Conformational changes induced in tobacco mosaic virus nucleic acid by ultraviolet radiation

Summary In order to study any Conformational changes associated with ultraviolet irradiation of TMV-RNA, methods of circular dichroism and absorbance-temperature profile were applied. RNA irradiated in water at 254 nm showed a distinct change in CD spectrum, but there was only a slight change accompanying irradiation in phosphate buffer.A small change in absorbance-temperature profile at 258 nm is associated with irradiation of RNA in water; RNA irradiated in phosphate buffer and Mg⁺⁺ solutions showed essentially no changes.It is concluded that conformational changes accompanying irradiation of RNA in water are greater than those taking place in phosphate or magnesium solutions, and the enhanced change in water is related to the larger quantum yield for inactivation found in water than in buffer solutions.Photochemistry of Macromolecules XXXIII, supported in part by the U.S. Atomic Energy Commission, contract AT(11-1)-34, Project 116.     

The ultraviolet light and photosensitized inactivation of tobacco mosaic virus

The quantum yield for the inactivation of tobacco mosaic virus has been determined at 253.7 mµ and found to be 4.3 x 10^–6. The possible significance of the observed one-hit process of inactivation has been discussed in terms of the kinetics and the rupture of model substances including nucleic acid. The ultraviolet light inactivation, which proceeds independent of oxygen, occurs without change in physicochemical properties, with the possible exception of an enhanced sensitivity to thermal denaturation. The photosensitized inactivation of virus by acriflavine has been found to proceed parallel with the destruction of the dye. The action was found to be dependent upon adsorbed dye, and the inactivation is enhanced by the presence of oxygen.     

The study of amorphous aggregation of tobacco mosaic virus coat protein by dynamic light scattering

The kinetics of heat-induced and cetyltrimethylammonium bromide induced amorphous aggregation of tobacco mosaic virus coat protein in Na(+)/Na(+) phosphate buffer, pH 8.0, have been studied using dynamic light scattering. In the case of thermal aggregation (52 degrees C) the character of the dependence of the hydrodynamic radius (R(h)) on time indicates that at certain instant the population of aggregates is split into two components. The size of the aggregates of one kind remains practically constant in time, whereas the size of aggregates of other kind increases monotonously in time reaching the values characteristic of aggregates prone to precipitation (R(h)=900-1500 nm). The construction of the light scattering intensity versus R(h) plots shows that the large aggregates (the start aggregates) exist in the system at the instant the initial increase in the light scattering intensity is observed. For thermal aggregation the R(h) value for the start aggregates is independent of the protein concentration and equal to 21.6 nm. In the case of the surfactant-induced aggregation (at 25 degrees C) no splitting of the aggregates into two components is observed and the size of the start aggregates turns out to be much larger (107 nm) than on the thermal aggregation. The dependence of R(h) on time for both heat-induced aggregation and surfactant-induced aggregation after a lapse of time follows the power law indicating that the aggregation process proceeds in the kinetic regime of diffusion-limited cluster-cluster aggregation. Fractal dimension is close to 1.8. The molecular chaperone alpha-crystallin does not affect the kinetics of tobacco mosaic virus coat protein thermal aggregation.     

Translational and rotational diffusion of tobacco mosaic virus from polarized and depolarized light scattering

The translational and rotational dynamics of tobacco mosaic virus in sodium phosphate buffer (pH =7.5) solutions has been investigated by polarized and depolarized light scattering Rayleigh linewidth studies. For concentrations ranging from 1.75 × 10^?4 g ml^?1 to 0.25 × 10^?4 g ml^?1 the translational diffusion coefficient (D_T) has been found to be slightly concentration dependent and extrapolation to zero concentration gives D₀^20°C = 0.34 ± 0.01 × 10^?7 cm²S^?1. A full analysis of the polarized spectra obtained at high and low scattering angles and the depolarized spectra at near zero scattering angles has enabled these techniques to be compared and the rotational diffusion constant D_R to be determined. At a solution concentration of 1.75 × 10^?4 g ml^?1 a mean value is found to be D_R^20°C = 350 ± 30s^?1. These values of D_T and D_R are in approximate agreement with calculations based on models of the tobacco mosaic virus molecule as a cylindrical rod.     

Teaching light scattering spectroscopy: the dimension and shape of tobacco mosaic virus.

The tobacco mosaic virus is used as a model molecular assembly to illustrate the basic potentialities of light scattering techniques (both static and dynamic) to undergraduates. The work has two objectives: a pedagogic one (introducing light scattering to undergraduate students) and a scientific one (stabilization of the virus molecular assembly structure by the nucleic acid). Students are first challenged to confirm the stabilization of the cylindrical shape of the virus by the nucleic acid, at pH and ionic strength conditions where the coat proteins alone do not self-assemble. The experimental intramolecular scattering factor is compared with the theoretical ones for several model geometries. The data clearly suggest that the geometry is, in fact, a rod. Comparing the experimental values of gyration radius and hydrodynamic radius with the theoretical expectations further confirms this conclusion. Moreover, the rod structure is maintained over a wider range of pH and ionic strength than that valid for the coat proteins alone. The experimental values of the diffusion coefficient and radius of gyration are compared with the theoretical expectations assuming the dimensions detected by electron microscopy techniques. In fact, both values are in agreement (length approximately 300 nm, radius approximately 20 nm).     

Calcium and potassium ion binding by tobacco mosaic virus ribonucleic acid

Calcium and potassium ion titration experiments were performed on solutions of tobacco mosaic virus RNA using ion-specific electrodes. The data obtained were analyzed using Scatchard and Klotz plots for the number of binding sites per nucleotide (n), and the apparent stability constant for complex formation, beta Me. The experimental design also allowed for the determination of the number of protons released per metal ion bound, chi. The calcium ion titration in water yielded values of 0.45 for n, 6.03 for log beta Ca and 0.24 for chi. When this titration was repeated in 0.01 M-KCl, the values were found to be 0.11 for n, 5.08 for log beta Ca and zero for chi. An aqueous potassium titration was also performed, with values for n, log beta K and chi of 0.25, 2.96 and less than 0.10, respectively.