Submillisecond unfolding kinetics of apomyoglobin and its pH 4 intermediate

Submillisecond mixing experiments and tryptophan fluorescence spectroscopy are used to address two questions raised in earlier stopped-flow studies of the folding and unfolding kinetics of sperm whale apomyoglobin. A study of the pH 4 folding intermediate (I) revealed, surprisingly, that its folding and unfolding kinetics are measurable and fit the two-state model except for a possible burst phase in unfolding. Submillisecond mixing experiments confirm the unfolding burst phase and show that its properties are consistent with the recently discovered interconversion between two forms of I, Ia equilibrium Ib. In urea-induced unfolding, Ib is converted to Ia before Ia unfolds, and the unfolding kinetics of Ia fit the two-state model when the burst phase is assigned to Ib-->Ia. The second question is whether the Ia, Ib intermediates accumulate transiently when the native protein (N) unfolds to the acid unfolded form (U). Earlier work showed that Ia and Ib accumulate when U refolds to N at pH 6.0 and the results fit the linear folding pathway U equilibrium Ia equilibrium Ib equilibrium N. We report here that either or both Ia and Ib accumulate transiently when N unfolds to U at pH 2.7 and that the position of the rate-limiting step in the pathway changes between unfolding at pH 2. 7 and refolding at pH 6.0. In unfolding as in refolding, we do not detect a fast track that bypasses the Ia, Ib intermediates.