Dynamic light scattering from thin rigid rods: Anisotropy of translational diffusion of tobacco mosaic virus

An Exact theoretical expression for the apparent diffusion coefficient D_app(K) of a thin rigid rod with arbitrary anisotropy of its translational diffusion diffusion coefficient is derived from the first cumulant of its dynamic structure factor. D_app(K) is predicted to reach a limiting plateau value at extermely large values of KL, where K is the scattering vector and L the rod length. Howerver, that limiting plateau value is approached only very slowly along a quasi-plateau with a very gradual slope. Dynamic light-scattering studies have been performed on tobacco mosaic virus from K² = (0.4–20) × 10¹⁰ cm^?2 using 632-8-nm laser radiation. The present data yield D₀ = (4.19 ± 0.10) × 10^?8 cm²/s (corrected to 20,w conditions) and, with literature data to establish L = 2980 Å and the rotational diffusion coefficient D_R = 318s^?1, yield also Δ ≡ D_∥ ? D_⊥ = (1.79 ± 0.38) × 10^?8 cm²/s. The experimental data closely follow the curve of D_app(K) vs K² calcuated for these parameters. The present value of D₀ substantially exceeds all previous dynamic light-scattering values, but is in good aggreement with previous sedimentation data, which were confirmed for the presemt sample. The anisotropy ratio Δ/D₀ = 0.43 ± 0.09 is in accord with theoretical predictions based on the modified Kirkwood algorithm, despite the fact the D₀ lies significantly below its corresponding theoretical value. The present data largely predlude the possibility that both D₀ and Δ/D₀ could simultaneously match their theoretical predictions. We present a detailed comparison of the experimental data with the calculations of Tirado and Garcia de la Torre based on the modified Kirkwood algorithm and with the Broersma formulas.     

A rotational diffusion coefficient of the 70S ribosome determined by depolarized laser light scattering

We have obtained a rotational diffusion coefficient of the 70S ribosome isolated from Escherichia-coli (MRE-600), from the depolarized light scattering spectrum measured by photon correlation spectroscopy. The intensity correlation function of depolarized scattered light contains contributions due to multiple scattered and anisotropy scattered light from the ribosomal particle. We discuss extensively the subtraction procedure used to obtain the rotational correlation time from the experimental correlation function. We have also obtained the translational diffusion coefficient from the same sample by determining the polarized correlation function. The hydrodynamic radius determined from the rotational diffusion coefficient is only slightly larger than the radius obtained from the translational diffusion coefficient. Therefore the ribosomal particle has a non-spherical shape. This conclusion, however, could be impaired by the effect of free draining of the ribosome.     

A rotational diffusion coefficient of the 70S ribosome determined by depolarized laser light scattering.

We have obtained a rotational diffusion coefficient of the 70S ribosome isolated from Escherichia-coli (MRE-600), from the depolarized light scattering spectrum measured by photon correlation spectroscopy. The intensity correlation function of depolarized scattered light contains contributions due to multiple scattered and anisotropy scattered light from the ribosomal particle. We discuss extensively the subtraction procedure used to obtain the rotational correlation from the time from the experimental correlation function. We have also obtained the translational diffusion coefficient from the same sample by determining the polarized correlation function. The hydrodynamic radius determined from the rotational diffusion coefficient is only slightly larger than the radius obtained from the translational diffusion coefficient. Therefore the ribosomal particle has a non-spherical shape. This conclusion, however, could be impaired by the effect of free draining of the ribosome.     

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).     

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.     

Rod-like cholesterol micelles in aqueous solution studied using polarized and depolarized dynamic light scattering.

Micelles of cholesterol in aqueous solution have been investigated using polarized and depolarized dynamic light scattering. They are shown to be highly extended and characterized by a narrow size distribution. It is shown that a rod-like model is applicable with length, L = 580 nm. Determination of the rotational diffusion coefficient by analysis of the autocorrelation function gave a value of theta = 150 s-1, which is close to the calculated value for the rod with this dimension. Depolarized dynamic light scattering measurements as a function of angle gave a value of 110 s-1.     

Influence of double scattering in determination of rotational diffusion coefficients by depolarized dynamic light scattering: Application to the bacteriophages T7 and T4B

Dynamic light scattering experiments were performed on solutions of the bacteriophages T7 and T4B in order to obtain the rotational diffusion coefficients of these phages. Correlation functions were determined from the depolarized intensity scattered in the forward direction. The apparatus used in this study is described in detail. Particular attention is paid to the minimalization of the depolarized intensity due to double scattering. If double scattering cannot be neglected, the correlation function of the depolarized field is the sum of the correlation functions resulting from single and double scattering. It is shown that by correcting for double scattering, it is then possible to obtain the rotational diffusion coefficient of the macromolecules. Although the optical anisotropy of both T4B (retracted fibers) and T7 is very small, the experimental conditions could be chosen in such a way that no depolarized scattering due to double scattering was observed. The measured rotational diffusion coefficients for T4B and T7 are D = 258 ± 12 and 4528 ± 100 sec^?1, respectively. These values compare very well with those obtained by electric birefringence experiments.     

