Small-angle x-ray studies on malate synthase from baker's yeast.

Malate synthase was investigated in solution by the small-angle X-ray scattering technique. The substrate-free enzyme was shown to have a molecular weight of 186000, a radius of gyration of 3.96 nm, a maximum particle diameter of 11.2 nm, a volume of 343 nm³, a radius of gyration of the thickness of 1.04 nm, and an axial ratio of 1:0.33. The enzyme molecule undergoes small changes in overall structure upon binding substrates. Investigation of the enzyme under prolonged exposure to X-rays led to an aggregation of the enzyme and allowed statements concerning the way of aggregation and factors influencing aggregation.     

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.     

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 study on bovine porphobilinogen synthase (5-aminolaevulinate dehydratase)

The quaternary structure of the native (zinc) porphobilinogen synthase (5-amino-laevulinate dehydratase) from bovine liver and its lead-substituted derivative is studied in solution by small angle X-ray scattering. In spite of the profound inhibitory effect of lead ions in the enzyme they do not produce a change in the quaternary structure detectable by small angle X-ray scattering. The most important molecular parameters of the native enzyme were found to be: radius of gyration Rg = 4.04 +/- 0.04 nm and maximum dimension Dmax = 12.0 +/- 0.5 nm. The corresponding values for the lead derivative are: Rg = 4.26 +/- 0.1 nm and Dmax = 12.5 +/- 0.5 nm. The quaternary structure of the enzyme in solution is described by a model, which fits the experimental scattering and distance distribution function.     

Protein S1 from Escherichia coli ribosomes: an improved isolation procedure and shape determination by small-angle X-ray scattering.

Ribosomal protein S1 from Escherichia coli was studied in solution by small-angle X-ray scattering and the following parameters were obtained. The radius of gyration R = 8.0 +/- 0.2 nm; largest diameter D = 28 nm; molecular weight = (8--9) x 10(4). The data also yielded (with the assumption of a rigid particle with almost constant electron density) two radii of gyration of cross-section Rq1 = 2.5 +/- 0.1 nm and Rq2 = 1.05 +/- 0.05 nm and molecular volume = 140 nm3. The experimental scattering curve of S1 was compared with the theoretical scattering curves for several rigid triaxial homogeneous bodies and the closest fit was given by that of a flat elliptical cylinder with the dimensions of 4.5 nm and 0.88 nm for the two semiaxes and 26.5 nm for height. The results from the present X-ray scattering studies and those from limited proteolytic digestion of protein S1 [J. Mol. Biol. 127, 41--54, (1979)] support the notion that the structure of protein S1 is organized into two distinct subdomains within its elongated overall shape. Protein S1 was purified for this study by an efficient procedure which yielded 12 mg S1/g ribosomes. The isolated protein was fully active in functional tests both before and after X-ray irradiation.     

Quantitative NMR studies of high molecular weight proteins: application to domain orientation and ligand binding in the 723 residue enzyme malate synthase G

A high-resolution multidimensional NMR study of ligand-binding to Escherichia coli malate synthase G (MSG), a 723-residue monomeric enzyme (81.4 kDa), is presented. MSG catalyzes the condensation of glyoxylate with an acetyl group of acetyl-CoA, producing malate, an intermediate in the citric-acid cycle. We show that despite the size of the protein, important structural and dynamic information about the molecule can be obtained on a per-residue basis. 15N-1HN residual dipolar couplings and carbonyl chemical shift changes upon alignment in Pf1 phage establish that there are no significant domain reorientations in the molecule upon ligand binding, in contrast to what was anticipated on the basis of both the X-ray structure of the glyoxylate-bound form of the enzyme and structural studies of a related set of proteins. The chemical shift changes of 1HN, 15N and 13CO nuclei upon binding of pyruvate, a glyoxylate-mimicking inhibitor, and acetyl-CoA have been mapped onto the three-dimensional structure of the molecule. Binding constants of pyruvate, glyoxylate, and acetyl-CoA (in the presence of pyruvate) have been measured, along with the kinetic parameters for glyoxylate and pyruvate binding. The on-rates of pyruvate and glyoxalate binding, approximately 1.2 x 10(6)M(-1)s(-1) and approximately 2.7 x 10(6)M(-1)s(-1), respectively, are significantly lower than what is anticipated from a simple diffusion-controlled process. Some structural implications of the chemical shift perturbations upon binding and the estimated ligand on-rates are discussed.     

Small-angle X-ray scattering study by synchrotron orbital radiation reveals that high molecular weight subunit of glutenin is a very anisotropic molecule.

