POLYMERIZATION-DEPOLYMERIZATION OF TOBACCO MOSAIC VIRUS PROTEIN .12. FURTHER STUDIES ON ROLE OF WATER

The endothermic polymerization of tobacco mosaic virus protein depends upon entropic or “hydrophobic” interactions between the protein subunits leading to a definite water release during the association process. As shown by previous pH change experiments, the amount of water released upon polymerization can be estimated from equilibrium dialysis in a quartz spring balance, using glycerol as the solvent component of high density. When, because of the endothermic character of the polymerization reaction, polymerization is brought about by change of temperature, the amount of water lost by the protein is found to be 0.033 ± 0.00035 g/g of protein. This figure corresponds to about 96 moles/mole of tobacco mosaic virus protein trimer (molecular weight 52,500). To eliminate temperature effects on the spring balance as well as effects caused by unspecific changes of the partial specific volume of the protein, intact virus, tobacco mosaic virus, was used as reference. The temperature dependences of the partial specific volumes of both tobacco mosaic virus and its coat protein (outside the polymerization range) as measured with pycnometers and dilatometers are indentical, showing a linear temperature coefficient of 0.00025 cc/g°C in buffer as well as in glycerol solutions of different concentration (pH 6.7, μ = 0.05, 0 ≦ Cg1 ≦ S 25% w/v). The change of the partial specific volume attributable to polymerization can be estimated from dilatometric experiments to be +0.0049 ± 0.0003 ml/g. When spring balance data are interpreted on the assumption of no change in ion binding, a value of 0.0041 ± 0.0008 ml/g is obtained for this quantity. For this and other reasons, it is concluded that the effects of ion binding are small. In order to determine conformational changes upon the transition from aqueous to glycerol-containing buffers or under different conditions of temperature and pH, optical rotatory dispersion and ultraviolet difference spectra were analyzed. Temperature as well as pH changes lead to small differences in the chromophore interactions which are reversible as long as denaturation is excluded. Tyrosine seems to participate in the polymerization and in the protein-solvent interaction. No changes in the gross structure seem to occur as shown by electron microscopy of the polymerized protein in buffer and in 25% glycerol solution. © 1969, American Chemical Society. All rights reserved.