvis

The erythrocruorin from the aquatic snail Helisoma trivolvis was studied in sodium phosphate buffer at pH 6.7 by small angle X-ray scattering. The following molecular parameters were determined: radius of gyration 9.4 ± 0.1 nm and maximum dimension 29 ± 1 nm. A model which fits the experimental data well is presented. The overall shape is best described by a slightly ellipsoidal shape with a hole in the centre. A model consisting of 12 subunits forming a slightly ellipsoidal shape fits very well all scattering data.     

Small-angle X-ray scattering studies of 7S and 11S globulins from pea (Pisum sativum)

Small-angle X-ray scattering studies have been conducted on solutions of 11S and 7S globulins isolated from peas (Pisum sativum cv. Filby), and the radii of gyration and molecular weights determined. The general features of the scattering curves were similar to those reported for other seed storage proteins.     

Small-angle X-ray scattering studies of the intact eye lens: Effect of crystallin composition and concentration on microstructure

Background

The cortex and nucleus of eye lenses are differentiated by both crystallin protein concentration and relative distribution of three major crystallins (α, β, and γ). Here, we explore the effects of composition and concentration of crystallins on the microstructure of the intact bovine lens (37 °C) along with several lenses from Antarctic fish (− 2 °C) and subtropical bigeye tuna (18 °C).

Methods

Our studies are based on small-angle X-ray scattering (SAXS) investigations of the intact lens slices where we study the effect of crystallin composition and concentration on microstructure.

Results

We are able to distinguish the nuclear and cortical regions by the development of a characteristic peak in the intensity of scattered X-rays. For both the bovine and fish lenses, the peak corresponds to that expected for dense suspensions of α-crystallins.

Conclusions

The absence of the scattering peak in the nucleus indicates that there is no characteristic wavelength for density fluctuations in the nucleus although there is liquid-like order in the packing of the different crystallins. The loss in peak is due to increased polydispersity in the sizes of the crystallins and due to the packing of the smaller γ-crystallins in the void space of α-crystallins.

General significance

Our results provide an understanding for the low turbidity of the eye lens that is a mixture of different proteins. This will inform design of optically transparent suspensions that can be used in a number of applications (e.g., artificial liquid lenses) or to better understand human diseases pathologies such as cataract.     

Small-angle X-ray study on the structure of ribulose-1,5-bisphosphate carboxylase-oxygenase from Rhodospirillum rubrum

The quaternary structure of ribulose-1,5-bisphosphate carboxylase-oxygenase (rubisco) from Rhodospirillum rubrum, an enzyme consisting of two large subunits, L₂, was investigated by small-angle X-ray scattering. In the presence of HCO₃^-and Mg²⁺, rubisco is in the active state and displays a radius of gyration of 2.96 nm, a maximum diameter of 9.5 nm and a volume of 170 nm³. A model is presented where the subunits are arranged back-to-back, rotated relative to each other by 90°, and shifted by 1.3 nm. Upon inactivation by removal of HCO₃^-and Mg²⁺, the model swells slightly without any distinct changes in configuration. This contrasts with our previous observations with rubisco from Alcaligenes eutrophus, an enzyme composed of small (S) and large (L) subunits, L₈S₈, where inactivation gives rise to substantial changes in configuration.Abbreviations RuBP Ribulose-1,5-bisphosphate - 3-PGA 3-phosphoglyceric acid     

Small-angle X-ray scattering studies of the structure of nucleosome core histone complexes in solution

The nucleosome core histone complex in solution at 2 M NaCl and pH 7 has a radius of gyration R_s, of 3.48 nm and a maximum dimension, L, of 12 nm. Its shape is disc-like with a mean thickness of 3 nm. The radius of gyration determined by us is of the same value as the radius of gyration of the complex in intact core particles (Braddock) et al., Biopolymers 1981, 20, 327). Thus, we conclude that the basic histone tails of the protein complex project about 2 nm from its central part.     