Small-angle X-ray scattering of one high molecular weight (HMW) subunit of wheat glutenin was measured at protein concentration ranges from 1.0 to 10.0 mg/ml. The radius of gyration of whole particles, RO, in aq. 50% (v/v) 1-propanol and 0.1M acetic acid was 16.6 +/- 0.1nm and 22.8nm, respectively, and the corresponding radius of gyration of the cross-section, RC, was 2.82 +/- 0.02 nm and 2.23 +/- 0.01 nm, which indicate that the glutenin HMW subunit exists as very anisotropic particles in both solutions. The RO and RC values of the subunit, and the drastic decrease in scattered intensity at small angles that occurs in the acetic acid solution with relatively low protein concentration are completely explained in terms of rod-like molecules of the glutenin HMW subunit.     

Size of a human serum albumin molecule in solution]

The size of a human serum albumin molecule in aqueous solution containing 150 mM NaCl was studied using small-angle neutron scattering. The molecular radius of gyration was estimated to be 27.4 +/- 0.35 A. The compact sphere should have a smaller radius of gyration, whereas the popular human serum albumin model, a "cigar" 136 A long, should correspond to a greater radius of gyration. Possible shapes of the human serum albumin molecule which are in accordance with the results obtained, are the following: an extended ellipsoid less than 110 A of length or a nonsymmetrical oblate ellipsoid with a diameter of 85 A. The oblate ellipsoid might be close to the heart"-shaped structure of the crystalline human serum albumin molecule. The size of the albumin molecule does not change significantly as pH increases to 8.9. The possibility of the dynamic coexistence of various human serum albumin conformers in solution is discussed.     

On the subunit structure of crotoxin: hydrodynamic and shape properties of crotoxin, phospholipase A and crotapotin.

This paper reports physical-chemical properties of the subunit structure of crotoxin, phospholipase A and crotapotin. The native crotoxin has a sedimentation coefficient of 3S and a radius of gyration of R_g = 16.5 Å and a molecular weight of 30,900. Dissociation of the 3S particle results in two proteins of unequal size with sedimentation coefficients of 1.5 S (crotapotin) and 1S (phospholipase A). These dissociated species and the reconstituted complex were investigated by means of hydrodynamic methods including small angle X-ray scattering. The actual frictional ratios were obtained indicating that crotoxin is a sphere with a Stokes' radius of R_o = 22.5 Å and an axial ratio of 1:3, whereas phospholipase A, depending on the degree of association, has a radius of gyration of R_g = 32.4 Å and a high axial ratio of 1:14 for the monomer. Crotapotin has a radius of gyration of R_g = 12.4 Å, indicating an oblate ellipsoid of revolution of an axial ratio of 1:4. Evidently, the crotoxin complex consists of one highly asymmetric molecule (phospholipase A) and an oblate ellipsoid (crotapotin), which reconstitutes to a spherical 3S-particle (crotoxin).     

Small-angle X-ray study on the quaternary structure of erythrocruorin from Caenestheria inopinata.

The erythrocruorin of the clam shrimp Caenestheria inopinata was studied in sodium phosphate buffer at pH 6.8 by small-angle X-ray scattering. The following molecular parameters were determined: radius of gyration 4.77 +/- 0.05 nm, maximum dimension 14.0 +/- 0.5 nm and a volume of 640 +/- 40 nm3. A model which fits the experimental data well is presented. The model is composed of 10 subunits arranged symmetrically in two layers, whereby five subunits are always forming a ring.     

Studies by small-angle x-ray scattering of the quaternary structure of dissociation products of the beta-haemocyanin of Helix pomatia.

Helix pomatia beta-haemocyanin was split into dissociation products by varying the pH and the ionic strength. The purity of the solution was checked in an ultracentrifuge. Two defined dissociation products were studied in solution by small-angle X-ray scattering. In Tris-HC1 buffer, pH 8.0 and ionic strength 1 M, the following parameters of the dissociation product (tenths) could be determined: molecular weight = 7 x 10(5), volume = 1350 nm3, radius of gyration = 9.0 nm, maximal distance = 28.3 nm, radius of the spherical subunits about 2.6 nm, number of the subunits approximately 19. Tris-HC1 buffer, pH 8.7 and ionic strength 0.01 M, yielded dissociation products (twentieths) with the following parameters: molecular weight = 3.5 x 10(5), volume = 635 nm3, radius of gyration = 7.5 nm, maximal distance = 21.9 nm, radius of the spherical subunits about 2.5 nm. With this information, the assumption that the larger fragment was double the smaller one and the latest biochemical and morphological results, theoretical scattering curves of models were calculated and compared with the experimental curves. Two models are suggested which argee well with the dissociation products in radius of gyration and scattering.     

