Bo Sjöberg

Small‐angle X‐ray scattering and crosslinking study of the proteins L7/L12 from Escherichia coli ribosomes

ribosomes there is only one protein that occurs in more than one copy [ 11. This is the protein L12 and its cr-aminoacetylated form, L7 [2] . The total sum of these two components appears to be at least three and possibly four copies per ribosome [1,3,4] and the fraction of L12 found varies from 25% to 85% during the growth cycle of the bacteria [S] .

Small-angle X-ray scattering study of the 5S RNA binding proteins L18 and L25 from Escherichia coli ribosomes

The elucidation of the structure and function of ribosomes at the molecular level is a major objective in molecular biology. The specific complex formation between 5S RNA and a few ribosomal proteins is a part of this general problem and consequently has received much attention [1-6] . Nevertheless, we are very far from a complete understanding of the mechanisms involved in the ribosomal protein-nucleic acid interaction, and this will eventually require a thorough knowledge of the structures of 5S RNA, the binding proteins, and their complexes. Since, as yet, none of these ribosomal components have been crystallized, and consequently the prospects of X-ray crystallographic analyses are curtailed, we have initiated a small-angle X-ray scattering study of these components. In a previous report, a tertiary model for 5S RNA was described [7]. This report describes the shapes of the 5S RNA-binding proteins L18 and L25 [81. Proteins L18 and L25, like many other ribosomal proteins, exhibit low solubility in aqueous solution and tend to aggregate; as a result, it was necessary to study the monomeric forms of the proteins at rather low concentrations. This limited the amount of information that could be gained about the protein structures. 2. Materials and methods

Small-angle X-ray scattering study of the interaction between lysine transfer RNA ligase from yeast and transfer RNA*

The main complex formed in solution between lysine:tRNA ligase from yeast and tRNA is composed of two enzyme molecules and one molecule of tRNA as shown by an equilibrium analysis of small angle X-ray scattering data recorded at 21°C in the pH range 6·6 to 7·4. The stability of the complex with both cognate and non-cognate tRNA decreases with increasing Mg 2+ concentration. The two types of complexes differ, however, in as much as the stability of the non-cognate complex is independent of pH while the stability of the cognate complex decreases with increasing pH. Analysis of the small-angle X-ray scattering from the complex between enzyme and cognate tRNA gave a radius of gyration of 57·4 A, a molecular weight of 236,000 and a volume of 600,000 A 3 , in agreement with the theoretical values calculated for a complex containing two enzyme molecules and one tRNA (molecular weight 252,600 and volume 631,000 A 3 ). A comparison of the experimental data with theoretical scattering curves computed for different triaxial bodies suggests that the two ellipsoid protein molecules interact with their longest semi-axes at right angles to each other, and that within the angle formed, partly engulfed by the protein ellipsoids, is the supposedly L-shaped tRNA molecule.

Small‐angle X‐ray scattering study of the proteins S1, S8, S15, S16, S20 from Escherichia coli ribosomes

A series of biochemical data indicates that the ribosomal proteins 54, S8, S15 and 520 are specifically bound to 16s RNA, for a review see ref. ill. However, very little is known regar~g how these proteins interact with the nucleic acid. One approach to a better understanding of this interaction may be to first analyse the conformation of the individual components and then to analyse the specific rRNAprotein complexes by using the small-angle X-ray scattering method. As the first step of this general study, we have characterized both S4 and its binding area on 16s RNA from their small-angle X-ray scattering curves (2,3f. Here, we report the results from similar studies on the proteins Sl, 58, S15, S16 and SZO. The results indicate that these proteins are elongated: the proteins S8, Sl5 and S16 appear to have a largest dimension of about 100 1p and they are similar to the L18 and L25 proteins [4] ; the protein Sl is more elongated and appears to have a maximum dimension of about 260 A . It should be noted that all the proteins were prepared by a new, gentle method [S] that appears to preserve their native conformations, cf. [6].

The solution shapes of IgG3 immunoglobulin and its Fch and Fc fragments. A small-angle x-ray scattering study

A homogeneous IgG3 protein and its corresponding Fch and Fc fragments have been studied in solution using the small-angle X-ray scattering method. The Fch and Fc fragments were produced by short digestion of IgG3 with papain and trypsin. The results indicate that the overall shape of the IgG3 molecule in solution can best be described as an elliptical cylinder with a total length of 29 nm and with a cross-section having the semiaxes 3.8 and 0.9 nm. Thus, the overall shape of IgG3 is considerably different from the Y-shape normally adopted for the IgG1 molecule. The analysis of the data obtained for the Fch and Fc fragments also yields elliptical cylinders with almost the same dimensions of the cross-section but with shorter total lengths, 11.4 and 6.7 nm, respectively. The molecular weights of the IgG3 protein and the Fch and Fc fragments were determined to be 1.8 x 10(5), 0.61 x 10(5), and 0.50 x 10(5), respectively.