Small-angle X-ray studies of Lumbricus terrestris haemoglobin

Small-angle X-ray scattering of Lumbricus terrestris haemoglobin was measured in dilute solutions in 0.1 M Tris HCl buffer, pH 7.0. The following molecular parameters were determined: radius of gyration 11.2 nm, volume 7700 nm³, maximum diameter 29 nm, molecular weight 3.95 × 10⁶. The experimental scattering curve was compared with the scattering curves and distance distribution functions calculated for various models. The overall shape of the haemoglobin could be approximated by a hollow cylinder with the following dimensions: outer radius 13.5 nm, inner radius 5.4 nm, height 16.0 nm. The best fit was obtained with a model which consists of 12 large subunits arranged in two superimposed hexagonal rings with a number of smaller subunits between the large subunits and in the centre of the molecule.     

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.     

1964: The first model for the shape of a transfer RNA molecule. An account of an unpublished small-angle X-ray scattering study

The shape of non-fractionated Escherichia coli transfer RNA molecules in solution was investigated using small-angle X-ray scattering during the years 1960-1962 at the Centre de Recherche sur les Macromolécules in Strasbourg. The innermost region of the scattering curve yielded the average molecular weight (Mr) and the radius of gyration (Rg) of the particles, whereas the experimental data at large angles could be approximated at best by the scattering curve of a kinked rod-shaped molecule. The simplest model that was compatible with Mr, Rg, and the mass per unit length of the rod was a boomerang-shaped particle made of two double helical stems connected by a sharp kink. This model that eventually proved similar to the high-resolution L-shaped structure, was presented in my Ph.D. dissertation (J. Witz, Etude de la structure de quelques polynucléotides en solution par diffusion centrale des rayons X, Ph.D. dissertation, University of Strasbourg, France, 1964) but has never been published in detail. It is the purpose of this note to recall this story.     

Small-angle X-ray scattering studies of Escherichia colil-asparaginase

Small angle X-ray scattering studies on Escherichia colil-asparaginase solutions show that the enzyme has a radius of gyration of 34.0 Å ± 0.5 Å at pH 7. The radius of gyration of the dissociated monomer is 16.0 Å ± 1.0 Å; it has the general shape of a prolate ellipsoid with an axial ratio of 1.4. A tetramer of four such ellipsoids arranged with 222 symmetry gives good agreement between measured and calculated radii of gyration if the distance between subunit centers is 43 Å. The tetramer dissociates on dilution below 1% and at pH values below 3.0. Acid-induced denaturation at pH 2.0 is irreversible in contrast to the reversible guanidine-HCl-induced denaturation.     

Structure of clathrin-coated vesicles from small-angle scattering experiments

Previously published small-angle neutron and X-ray scattering data from coated vesicles, reassembled coats, and stripped vesicles have been analyzed in terms of one common model. The neutron data sets include contrast variation measurements at three different D20 solvent concentrations. The model used for interpreting the data has spherical symmetry and explicitly takes into account polydispersity, which is described by a Gaussian distribution. Å constant thickness of the clathrin coats is assumed. The fitting of the model shows that the coated vesicles consist of a low-density outer protein shell (clathrin) and a central protein shell (accessory polypeptides and receptors) of approximately six times higher density. For the X-ray scattering and neutron contrast variation data, the polydispersity of the samples is of the order of 90 Å (full-width-at-half-maximum value) and the average outer radius is approximately 400 Å. The inner high-density shell has inner and outer radii of 115 and 190 Å, respectively. Å simultaneous fit to the three neutron contrast variation data sets identifies the lipid membrane with a thickness of 40 Å and an outer radius of 196 Å. Thus, the membrane and the high-density protein shell overlap in space, which shows that the lipid membrane contains protein. The molecular mass of the average particle is 27 × 106 Da. The coated vesicles consist, on average, of approximately 85 % protein and 15 lipids. About 40% of the protein mass is situated in the central high-density shell, which gives a large amount of protein in the lipid membrane. The densities of the central shell and the lipid membrane show that the hydration is small in the central region. Å comparison of the total mass, the mass distribution, and the structure of the average-size particles with the barrel structure shows that the accessory polypeptides are incorporated in the lipid membrane. The results from the neutron data for the reassembled coats show that the structure of these particles is very similar to the structure of the native coats. The main difference is a higher density of the central protein shell, which shows that the membrane is replaced by protein in the reassembled coats.     