A model of the quaternary structure of the Escherichia coli F1 ATPase from X-ray solution scattering and evidence for structural changes in the delta subunit during ATP hydrolysis.

The shape and subunit arrangement of the Escherichia coli F1 ATPase (ECF1 ATPase) was investigated by synchrotron radiation x-ray solution scattering. The radius of gyration and the maximum dimension of the enzyme complex are 4.61 +/- 0.03 nm and 15.5 +/- 0.05 nm, respectively. The shape of the complex was determined ab initio from the scattering data at a resolution of 3 nm, which allowed unequivocal identification of the volume occupied by the alpha3beta3 subassembly and further positioning of the atomic models of the smaller subunits. The delta subunit was positioned near the bottom of the alpha3beta3 hexamer in a location consistent with a beta-delta disulfide formation in the mutant ECF1 ATPase, betaY331W:betaY381C:epsilonS108C, when MgADP is bound to the enzyme. The position and orientation of the epsilon subunit were found by interactively fitting the solution scattering data to maintain connection of the two-helix hairpin with the alpha3beta3 complex and binding of the beta-sandwich domain to the gamma subunit. Nucleotide-dependent changes of the delta subunit were investigated by stopped-flow fluorescence technique at 12 degrees C using N-[4-[7-(dimethylamino)-4-methyl]coumarin-3-yl]maleimide (CM) as a label. Fluorescence quenching monitored after addition of MgATP was rapid [k = 6.6 s-1] and then remained constant. Binding of MgADP and the noncleavable nucleotide analog AMP . PNP caused an initial fluorescent quenching followed by a slower decay back to the original level. This suggests that the delta subunit undergoes conformational changes and/or rearrangements in the ECF1 ATPase during ATP hydrolysis.     

An elongated model of the Xenopus laevis transcription factor IIIA-5S ribosomal RNA complex derived from neutron scattering and hydrodynamic measurements.

The precise molecular composition of the Xenopus laevis TFIIIA-5S ribosomal RNA complex (7S particle) has been established from small angle neutron and dynamic light scattering. The molecular weight of the particle was found to be 95,700 +/- 10,000 and 86,700 +/- 9000 daltons from these two methods respectively. The observed match point of 54.4% D2O obtained from contrast variation experiments indicates a 1:1 molar ratio. It is concluded that only a single molecule of TFIIIA, a zinc-finger protein, and of 5S RNA are present in this complex. At high neutron scattering contrast radius of gyration of 42.3 +/- 2 A was found for the 7S particle. In addition a diffusion coefficient of 4.4 x 10(-11) [m2 s-1] and a sedimentation coefficient of 6.2S were determined. The hydrodynamic radius obtained for the 7S particle is 48 +/- 5 A. A simple elongated cylindrical model with dimensions of 140 A length and 59 A diameter is compatible with the neutron results. A globular model can be excluded by the shallow nature of the neutron scattering curves. It is proposed that the observed difference of 15 A in length between the 7S particle and isolated 5S RNA most likely indicates that part(s) of the protein protrudes from the end(s) of the RNA molecule. There is no biochemical evidence for any gross alteration in 5S RNA conformation upon binding to TFIIIA.     

Small-angle X-ray studies of the human immunoglobulin molecule Kol.

The conformation of the human immunoglobulin molecul Kol [IgG I, kappa2 gamma2, Gm(f)+] was studied by small-angle X-ray scattering in 0.15 M NaCl solution. The radius of gyration was found to be 5.84 +/- 0.04 nm, the volume 329 +/- 15 nm3 and the molecular weight 150 000 +/- 10 000. Information on the overall shape was obtained by comparing the experimental scattering curve with the calculated curves for various models. The models were obtained by arranging the models found for the Fab and Fc fragments of the same immunoglobulin molecule in a different manner. A model which fits all the date and the form of the experimental scattering curve is presented.     

Structure and RNA content of the prosomes.

Duck erythroblasts prosomes were analysed by small angle neutron scattering (SANS), dynamic light scattering and (cryo-)electron microscopy. A molecular weight of approximately 720,000 +/- 50,000, a radius of gyration of 64 +/- 2 A and a hydrodynamic radius of approximately 86 A were obtained. Electron micrographs show a hollow cylinder-like particle with a diameter of 120 A, a height of 170 A and a diameter of 40 A for the cavity, built of four discs, the two outer ones being more pronounced than those in the center. Results from SANS indicate less then 5% of RNA in the purified prosomes, but nuclease protection assays confirm its presence.     