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.     

Dynamic light scattering from collagen solutions. I. Translational diffusion coefficient and aggregation effects

Solutions of native collagen extracted from rat tail tendons in neutral salt solution have been studied by dynamic light scattering. The spectra obtained are consistent with the presence in solution of both single rod-shaped collagen molecules and aggregates of molecules. No contribution to the spectrum has been detected at any scattering angle from rotational diffusion of single molecules, although a measurable broadening effect is expected at high angles. The translational diffusion coefficient D of single molecules, calculated from the broader spectral component, shows an anomalous dependence on collagen concentration with a maximum value of D_20,w = 8.6 ± 0.2 × 10^?12 m²/sec near the concentration 0.04% by weight. Above 0.05% D falls linearly with increasing concentration and takes the value D _20,w = 8.1 ± 0.2 × 10^?12 m²/sec at 0.064% collagen.     

Dark and light green tissues of tobacco leaves systemically infected with tobacco mosaic virus

There are significant changes in the structure of the upper tobacco (Nicotiana tabacum L.) leaves systemically infected with tobacco mosaic virus (TMV) especially in the light green tissue (LGT). Dark green areas (DGI) had intermediate status between healthy tissue and LGT. DGI contained significantly less infectious TMV and viral antigen than the LGT. The DGI, LGT and healthy tissues did not differ in the permeability of cell membranes and in the set of acidic pathogenesis-related (PR) proteins but the total content of PR-proteins in the healthy plants was higher than in the infected ones with the DGI being intermediate between healthy tissue and LGT. The crude leaf extracts from DGI and LGT showed less total ribonuclease activity and ribonuclease isozymes in comparison with control.     

Interaction of tobacco mosaic virus and tobacco mosaic virus protein with bovine serum albumin.

Bovine serum albumin (BSA) causes tobacco mosaic virus (TMV) to crystallize at pH values where both have negative charges. The amount of albumin required to precipitate the virus varies inversely with ionic strength of added electrolyte. At pH values above 5, the precipitating power is greatest when BSA has the maximum total, positive plus negative, charge. Unlike early stages of the crystallization of TMV in ammonium sulfate-phosphate solutions, which can be reversed by lowering the temperature, the precipitation of TMV by BSA is not readily reversed by changes in temperature. The logarithm of the apparent solubility of TMV in BSA solutions, at constant ionic strength of added electrolyte, decreases linearly with increasing BSA concentration. This result and the correlation of precipitating power with total BSA charge suggest that BSA acts in the manner of a salting-out agent. The effect of BSA on the reversible entropy-driven polymerization of TMV protein (TMVP) depends on BSA concentration, pH, and ionic strength. In general, BSA promotes TMVP polymerization, and this effect increases with increasing BSA concentrations. The effect is larger at pH 6.5 than at pH 6. Even though increasing ionic strength promotes polymerization of TMVP in absence of BSA, the effect of increasing ionic strength from 0.08 to 0.18 at pH 6.5 decreases the polymerization-promoting effect of BSA. Likewise, the presence of BSA decreases the polymerization-promoting effect of ionic strength. The polymerization-promoting effect of BSA can be interpreted in terms of a process akin to salting-out. The mutual suppression of the polymerization-promoting effects of BSA and of electrolytes by each other can be partially explained in terms of salting-in of BSA.     

Interaction of cucumber mosaic virus and potato virus y with tobacco mosaic virus

A study was performed on the interaction of cucumber mosaic virus (CMV) of potato virus Y (PVY) with tobacco mosaic virus (TMV). Interference was evaluated using tobacco plantsNicotiana tabacum cv. Java responding to CMV and PVY with a systemic infection and to TMV with local necrotic lesions. The decrease in TMV — induced lesion number gave evidence of a decrease in susceptibility caused by the previous infection with CMV or PVY, the decrease of lesion enlargement demonstrated a decreased TMV reproduction in the plants previously infected with CMV or PVY. The interference concerned was incomplete, as evaluated from reproduction of the challenging TMV and from the decrease in susceptibility of the host to TMV brought about by the first infection with CMV or PVY.     

Electroporation-mediated infection of tobacco leaf protoplasts with tobacco mosaic virus RNA and cucumber mosaic virus RNA

Conditions were established for the introduction of both tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV) RNAs into tobacco mesophyll protoplasts by electroporation. The proportion of infected protoplasts was quantified by staining with viral coat protein-specific antibodies conjugated to fluorescein isothiocyanate. Approximately 30–40% of the protoplasts survived electroporation. Under optimal conditions, up to 75% of these were infected with TMV-RNA. Successful infection was demonstrated in 19 out of 20 experiments. Optimal infection was achieved with several direct current pulses of 90 sec at a field strength of 5 to 10 kV/cm. Changing the position of the protoplasts within the chamber between electric pulses was essential for achievement of high rates of infection. Optimal viral RNA concentration was about 10 g/ml in a solution of 0.5 M mannitol without buffer salts.