On the quaternary structure of native rabbit muscle phosphofructokinase.

Small angle X-ray scattering measurements on solutions of native rabbit muscle phosphofructokinase (EC 2.7.1.11; ATP; D-fructose-6-phosphate 1 phosphotransferase) show that the dimer has a radius of gyration of 32.5 Å and a molecular weight of 160,000, and that the biologically active tetramer has a radius of gyration of 51.5 Å and a molecular weight of 320.000. A possible model was calculated from scattering curves of the dimer and tetramer suggesting two hollow cylinders with cell dimensions for the dimer of a height of 78.0 Å and a long half axis of 38.0 Å, and for the tetramer of a height of 155.0 Å and an outer radius of 35.0 Å. The tetramer is formed along the 78.0 Å axis of the dimer by means of an end-to-end aggregation. The overall particle dimensions of the protomer of molecular weight 80,000 is calculated to be 35.0 × 30.0 × 55.0 Å, assuming an elliptical molecule. The distance between the centers of the two dimeric units within the tetramer is 104.5 ± 1.5 Å.     

Escherichia coli lac repressor is elongated with its operator DNA binding domains located at both ends

From small-angle X-ray scattering experiments on solutions of Escherichia coli lac repressor and repressor tryptic core, we conclude that the domains of repressor that bind to operator DNA lie at the ends of an elongated molecule. The addition of the inducer, isopropyl-β-d-thiogalactoside, to either repressor or core does not produce a measurable structural change, since the radius of gyration of repressor is 40.3 ± 1.9 Å without and 42.2 ± 1.7 Å with isopropyl-β-d-thiogalactoside; the core radius of gyration is 35.4 ± 1.1 Å without ligand and 36.3 ± 1.1 Å with isopropyl-β-d-thiogalactoside. In the context of data from single crystals of repressor and core, the measured radii of gyration are shown to be consistent with a core (or repressor) molecule of dimensional anisotropy 1: (1.5 to 2.0): (3.0 to 4.0). The 5 Å difference in radius of gyration between native and core repressor is interpreted to mean that the amino terminal 59 residues (headpieces) lie at the ends of an elongated repressor molecule. This structure implies that the repressor may have DNA binding sites, consisting of two adjacent headpieces, on each end of the molecule and this binds to the DNA with its long axis perpendicular to the DNA.     

5.5 Å crystallographic structure of penicillin β-lactamase and radius of gyration in solution

An electron density map of crystalline R-TEM Escherichia coli β-lactamase (penicillinase) has been calculated from X-ray diffraction data at 5.5 Å resolution with protein phases based on Friedel mates from a high-quality samarium derivative. The mean figure of merit for 854 independent reflections is 0.75. The monomeric molecule is slightly ellipsoidal and contains one and possibly two regions of α-helix which are 25 Å long. The Crystallographic search for the substrate binding site has so far been inconclusive. The radius of gyration of the enzyme in solution at pH 7 is 17.1 ± 1.0 Å from small-angle X-ray scattering measurements. This compares with 18.6 å calculated from the low-resolution electron density map of the molecule in the crystal.     

Structure and mechanism of action of riboflavin-binding protein: Small-angle X-ray scattering,sedimentation, and circular dichroism studies on the holo- and apoproteins

