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The different partially folded states of the capsid protein that appear in the disassembly pathway of cowpea severe mosaic virus (CPSMV) were investigated by examining the effects of hydrostatic pressure, sub-zero temperatures and urea. The conformational states of the coat protein were analyzed by their intrinsic fluorescence, binding of bis(8-anilinonaphthalene-1-sulfonate) (bis-ANS) and susceptibility to trypsin digestion. CPSMV could be disassembled by pressure at 2.5 kbar. Intrinsic fluorescence and hydrodynamic measurements showed that pressure-induced dissociation was completely reversible. Virus pressurization in the presence of ribonuclease revealed that viral RNA was not exposed, since it was not digested by the enzyme, suggesting the maintenance of protein-nucleic acid interactions under pressure. When the temperature was decreased to -10 degrees C under pressure, CPSMV disassembly became an irreversible process and in this condition, viral RNA was completely digested by ribonuclease. These results suggest a relationship between protein-RNA interactions and CPSMV assembly. Bis-ANS binding and trypsin digestion of coat proteins revealed that they assume a different conformation when they are denatured by low temperatures under pressure or than when they are denatured by urea at atmospheric pressure. The results indicate that the coat proteins can exist in at least four states: (1) The native conformation in the virus capsid; (2) bound to RNA when the virus is dissociated by pressure at room temperature, assuming a conformation that retains the information for reassembly; (3) free subunits in a molten-globule conformation when the virus is dissociated by low temperature under pressure; and (4) free subunits completely unfolded by high concentrations of urea., (Copyright 1997 Academic Press Limited.)

Hydration forces are believed to play a determining role in protein folding. We have examined the contribution of water for the stability of the native dimer state of Arc repressor, a DNA-binding protein. Hydrostatic pressure was utilized to convert Arc repressor protein from a native state to a denatured, molten-globule state at decreasing concentrations of water. The volume change associated with Arc denaturation fell linearly with the increase in concentration of glycerol, whereas the free energy of the reaction increased. The pressure that promotes 50% denaturation (p1/2) increased in direct proportion to the concentration of glycerol or the decrease of water. Extrapolated to zero concentration of water, the data indicate that pressure denaturation would not occur without water. It is concluded that water plays a crucial role in decreasing the stability of a protein to a level that is compatible with its biological properties.

In the absence of urea, pressures up to 2.5 kbar promote only 10% dissociation of the whole particles of R17 bacteriophage. In the presence of concentrations of urea between 1.0 and 5.0 M, pressure promotes complete, reversible dissociation of the virus particles. At the lower urea concentrations reversible dissociation of R17 virus particles shows no dependence on protein concentration indicating a high degree of heterogeneity of the particles, but higher urea concentrations, 2.5 to 5.0 M, result in progressive restoration of the protein concentration dependence of the pressure dissociation. At still higher urea concentrations, 5.0 to 8.0 M, irreversible dissociation of virus takes place at atmospheric pressure. In contrast, the dissociation of the isolated dimers of the capsid protein was dependent on protein concentration to the extent predicted for a stochastic equilibrium, and dimers were much less stable than the whole virus both to dissociation by pressure or urea. In contradistinction, the reversible whole-virus dissociation observed at urea concentrations below 2.5 M appears to be a typical deterministic equilibrium, without appreciable dynamic exchange between whole particle and subunits during the lengthy experiments. The experiments demonstrate that the "thermodynamic individuality" of the virus particles arises in conformational differences in the assembled viruses, and that there is a direct relation between the stability of the particles and their heterogeneity.