X-ray scattering study on potato (Solanum tuberosum L.) cultivars during winter storage

The nanometer range structure of potato (Solanum tuberosum L.) tubers was examined by wide-angle, small-angle and ultra small-angle X-ray scattering methods. The crystallinity of starch, the lattice constants of the hexagonal lattice of amylopectin, the average crystallite size in the direction [100], the lamellar distance and the thickness of lamella stacks were determined from the data. A new achievement presented in this paper is that reasonable results for these parameters of potato starch were obtained by carrying out experiments on slices and mashes of raw potato tubers. The effects of sample preparation were also investigated by doing experiments on air-dried and re-hydrated potato samples, and on isolated potato starch as well. Changes in the structure of three different cultivars grown in Finland (S. tuberosum cv. Satu, Saturna and Lady Rosetta) were studied monthly from August to May. The physiological ageing caused changes in the crystallinity and in the crystal structure. The mean values (±SD) were determined from the data measured between September and January (30 samples). The lattice constants a=18.4±0.06 and c=10.4±0.04 Å, the crystallinity of starch 24±2% and the crystallite size 118±10 Å were obtained. The lamellar distance was 97±3 Å and the thickness of lamella stacks 513±6 Å. The structural parameters did not vary significantly between Satu, Saturna and Lady Rosetta. For comparison, two cultivars grown in the Netherlands were studied in December. The Dutch cultivars showed the same structural parameters as the Finnish cultivars.     

Molecular modeling and small angle X-ray scattering studies of Hoplosternum littorale cathodic haemoglobin

Considerable interest is currently focused on fish haemoglobins in order to identify the structural basis for their diversity of functional behavior. Hoplosternum littorale is a catfish that presents bimodal gill (water)/gut (air)-breathing, which allows this species to survive in waters with low oxygen content. The hemolysate of this fish showed the presence of two main haemoglobins, cathodic and anodic. This work describes structural features analyzed here by integration of molecular modeling with small angle X-ray scattering. Here is described a molecular model for the cathodic haemoglobin in the unliganded and liganded states. The models were determined by molecular modeling based on the high-resolution crystal structure of fish haemoglobins. The structural models for both forms of H. littorale haemoglobin were compared to human haemoglobin.     

Synchrotron radiation wide-angle X-ray scattering studies of glycinin solutions

X-ray scattering studies of glycinin solutions have been extended to higher scattering angles of 2θ 0.15 rad. Reproducible, high quality data in the angular range 0.38–6.29 nm⁻¹ have been obtained. Scattering curves show features hitherto unreported that reveal details not only of overall shape and size of the protein but also its internal structure. Analysis suggests that currently accepted models for this protein based on the assumptions of equal-sized, touching spheres are inadequate to describe experimental observations.     

Small-angle X-ray scattering studies on the X-ray induced aggregation of malate synthase