Riboflavin-binding protein, a transport protein occurring in egg whites, binds riboflavin tightly at pH values above 4.5 but releases it readily at pH values below 4.0. Structural aspects of this biologically important binding were studied by several methods. Analysis of sedimentation equilibrium data gave an average molecular weight of 32,500 ± 1000 for all forms of the protein and showed the absence of changes in quaternary structure when riboflavin was bound at neutral pH or released at pH 3.7. Sedimentation velocity showed no change in tertiary structure on binding at pH 7.0 but revealed a significant change in sedimentation constant at pH 3.7. While circular dichroism showed no appreciable change in secondary structure, it gave evidence of a marked change in the aromatic region at the lower pH. Small-angle X-ray scattering, going from the holoprotein at neutral pH to the apoprotein at low pH, showed a small but significant increase in radius of gyration (19.8 ± 0.2 vs 20.6 ± 0.1 Å) with slightly decreased anisotropy and with substantial increases in molecular volume (55,600 ± 530 vs 66,500 ± 240 ų), surface (11,840 ± 120 vs 13,470 ± 140 Å), and hydration (0.27 ± 0.01 vs 0.38 ± 0.01 g H₂O/g dry protein). Hydration values were obtained from small-angle X-ray scattering in two different ways for comparison with those calculated from sedimentation coefficients by way of frictional coefficients (derived from two different dimensionless ratios based independently on the structural small-angle X-ray scattering data). For either form of the protein, the surface calculated from an ellipsoidal model could account for only about 62% of the surface found experimentally. The excess surface was ascribed to topographic features of the molecule. Relative changes in this new parameter, together with the circular dichroism data and the known association of riboflavin binding with aromatic residues, suggested the opening of an aromatic-rich cleft concomitant with the release of riboflavin as a consequence of lowered pH.     

Solution scattering studies of dimeric and tetrameric spectrin

The structure of spectrin dimers and tetramers in solution has been examined by light, low-angle X-ray and neutron scattering. The results show a good correspondence between the solution dimensions of these molecules and their appearance in the electron microscope after shadowing. The scattering profiles are not compatible with an extended rod-like character, but reflect the presence of a considerable degree of bending. The radii of gyration of the dimer and tetramer were determined to be 170 and 375 Å and the cross-section radii of gyration 14 and 12.3 Å. respectively. Both are thus long. thin. rather bent molecules, and the tetramer is twice the length of the dimer.     

Evidence for calmodulin inter-domain compaction in solution induced by W-7 binding

Small-angle X-ray scattering and nuclear magnetic resonance were used to investigate the structural change of calcium-bound calmodulin (Ca²⁺/CaM) in solution upon binding to its antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). The radius of gyration was 17.4±0.3 Å for Ca²⁺/CaM-W-7 with a molar ratio of 1:5 and 20.3±0.7 Å for Ca²⁺/CaM. Comparison of the radius of gyration and the pair distance distribution function of the Ca²⁺/CaM-W-7 complex with those of other complexes indicates that binding of two W-7 molecules induces a globular shape for Ca²⁺/CaM, probably caused by an inter-domain compaction. The results suggest a tendency for Ca²⁺/CaM to form a globular structure in solution, which is inducible by a small compound like W-7.     

The conformation of polyriboadenylic acid at low temperature and neutral pH. A single-stranded rodlike structure.

The conformation of single-stranded polyrA in aqueous solution has been measured at temperatures down to ?12°C. The radius of gyration of low-molecular-weight polyrA varies very little with temperature in this range. By studying the dependence of radius of gyration on temperature for several polyrA fractions, we show that the dependence of the radius upon chain length is consistent with formation of a single-stranded rodlike structure at low temperature. The structure has an approximate length of 3.2 Å/nucleotide.     

Sizes of two amino acyl-tRNA synthetase complexes from E. coli with their cognate tRNAs.

The complexes of valyl-tRNA synthetase with tRNA_I^Val and arginyl-tRNA synthetase with tRNA_II^Arg from $\text{E. coli}$ were studied by light scattering measurements and analytical ultracentrifugation of concentrations as low as 40 μg/ml. The molecular weights determined from these studies were 260,000 ± 2,000 for the valyl-tRNA synthetase·tRNA complex, and 310,000 ± 1,500 for the arginyl-tRNA synthetase·tRNA complex at pH 7.1. The stoichiometry for the complexes are apparently 2:1 for valyl-tRNA synthetase and tRNA and 4:1 in the case of the arginyl-tRNA synthetase and tRNA. From the angular dependence of the scattered intensity a radius of gyration of 54.5 Å for the complex between valyl-tRNA synthetase and tRNA was found, whereas for the other complex a value of 59.1 Å was found.     

