Tertiary and quaternary structure of tobacco mosaic virus and protein. I. Effect of pH on fluorescence and 2-p-toluidinylnaphthalene-6-sulfonate binding

Tobacco mosaic virus and its protein exhibit pH-dependent changes in their wavelength and intensity of maximum emission, λ_max,pro and I_pro, respectively. The binding of 2-p-toluidinylnaphthalene-6-sulfonate (TNS) to tobacco mosaic virus protein or tobacco mosaic virus produces a strong increase in its fluorescence intensity, I_TNS. This binding is pH dependent. The protein is severely denatured at pH >; 10.5 or < 3 as indicated by large increases in λ_max,pro. Strong increases in I_TNS occur concomitantly with these increases in λ_max,pro, indicating increased binding of TNS upon denaturation. Presumably hydrophobic sites, exposed on denaturation, provide effective binding sites for TNS. Heat denaturation of tobacco mosaic virus protein at pH 7 led to similar increases in λ_max,pro and I_TNS. In the pH range where aggregation of the protein occurs, pH 6-7, λ_max,pro remains constant and only minor changes in I_pro and I_TNS occur. Major conformational changes do not appear to accompany aggregation. No significant changes in λ_max,pro, I_pro and I_TNS occur between pH 6 and 7 when the native virus is titrated. The changes in I_pro and I_TNS for tobacco mosaic virus protein in this pH range are apparently associated with the aggregation of the protein. TNS is bound more weakly to the virus than to the protein at all pH values.