Unfolding of apomyoglobin from Aplysia limacina: The effect of salt and pH on the cooperativity of folding

The equilibrium unfolding pathway of Aplysia apomyoglobin has been studied under various solvent conditions. The protein exhibits a single unfolding transition in acid in contrast to the two transitions observed for the mammalian apomyoglobins with which it shares a common fold but a low level of sequence identity (24%). This acid-unfolded species has considerable residual structure as evidenced by both tryptophan fluorescence and far-UV CD spectroscopy. It remains 40% α-helical under low salt conditions (2 mM citrate, 4°C); the folded form is 65% helical. A similar species is observed for the mammalian globins in mild acid conditions. Titration with GdnHCl at pH 7 reveals two unfolding transitions, the first having common features with that observed in acid and the second resulting in a completely unfolded state. Under the same conditions, urea unfolds the protein completely in an apparently single cooperative transition. Assuming a simple three-state model (F⇆I⇆U), data from GdnHCl and urea titrations over a range of pH conditions were used to derive values for the apparent stability (ΔG(w(app))) and solvent accessibility (n((app))) of the folded (F) and intermediate (I) forms of the protein. Urea titrations were then repeated over a range of KCl concentrations in order to understand the contribution of Cl- to the different unfolding activity of GdnHCl. A three-state scheme is justified when changes in ΔG(w(app)) occur without changes in n((app)). The change in free energy of folding of I⇆F (ΔG(w(F/I))) decreases to 0 at pH 4 as expected from the acid unfolding curve. ΔG(w(I/U)) reaches its maximum at pH 4.5, the isoelectric point of the protein. Variations of this value with pH and chloride are as much as 3 kcal mol-1 and correlate closely with changes in n((app)) although there is no change in the α-helical content of I across the pH range. This observation is interpreted here as a deviation of the unfolding of the I state of Aplysia apomyoglobin from a cooperative behaviour.