Neutron scattering studies of escherichia coli tyrosyl-trna synthetase and of its interaction with trna tyr

Small-angle neutron scattering studies of Escherichia coli tyrosyl-tRNA synthetase indicate that in solution this enzyme is a dimer of M_r, 91 (±6) × 10³ with a radius of gyration R_G of 37.8 ± 1.1 Å.The increase in the scattering mass of the enzyme upon binding tRNA^Tyr has been followed in 20 mm-imidazole · HCl (pH 7.6), 10 mm-MgCl₂, 0.1 mm-EDTA, 10 mm-2-mercaptoethanol, 150 mm-KCl. A stoichiometry of one bound tRNA per dimeric enzyme molecule was found. The R_G of the complex is equal to 41 ± 1 Å. Titration experiments in 74% ²H₂O, close to the matching point of tRNA, show an R_G of 38.5 ± 1 Å for the enzyme moiety in the complex. From these values, a minimum distance of 49 Å between the centre of mass of the bound tRNA and that of the enzyme was calculated.In low ionic strength conditions (20 mm-imidazole-HCl (pH 7.6), 10 mm-MgCl₂, 0.1 mm-EDTA, 10 mm-2-mercaptoethanol) and at limiting tRNA concentrations with respect to the enzyme, titrations of the enzyme by tRNA^Tyr are characterized by the appearance of aggregates, with a maximum scattered intensity at a stoichiometry of one tRNA per two enzyme molecules. At this point, the measured M_r and R_G values are compatible with a compact 1:2, tRNA: enzyme complex. This complex forms with a remarkably high stability constant: (enzyme:tRNA:enzyme)/(enzyme:tRNA)(enzyme) of 0.1 to 0.3(× 10⁶) m^?1 (at 20 °C). Upon addition of more tRNA, the complex dissociates in favour of the 1:1, enzyme:tRNA complex, which has a higher stability constant (1 to 3 (× 10⁶) m^?1).     

Small-angle x-ray scattering study of lysine transfer RNA ligase from yeast

The scattered X-ray intensities from dilute solutions of lysine transfer RNA ligase, in 0.1 m-phosphate buffer at pH 7.0, have been measured at 21 °. The radius of gyration R (37.5 Å), the molecular weight M (114,000), and the volume V (295,000 ų) were determined.A comparison between the scattering curves obtained from the enzyme and the theoretical scattering curves of different triaxial bodies shows that the shape of the molecule can be represented by an oblate ellipsoid with the semiaxes A = 62.7, B = 50.1 and $\text{C = 23.5}\text{A}\text{?}$.     

Changes in the x-ray solution scattering of aspartate transcarbamylase following the allosteric transition.

Aspartate transcarbamylase (Escherichia coli) has been studied by X-ray solution scattering in the s range 0.002 to 0.06 Å^?1. The spectra display sharp maxima and minima whose positions and amplitudes show considerable changes upon ligation with the transition state analogue N-(phosphonacetyl)-l-aspartate. The magnitude of the change in diffraction pattern is so large that X-ray solution scattering should be a useful technique for studying the proportions of different quaternary forms in solutions of this enzyme. In particular, the kinetics of the allosteric transition appear to be within the reach of X-ray diffraction experiments.Some structural parameters of the allosteric transition were obtained from the diffraction patterns. The radius of gyration of the native enzyme is 45.9 ± 0.5 Å, and after ligation it increases to 48.4 ± 1.0 Å. At the same time, the peak of the pair distribution function is shifted from 58 Å to 63 Å. These changes indicate that the molecule swells after the allosteric transition to the R form. However, the maximum distance (from the pair distribution function) does not increase after ligation, and may even decrease slightly. Some probable subunit movements during allosteric activation are discussed.     

Conformational changes in arginine kinase upon ligand binding seen by small-angle X-ray scattering

Small-angle X-ray scattering is used to study the effects of substrate binding to lobster arginine kinase in solution. We measure the radius of gyration of the enzyme in the absence and in the presence of ligands. We find that the radius of gyration decreases by 1.20 ± 0.25 Å upon binding ADP-Mg and L-arginine to form the ternary complex. The same decrease is also observed upon binding ADP-Mg alone or ATP-Mg. These results indicate a large conformational change consistent with the hinge motion of domains observed in other phosphokinases.     