Molecular structure of malate synthase and structural changes upon ligand binding to the enzyme

Malate synthase has a molecular weight of about 170 000 as shown by ultracentrifugation, sucrose gradient centrifugation, and thin layer gel-chromatography. High dilution, extremes of pH, succinylation, and treatment with sodium dodecylsulfate suggest the enzyme to be a tetramer. The CD spectrum is typical for a globular protein with moderate helical content (～30 %), and shows anomalous Cotton effects at 250–290 nm. Binding of substrates (acetyl-CoA, glyoxylate) or the substrate analog pyruvate causes slight conformational changes which are reflected in alterations of the CD bands in the range of aromatic absorption; binding of Mg²⁺ causes no structural effects, suggesting the metal ion to be involved in enzymatic catalysis rather than structural alterations.     

The conformation of a large RNA fragment from the E.coli ribosomal 16S-RNA. An X-ray and neutron small-angle scattering study.

A large 12S RNA fragment which constitutes the 5' two-thirds of 16S-RNA from the E. coli 30S subunit has been investigated by small-angle X-ray and neutron scattering. The results indicate that in reconstitution buffer the 12S-RNA fragment has a molecular weight of 270,000 +/- 20,000 and a radius of gyration of 7.1 nm. The scattering data are compatible with the RNA being folded into two major domains with the shapes of two adjacent, quite similar cylinders.     

Solution structure of human plasma fibronectin using small-angle X-ray and neutron scattering at physiological pH and ionic strength

Human plasma fibronectin has been investigated at physiological pH and ionic strength, by using small-angle X-ray and neutron scattering techniques. The results indicate that the molecule is disc shaped with an axial ratio of about 1:10. In fact, an ellipsoid of revolution with semiaxes a = 1.44 nm and b = c = 13.8 nm is in agreement with the experimental scattering data, and can also fully explain the rather extreme hydrodynamic parameters reported for fibronectin. The X-ray data gave a radius of gyration of 8.9 nm and a molecular weight of 510,000, whereas the neutron data gave slightly larger values, 9.5 nm and 530,000, respectively. From the volume of the best fitting ellipsoid we obtain a degree of hydration of 0.61 g H2O/g protein (dry weight). Neutron data, recorded at different D2O concentrations in the solvent, gave a match point of 43% D2O, which indicates that approximately 80% of the hydrogens bound to oxygen and nitrogen are exchangeable.     

Small-angle x-ray scattering study of metal ion-induced conformational changes in Serratia protease.

Metal ion-induced conformational changes in Serratia protease which contains one zinc ion per molecule were investigated by the small-angle x-ray scattering method. The molecule is an elongated ellipsoid of approximately 110 x 40 x 40 A with a large cleft in its central region. Comparisons of the native (zinc-enzyme) with the zinc-free (apoenzyme) enzyme and with the zinc-replated metalloenzyme show small but significant differences in their radii of gyration, maximum particle dimensions, and intraparticle pair-distance distributions. The radius of gyration and maximum particle dimension of the native enzyme are almost the same as those of the cobalt-enzyme but are shorter and longer, respectively, than those of the apo- and cadmium-enzymes. Simulation analysis based on the intraparticle pair-distribution function showed that these modified enzymes are comparable with the native enzyme in overall structure, and, except for the cobalt-enzyme, differ in cleft size. The residual enzymatic activity of the cobalt-enzyme is the same as that of the native enzyme, but the apo- and cadmium-enzymes have considerably less activity. The size of the cleft therefore is strictly controlled to ensure optimal enzyme activity, and the position and coordination behavior of the zinc ion in the cleft appears to be essential both for biological functioning and for the maintenance of the gross tertiary structure.     

Neutron scattering study of the binding of tRNAPhe to Escherichia coli phenylalanyl-tRNA synthetase

Escherichia coli phenylalanyl-tRNA synthetase has been characterized by small-angle neutron scattering. In solution (20 mM imidazole hydrochloride, pH 7.6, 10 mM 2-mercaptoethanol, and 0.1 mM ethylenediaminetetraacetic acid), this enzyme has a molecular weight of 227K +/- 20K with a radius of gyration of 48.3 +/- 0.6 A, independent of the presence of MgCl2 up to 50 mM. The change of the scattering upon adding tRNAPhe to the enzyme has been followed with 10 mM MgCl2 present in the buffer. One enzyme molecule is capable of binding two tRNAPhe molecules with affinity constants larger than 10(6) M-1. Parallel titration experiments in 73% 2H2O, close to the matching point of tRNA, show that the RG of the enzyme is not changed by the binding of one or two tRNAPhe molecules. These results are compared with quasi-electric light scattering studies [Holler, E., Wang, C. C., & Ford, N.C., Jr. (1981) Biochemistry 20, 861-867] where the addition of either MgCl2 or tRNAPhe was shown to cause dramatic changes of the apparent translational diffusion constant of phenylalanyl-tRNA synthetase.