UNFOLDING OF APOMYOGLOBIN FROM APLYSIA-LIMACINA - THE EFFECT OF SALT AND ON ON THE COOPERATIVITY OF FOLDING

The equilibrium unfolding pathway of Aplysia apomyoglobin has been stu died under various solvent conditions. The protein exhibits a single u nfolding transition in acid in contrast to the two transitions observe d for the mammalian apomyoglobins with which it shares a common fold b ut a low level of sequence identity (24%). This acid-unfolded species has considerable residual structure as evidenced by both tryptophan fl uorescence and far-UV CD spectroscopy. It remains 40% alpha-helical un der low salt conditions (2 mM citrate, 4 degrees C); the folded form i s 65% helical. A similar species is observed for the mammalian globins in mild acid conditions. Titration with GdnHCl at pH 7 reveals two un folding transitions, the first having common features with that observ ed in acid and the second resulting in a completely unfolded state. Un der the same conditions, urea unfolds the protein completely in an app arently single cooperative transition. Assuming a simple three-state m odel (F <-> I <-> U), data from GdnHCl and urea titrations over a rang e of pH conditions were used to derive values for the apparent stabili ty (Delta G(w(app))) and solvent accessibility (n((app))) of the folde d (F) and intermediate (I) forms of the protein. Urea titrations were then repeated over a range of KCl concentrations in order to understan d the contribution of Cl- to the different unfolding activity of GdnHC l. A three-state scheme is justified when changes in Delta G(w(app)) o ccur without changes in n((app)). The change in free energy of folding of I <-> F (Delta G(w(F/I))) decreases to 0 at pH 4 as expected from the acid unfolding curve. Delta G(w(I/U)) reaches its maximum at pH 4. 5, the isoelectric point of the protein. Variations of this value with pH 4.5, the isoelectric point of the protein. Variations of this valu e with pH and chloride are as much as 3 kcal mol(-1) and correlate clo sely with changes in n((app)) although there is no change in the alpha -helical content of I across the pH range. This observation is interpr eted here as a deviation of the unfolding of the I state of Aplysia ap omyoglobin from a cooperative behaviour. (C) 1998 Academic Press Limit ed.