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 S4-RNA, the 16 S RNA binding site for the protein S4 from Escherichia coli ribosomes

The ribonucleic acid (16 S RNA) of the 30 S Escherichia coli ribosomal subunit binds many of the proteins in this subunit, such as, for instance, S4, S7, S8, S15 and S20 [1,2]. The 16 S RNA binding site for the S4 protein, S4-RNA, can be isolated in pure form [3-91 ; and it is indicated that this binding site consists mainly of two regions of approximately 150 and 160 nucleotides, originating from the 5’ terminal third of the 16 S RNA molecule [7]. These two regions, which are separated by about 120 nucleotides of the 16 S RNA sequence, seem to be stabilized by a specific RNA-RNA interaction [7] . Although the sequence of the S4-RNA region, prepared with both T1 and pancreatic ribonucleases, has been partially analysed [7,9], the correct molecular weight and the size and shape of S4-RNA are not known. We have analysed S4-RNA by using the small-angle X-ray scattering method; the results yield a molecular weight of 136 000 and a radius of gyration of 43.5 A. The X-ray scattering curve can be explained, in its proximal angular range, by the scattering from a twoparameter, uniform electron density model with a shape of an oblate ellipsoid and the dimensions of 132 X 132 X 32 a. 2. Materials and methods

Dodecylsulfate‐induced dissociation of human α2‐macroglobulin

The dodecylsulfate-induced dissociation of the tetrameric alpha 2-macroglobulin molecule from human plasma has been investigated by the small-angle neutron scattering (SANS) method. The great advantage with the SANS method is that, by using deuterated dodecylsulfate, and contrast variation by changing the D2O/H2O ratio of the solvent, we can selectively study just the protein part, or the dodecylsulfate part, of the protein-dodecylsulfate complex. More than a thousandfold excess of dodecylsulfate (on a molar basis) is needed in order to dissociate alpha 2-macroglobulin to particles with, on average, half the original molecular mass. By combining the SANS data with results obtained by the equilibrium dialysis technique it follows that, under these circumstances, approximately one thousand dodecylsulfate molecules are associated per alpha 2-macroglobulin molecule. From the significant increase in the radius of gyration, which accompanies the dissociation process, we can conclude that the dissociation is associated with a drastic change in conformation of the protein molecule. From measurements where the dodecylsulfate part of the complex dominates the SANS signal we also get an indication that the dodecylsulfate is randomly distributed along the polypeptide chain, rather than being arranged in large clusters at certain regions of the protein molecule. By fitting the parameters of a binding model to the experimental data we obtain the result that most of the more than one thousand bound dodecylsulfate molecules, necessary for dissociation, are involved in the change in conformation, and the dissociation process is, in fact, driven by the binding of a very few extra dodecylsulfate molecules to the dissociation products. These data indicate that the dodecylsulfate-induced dissociation of alpha 2-macroglobulin is probably more complicated than just breaking, for instance, a hydrophobic interaction.

The H+-induced dissociation of human plasma α2-macroglobulin

The dissociation of the tetrameric alpha 2-macroglobulin molecule into two half-molecular fragments, which occurs at pH less than 4.5, has been investigated using the small-angle neutron scattering method, and test of trypsin binding activity. Best fit with the relative forward scattering of neutrons is obtained for a model where the dissociation of the protein is driven by the uptake of H+ on altogether four acid-base groups, one per monomeric subunit of alpha 2-macroglobulin. These groups are not (or only slightly) accessible in the native tetramer, but become exposed to the solvent after dissociation of the protein. The H(+)-binding constant obtained for these groups, after dissociation of the protein, log K1 in the range 4.2-4.5, suggests that they are most probably carboxylate groups. From the about 10% increase in the radius of gyration, which occurs when lowering the pH from 4.5 to 2.0, we can conclude that the dissociation is associated with a change in structure of the protein. Tests of trypsin binding show that there is also an irreversible loss in trypsin binding activity, which is directly related to the fraction of dissociated protein. Thus, at pH less than 4.5, there is a transition of alpha 2-macroglobulin which results simultaneously in dissociation, disorganisation of the conformation of the subunits and loss in activity.

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

Abstract 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 A), the molecular weight M (114,000), and the volume V (295,000 A3) 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 C = 23.5 A .