Entropy-driven polymerization of the protein from the flavum strain of tobacco mosaic virus

Abstract

Protein of the flavum mutant of tobacco mosaic virus has the same number of positive groups as vulgare but aspartic acid at position 19 in vulgare is replaced by alanine, yielding the same net charge as E66 protein, that is, one less negative charge near pH 7 than vulgare protein. Comparative temperature-OD measurements of both flavum and vulgare proteins, prepared identically, were made at pH 5.8 to 6.6 at ionic strengths of 0.10 and 0.05. Another set of T-OD experiments was carried out at two pH values, 5.9 and 6.2, with the ionic strength varying from 0.025 to 0.15 in increments of 0.025. Compared to vulgare, flavum protein polymerizes at higher temperatures and exhibits qualitatively similar effects of pH and ionic strength. Hydrogen ion titrations were carried out from pH 8 to pH 5 and back to pH 8 in 0.10 m KCl at three temperatures, 4, 10, and 15 °C. The titration was completely reversible when carried out slowly. The isoionic point is about pH 5; the charge at pH 7.5 is −3. The hydrogen ion binding was correlated with the different aggregates present under different conditions of pH and temperatures as determined from sedimentation velocity measurements. It was found that H + ion binding correlates with the disappearance of the smallest aggregates present and does not depend on the type of higher polymer formed. From the analysis for the effect of ionic strength and pH on the characteristic temperature, T ∗ , defined as the temperature corresponding to an optical density increment of 0.01, values of ΔH ∗, ΔS ∗, K s ′, and ΔW el are obtained. The salting out constant, K s′, for flavum protein turns out to be considerably higher than for vulgare and E66 proteins. This is to be expected of a protein with a higher content of hydrophobic residues. In contrast, the electrical work term turns out to be close to the value obtained for E66 protein and about one-half of the value for vulgare, consistent with the lower net negative charge.