Alternative Models for Describing the Acid Unfolding of the Apomyoglobin Folding Intermediate

Abstract

The acid-induced unfolding of the pH 4 intermediate of apomyoglobin (I) is described by either of two models: (1) a Monod−Wyman−Changeux-based model (MWC) where salt bridges perturb the pK_a values of specific ionizable side chains, causing unfolding of I as these salt bridges are broken at low pH, and (2) the Linderstrøm-Lang smeared charge model (L-L), which attributes acid unfolding of I to charge repulsion caused by the accumulation of positive charge on the surface of the protein. Both models fit earlier acid unfolding data well, but they make differing predictions about the effects of electrostatic mutants, which have been made and tested. Deletions of positive charge within I are found to stabilize I, but disruptions of potential salt bridges have little effect. These results show that the acid unfolding of I (I ⇌ U) is largely caused by generalized charge effects rather than by the loss of specific salt bridges. Acid unfolding of the native form, which is caused largely by a single histidine with a severely depressed pK_a, is a sensitive indicator of changes in stability produced by mutations. In contrast, the I ⇌ U transition is caused by a number of groups with smaller pK_a perturbations and both models predict that the pH midpoint of the I ⇌ U transition is an insensitive indicator of stability. This result reconciles previous conflicting results, in urea and acid unfolding studies of hydrophobic contact mutants, by showing that changes in the stability of I are poorly detected by acid unfolding.