Neutron scattering study of the 13 S fragment of 16 S RNA and its complex with ribosomal protein S4

A fragment with a molecular weight of 170,000 and a sedimentation coefficient of 13 S which is capable of specifically binding ribosomal protein S4 has been obtained by digestion of Escherichia coli 16 S RNA with ribonuclease A. The 13 S fragment of 16 S RNA and its complex with protein S4 have been studied by different physical methods; in the first place, by neutron scattering. It has been shown that this fragment is very compact in solution. The radii of gyration of this fragment (50 ± 3 Å) and of protein S4 within the complex (17 ± 3 Å) coincide, within the limits of experimental error, with the radii of gyration for the free RNA fragment (47 ± 2 Å) and the free ribosomal protein S4 in solution (18 ± 2 Å). Hence the conclusion is drawn that the compactness of the RNA fragment and the ribosomal protein does not change on complex formation. The compact 13 S fragment of 16 S RNA is shown to be contrast-matched in solvent containing 70% ²H₂O which corresponds to a value for the partial specific volume of RNA of 0.537 cm³/g.     

Molecular size and shape of the native and reassociated forms of the extracellular haemoglobin of Tubifex tubifex

The extracellular haemoglobin of Tubifex tubifex and the product of its reassociation at neutral pH subsequent to dissociation at alkaline pH, were examined by small-angle X-ray scattering. The following molecular parameters were determined for the native and reassociated molecules, respectively: maximum diameter 30.0±1.0 and 32.0±1.0nm; radius of gyration 10.66±0.15 and 11.07±0.15 nm; molecular weight (3.09±0.15) × 10⁶ and (2.99±0.15) × 10⁶ dalton. Although the scattering curves of the native and reassociated haemoglobin possess similar shapes the distance distribution functions exhibit slight differences in their shape as well as in the position of their maximum. The best fit with the experimental distribution functions was obtained with models consisting of 12 spheres arranged in two hexagonal layers. In the case of the native haemoglobin each of the 12 spheres has a diameter of 9.3 nm while for the reassociated haemoglobin each of the 12 spheres has a diameter of 11.5 nm. The results suggest that although their molecular weights are the same, the reassociated molecule is slightly larger than the native molecule     

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 scattering (SAXS) study of the extracellular Hemoglobin of Glossoscolex paulistus: effect of pH, iron oxidation state, and interaction with anionic SDS surfactant

pH effects on the oligomeric structure of giant Glossoscolex paulistus extracellular hemoglobin in the oxyand met-forms have been studied as well as effects of the addition of anionic sodium dodecyl sulfate surfactant. A radius of gyration of 110 A is observed for a macromolecule. At 2 mm surfactant, the radius of gyration diminishes slightly for the oxy-form. However, the extrapolated initial scattering intensity (I0) decreases a factor of 2.5, indicating protein dissociation. At 20 mm surfactant, further I0 decrease is observed, with a reduction of radius of gyration to approximately 30 A consistent with dissociation into smaller subunits. At pH 9.0, the scattering curves are similar to that obtained for the protein in the presence of 20 mm surfactant at pH 7.0. A radius of gyration of approximately 35 A shows that the giant hemoglobin dissociation into small subunits also occurs at alkaline pH. From the I0 value, one can suggest that the tetramer is the main scatter at pH 9.0. At pH 7.0, the met-form dissociates to a larger extent at 2 mm surfactant as compared with the oxy-form, and the main scatters seem to be the 1/12 subunit. At pH 9.0, for the oxy-form, the addition of surfactant does not modify the scattering curve and a radius of gyration approximately 30 A is obtained, while for the met-form some kind of aggregation is observed. Our results give support to conclude that the iron oxidation state is an important factor modulating the oligomeric dissociation.