Two forms of the pH 4 folding intermediate of apomyoglobin

Abstract

The pH 4 folding intermediate of apomyoglobin exists in two forms (Ia, Ib) at equilibrium. Their ratio depends on pH, urea concentration and the presence or absence of a stabilizing anion (citrate, sulfate), and it does not depend on protein concentration. The Ia and Ib species are separated by a kinetic barrier and their interconversion can be monitored by tryptophan fluorescence in stopped-flow experiments. At pH 4.2, Ib is converted to Ia at low urea concentrations and urea unfolding gives the unfolding transition of Ia. During the refolding of native (N) apomyoglobin at pH 6, starting from the acid unfolded species (U), both Ia and Ib appear as transient intermediates and both Ia and Ib appear as transient intermediates in the acid-induced unfolding of N. The results are consistent with a linear folding and unfolding pathway: U⇆Ia⇆Ib⇆N. Apomyoglobin provides the opportunity to investigate at equilibrium the structures and properties of two different kinetic folding intermediates. A non-obligatory dimeric species of the pH 4 intermediate is formed slowly and contributes to the refolding kinetics at concentrations above 5 μM. The dimer dissociates slowly and during refolding at pH 6 it forms N in a later time range than does the monomer.