Tobacco Mosaic Virus Disassembly by High Hydrostatic Pressure in Combination with Urea and Low Temperature^,

Abstract

We investigated the effect of low temperature and urea combined with high pressure on tobacco mosaic virus (TMV). The evaluation of its aggregation state and denaturation process was studied using gel filtration, transmission electron microscopy, and spectroscopic methods. The incubation at 2.5 kbar induced 18% dissociation, and decreasing of temperature to -19 degreesC promoted additional dissociation to 72%, with stabilization of the dissociation products. Under such conditions, extensive denaturation did not occur. The apparent enthalpy and entropy of dissociation (Delta and TDelta) were -9.04 kcal/mol subunit and -15.1 kcal/mol subunit, respectively, indicating that the TMV association is an entropicly driven process. The apparent free energy of stabilization given by the presence of RNA is at least -1.7 kcal/mol subunit. Urea-induced dissociation of TMV samples and incubation at high-pressure promoted a higher degree of dissociation. The volume change of dissociation decreased in magnitude from -16.3 to -3.1 mL/mol of dissociated subunit, respectively, in the absence and presence of 2.5 M urea, suggesting exposure of the protein-protein interface to the solvent. High-pressure induced remarkable TMV denaturation in the presence of 2.5 M urea, with a volume change of -101 mL/mol of denatured subunit. The apparent enthalpy and entropy of denaturation (Delta and TDelta) by 1.75 M urea at 2.5 kbar was -11.1 and -10.2 kcal/mol subunit, respectively, demonstrating that the TMV protein coat presents an apparent free energy of denaturation by urea close to zero. Although the processes could not be assumed to be pure equilibria, these thermodynamic parameters could be derived by assuming a steady-state condition.

Inactivation of a Human Norovirus Surrogate by High-Pressure Processing: Effectiveness, Mechanism, and Potential Application in the Fresh Produce Industry

“…This finding will be helpful to determine an optimal pressure to treat food products with different pH values. Our observation is consistent with the previous reports for FCV (16) and tobacco mosaic virus (8,51), which were also more resistant to HPP at lower pH. In contrast, a significantly enhanced inactivation of HAV was found in acidic environments (35)(36)(37).…”

Probing conformational changes in orphan nuclear receptor: The NGFI-B intermediate is a partially unfolded dimer

“…11 incubation of the protein with increasing amounts of GndHCl resulted in a significant shift in the center of mass [37,38] and a bathochromic red-shift in the fluorescence emission maximum (from 334 nm to 360 nm), and also in a severe quenching of the fluorescence emission intensity (Figures 2A and 2C). In contrast with the CD results, the GndHCl induced unfolding as monitored by the intrinsic fluorescence unambiguously reveals an intermediate state at the GndHCl concentrations around 2.75 M and 3.5 M (Figure 3).…”

“…CD spectra were measured from samples in 1.0 mm path length quartz cuvettes, and were the average of 16 accumulations, using a scanning speed of 100 nm min - Intrinsic fluorescence spectra were obtained by excitation at 295 nm, and the emission spectra were monitored from 300 to 450 nm. Relevant information about macromolecule conformational flexibility can be obtained by considering the fluorescence spectral center of mass [37,38]. In this case, the fluorescence spectral center of mass (λ cm ) was calculated, for each concentration of chaotropic agents, according to the equation [37,38]:…”

“…Relevant information about macromolecule conformational flexibility can be obtained by considering the fluorescence spectral center of mass [37,38]. In this case, the fluorescence spectral center of mass (λ cm ) was calculated, for each concentration of chaotropic agents, according to the equation [37,38]:…”

Chemical Outbreak for Tobacco Mosaic Virus Control

“…In plants, virus-urea interaction has been investigated in TMV dissociation and subunit denaturation. Bonafe et al (1998) showed how the apparent free energy associated with denaturation by urea for TMV protein coat was very low, almost null. The apparent urea stoichiometry of dissociation and denaturation of further viral agents has been calculated (Santos et al, 2008).…”