Abstract
Protein folding is often difficult to characterize experimentally because of the transience of intermediate states, and the complexity of the protein–solvent system. Atomistic simulations, which could provide more detailed information, have had to employ highly simplified models or high temperatures, to cope with the long time scales of unfolding; direct simulation of folding is even more problematic. We report a fully atomistic simulation of the acid-induced unfolding of apomyoglobin in which the protonation of acidic side-chains to simulate low pH is sufficient to induce unfolding at room temperature with no added biasing forces or other unusual conditions; and the trajectory is validated by comparison to experimental characterization of intermediate states. Novel insights provided by their analysis include: characterization of a dry swollen globule state forming a barrier to initial unfolding or final folding; observation of cooperativity in secondary and tertiary structure formation and its explanation in terms of dielectric environments; and structural details of the intermediate and the completely unfolded states. These insights involve time scales and levels of structural detail that are presently beyond the range of experiment, but come within reach through the simulation methods described here. An implicit solvation model is used to analyze the energetics of protein folding at various pH and ionic strength values, and a reasonable estimate of folding free energy is obtained. Electrostatic interactions are found to disfavor folding.