Tobacco mosaic virus disassembly by high hydrostatic pressure in combination with urea and low temperature.
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.
Departamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas, SP, Brazil.
C F
CF
Bonafe
C M
CM
Vital
R C
RC
Telles
M C
MC
Gonçalves
M S
MS
Matsuura
F B
FB
Pessine
D R
DR
Freitas
J
J
Vega
D001274:Atmospheric Pressure; D003080:Cold Temperature; D006874:Hydrostatic Pressure; D007700:Kinetics; D008027:Light; D008854:Microscopy, Electron; D011489:Protein Denaturation; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D014027:Tobacco Mosaic Virus / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D014508:Urea; D014764:Viral Proteins / Q000378:metabolism / Q000648:ultrastructure; D019065:Virus Assembly / Q000502:physiology*
D014764:Viral Proteins; D014508:Urea