J.W. Anderegg

An x-ray scattering study of ferritin and apoferritin.

Horse spleen ferritin has been fractionated according to density, and hence according to iron content, by equilibrium centrifugation in cesium chloride density-gradients. The separated fractions, including iron-free apoferritin, have been studied in solution by small-angle X-ray scattering. The scattering from apoferritin agrees well with that of a hollow sphere with an outer radius of 61 A and an inner radius of 37 A. In order to investigate the scattering from the ferric hydroxide micelle independently of the protein coat which surrounds it, the ferritin molecules were put in a 53% sucrose solution which has an electron density matching that of the protein (0·410 electrons/A 3 ). This essentially eliminates the scattering from the protein. The ferric hydroxide micelle in “full ferritin”, the ferritin molecule with maximum iron content, was found to have a molecular weight of 418,000. The scattering curve for the “full ferritin” micelle agreed very well with that of a uniform sphere of 37 A radius; it does not agree with any of the spherical subunit structures which have been reported by many electron microscopists. As the micelles decrease in molecular weight, they become more asymmetric so that one dimension is always at least as large as the diameter of the hollow center of the protein coat.

Neutralization Of Poliovirus By A Monoclonal Antibody: Kinetics And Stoichiometry

First-order kinetics of neutralization have usually been interpreted as evidence that a single antibody, binding at a critical site, neutralizes the infectivity of a virus particle. In such a case, if all the binding sites were critical, an average of one antibody bound per virion would be required to reduce the infectivity of a virus sample to 37% (1/e) of its initial infectivity. However, in the work reported here using a monoclonal antibody to poliovirus which inactivated with first-order kinetics, an average of four bound antibodies were required. These results are consistent with two different models: one in which only one-fourth of the antibody binding sites on the virion are critical for neutralization; the other, in which none of the sites is critical, but neutralization takes place instead in a stepwise fashion in which each bound antibody reduces the infectivity by a factor of 3/4. The maximum binding capacity of the virion for this monoclonal antibody was approximately 30 molecules. Since the 60 protein subunits of the poliovirus capsid are related by 30 twofold axes of symmetry, it is proposed that each monoclonal antibody binds bivalently to two protein subunits related by a twofold axis. Such a binding mode would crosslink pentamers, the basic structures in picornaviral assembly and dissociation. It is proposed that pentamer crosslinking is an important element in neutralization by this monoclonal antibody. Another antibody, which may neutralize by a different mechanism, is also discussed briefly.

X-ray scattering study of ribosomes from Escherichia coli

Abstract The small-angle X-ray scattering curves for the 70, 50 and 30 s ribosomes from Escherichia coli have been measured. The radii of gyration determined were 125, 77 and 69 A for the 70, 50 and 30 s ribosome, respectively. Measurements of the relative molecular weights from the extrapolated forward-angle scattering indicate that the 30 s subunit has half the molecular weight of the 50 s subunit, but the sum of the 30 and 50 s molecular weight was found to be only 75% of the weight of the 70 s ribosome. This result might be influenced by a tendency for the 70 s particles to aggregate. The 50 s subunit was found to have an RNA or RNA-protein tail that detectably altered the shape of the scattering curve. Estimates of the dimensions of the ribosomes were made by comparing the scattering curves with theoretical curves calculated for uniform density ellipsoids and cylinders. The best fit for the 30 s data was with the curve for an ellipsoid of dimensions 55 A × 220 A × 220 A. The 50 s subunit without the tail fit best with an ellipsoid of dimensions 115 A × 230 A × 230 A. The 70 s ribosome was best approximated by an elliptical cylinder of dimensions 135 A × 200 A × 400 A. A possible model for the 70 s ribosome is discussed.

Effects of solvent environment and mode of preparation on the physical properties of ribosomes from Escherichia coli

Abstract Molecular weights of ribosomes and ribosomal subunits prepared by precipitation in (NH 4 ) 2 SO 4 , by washing in NH 4 Cl and with no wash, were compared. The former two methods apparently removed approximately 100,000 daltons of material from each of the subunits, as compared with those prepared with no wash. Physical characterization of the 70 s ribosomes and the 50 s subunits in 0.5 m -NH 4 Cl showed these particles to have sedimentation coefficients of 65 s and 45 s respectively in this medium. Sedimentation equilibrium studies showed the 65 s and 45 s particles to have essentially the same weight as the 70 s and 50 s particles. Small-angle X-ray scattering and viscosity studies indicated that the particles were not unfolded in 0.5 m -NH 4 Cl. However, a significant decrease in the density increment ( ∂ϱ ∂c ) resulted from placing the particles in this medium. This decrease is in part responsible for the decrease in the sedimentation coefficient and in the zero-angle X-ray scattering. When 45 s particles are placed in solutions of very low magnesium concentration the sedimentation coefficient decreases further to 39 s. Small-angle X-ray scattering studies show that some unfolding has occured in this latter transition.