Summary Malate synthase was investigated by the small-angle X-ray scattering technique in aqueous solution. Measurements extending for several hours revealed a continuous increase of the intensity in the innermost portion of the scattering curve. There is clear evidence that this increase was caused by an X-ray induced aggregation of enzyme particles during the performance of the small-angle X-ray scattering experiment. The monitoring of the aggregation process in situ by means of small-angle X-ray scattering led to a model of the way how the aggregation might proceed. The analysis of the scattering curves of malate synthase taken at various stages of aggregation established the retention of the thickness factor of the native enzyme and the occurrence of one and later on of two cross-section factors. The process of aggregation was also reflected by the increase of extension of the distance distribution function. According to these results, the first step of aggregation might be a linear side-by-side association of the oblate enzyme particles, a process which is followed by a twodimensional aggregation. An aggregation in the third dimension was not observed during the time covered by our experiment. The predominance of aggregation in only one or two dimensions was corroborated by comparison of appropriate theoretical scattering curves with the experimental curves. The theoretical scattering curves for this comparison were obtained by averaging over the properly weighted scattering curves calculated for various species of hypothetical aggregates. The time dependence of the apparent mean radius of gyration was used to compare the aggregation of enzyme samples that were irradiated under different experimental conditions. It turned out that by addition of dithiothreitol to the enzyme solutions as well as in the presence of the substrates (acetyl-CoA, glyoxylate) or of a substrate analogue (pyruvate) or of ethanol the rate of aggregation is reduced. Enzymic activity was found to decrease about exponentially with increasing X-ray dose. The presence of dithiothreitol or of the substrate glyoxylate or of the substrate analogue pyruvate protects the enzyme against X-ray induced inactivation. The substrate acetyl-CoA does not exhibit a comparable protective effect against inactivation. Measurements of enzymic activity and small-angle X-ray scattering on samples, which had been X-irradiated with a defined dose prior to the measurements, established two different series of efficiency for the protection of the enzyme against aggregation (pyruvate > glyoxylate > acetyl-CoA) and inactivation (glyoxylate > pyruvate > $$ " align="middle" border="0"> acetyl-CoA). The results showed that there is no direct relation between the extent of aggregation and the loss of enzymic activity.     

Small-angle X-ray Scattering of Apolipoprotein A-IV Reveals the Importance of Its Termini for Structural Stability

ApoA-IV is an amphipathic protein that can emulsify lipids and has been linked to protective roles against cardiovascular disease and obesity. We previously reported an x-ray crystal structure of apoA-IV that was truncated at its N and C termini. Here, we have extended this work by demonstrating that self-associated states of apoA-IV are stable and can be structurally studied using small-angle x-ray scattering. Both the full-length monomeric and dimeric forms of apoA-IV were examined, with the dimer showing an elongated rod core with two nodes at opposing ends. The monomer is roughly half the length of the dimer with a single node. Small-angle x-ray scattering visualization of several deletion mutants revealed that removal of both termini can have substantial conformational effects throughout the molecule. Additionally, the F334A point mutation, which we previously showed increases apoA-IV lipid binding, also exhibited large conformational effects on the entire dimer. Merging this study''s low-resolution structural information with the crystal structure provides insight on the conformation of apoA-IV as a monomer and as a dimer and further defines that a clasp mechanism may control lipid binding and, ultimately, protein function.     

Time-resolved X-ray scattering study of actin polymerization from profilactin

The polymerization of actin in solutions of purified calf spleen actin or profilactin (1–10 mg·ml^-1) was followed by synchrotron radiation X-ray solution scattering. At the concentration used, polymerization of actin from profilactin or actin occurs without any lag phase. It is shown by a combination of solution scattering, model calculations and electron microscopy that contrary to the conclusions from previous viscometry studies, filaments form without any lag phase in profilactin solution but aggregate in bundles or networks. This phenomenon is independent of the method used to induce polymerization: slow temperature increase, temperature jump in the presence of polymerizing salts or fast mixing with salt. This aggregation explains the lower final viscosity levels, as compared to actin solutions, observed during the polymerization of actin from profilactin.     

Small-angle X-ray scattering studies of oxidized and reduced cytochrome oxidase from Pseudomonas aeruginosa

Small-angle X-rays scattering experiments were performed with oxidized and reduced cytochrome oxidase purified from Pseudomonas aeruginosa. The radii of gyration were calculated to be 40.5 A for the oxidized form and 37.0 A for the reduced. The longest dimension of the oxidized enzyme was 120 A while for the reduced it was 100 A. The volume of the oxidized protein was observed to be slightly greater than that of the reduced. These data indicate that there is a contraction of the structure of the enzyme during reduction of its constituent heme groups.