A neutralizing monoclonal antibody against poliovirus and its reaction with related antigens

Abstract A neutralizing monoclonal antibody against type 1 poliovirus reacted, not only with native virions (N-antigen) as expected, but also with naturally occurring RNA-free proteins shells (70 S) and with 14 S assembly subunits previously thought to lack N-antigenic sites. The latter finding has important implications for development of subunit vaccines for poliovirus and probably for other picornaviruses as well.

An X-ray Scattering Study of Bromegrass Mosaic Virus

X-ray scattering data and electron microscope observations are presented for bromegrass mosaic virus. Its radial density distribution is obtained from the Fourier transform of the amplitudes of the scattered x-rays. The results indicate that the virus is 260 A in diameter, it has an almost empty central cavity which is about 80 A in diameter, and the regions occupied by RNA and protein are approximately equal in average density. Electron micrographs of negatively stained preparations also give an outside diameter of 260 A and indicate that there is a central region about 90 A in diameter into which uranyl acetate can penetrate. Positively stained preparations indicate that the nucleic acid is concentrated in a shell-shaped region which is in turn surrounded by a shell of protein. In order for the RNA and protein regions to have the same average electron density the RNA must have a hydration of 1.29 gm of water per gm of RNA and the protein must have a hydration of 0.24 gm of water per gm of protein.

An X-ray scattering study of the bacterial virus R17

An X-ray scattering study has been made on solutions of the RNA-containing bacteriophage R17. The radius of gyration of the virus was found to be 128 A. The scattered intensity shows regularly spaced maxima and minima out to a scattering angle of 75 milliradians, corresponding to a Bragg spacing of about 20 A. The radial density distribution calculated by Fourier inversion of the scattering amplitudes indicates that the virus has a mean outer radius of 133 A, an outer shell of 30 to 40 A thickness, an inner region of somewhat lower electron density and a small hollow region of about 15 A radius at the center of the virus. Presumably the outer shell is protein, the inner region contains the RNA and the hollow center contains only solvent. The above dimensions, together with the dry molecular weight and partial specific volume of the virus, indicate that there is associated with the virus 1·0 gram of water per gram of virus.

An X-ray investigation of the sizes and hydrations of three spherical virus macromolecules in solution

Abstract We have measured the small-angle X-ray scattering from dilute aqueous solutions of southern bean mosaic virus, tobacco necrosis virus, and tomato bushy stunt virus. All of these virus particles are nearly spherical. Values of the sphere diameter obtained by a comparison of our curves with curves of the exact scattering function for spheres are SBMV = 286 A, TNV = 280 A and TBSV = 309 A. A comparison of these values with other molecular constants strongly suggests that they are internally hydrated. The effects upon the experimental curves of interparticle interference and of slit dimensions for the X-ray collimating system are considerable and have been taken into account.

Properties of poliovirus propagated in medium containing cesium chloride: Implications for picornaviral structure

Abstract When propagated in cesium-rich medium, type 1 poliovirus incorporated enough cesium atoms to shift its buoyant density from 1.34 to an upper limit of about 1.40 g/ml. Studies with radioactive 137 Cs indicated that this upper density limit corresponded to about 4200 cesium atoms per virion. The incorporated cesium was tightly bound, exchanging out of virions at a negligible rate at temperatures below 22°C. Cesium-loaded virions were normal with respect to specific infectivity, neutralizability by specific antisera, and electrophoretic profile of coat protein. Natural top component bound fewer than 20 cesium atoms per particle under the same conditions. These results demonstrate that the protein coat of poliovirus is impermeable to cesium ions. However, if the RNA of the virus is exposed to cesium ions while the virus is being assembled, the poliovirus binds approximately the same number of cesium ions as human rhinovirus. This similarity in cesium binding suggests that poliovirus and human rhinovirus may have the same fraction of their RNA neutralized by coat protein.

Comparisons of the low resolution structure of several small RNA-containing viruses.

A comparison is made of the structure of five small RNA-containing viruses and their accompnaying particles. The data obtained by a small-angle X-ray scattering at low resolution indicate that the radial distributions of electron density are quite similar for particles with similar percentage of RNA. Evidence is also presented indicating that the RNA probably penetrates the wall of the protein shell of most if not all of the virus particles.