Direct observation of microscopic reversibility in single-molecule protein folding.

Both folded and unfolded conformations should be observed for a protein at its melting temperature (T(m)), where DeltaG between these states is zero. In an all-atom molecular dynamics simulation of chymotrypsin inhibitor 2 (CI2) at its experimental T(m), the protein rapidly loses its low-temperature native structure; it then unfolds before refolding to a stable, native-like conformation. The initial unfolding follows the unfolding pathway described previously for higher-temperature simulations: the hydrophobic core is disrupted, the beta-sheet pulls apart and the alpha-helix unravels. The unfolded state reached under these conditions maintains a kernel of structure in the form of a non-native hydrophobic cluster. Refolding simply reverses this path, the side-chain interactions shift, the helix refolds, and the native packing and hydrogen bonds are recovered. The end result of this refolding is not the initial crystal structure; it contains the proper topology and the majority of the native contacts, but the structure is expanded and the contacts are long. We believe this to be the native state at elevated temperature, and the change in volume and contact lengths is consistent with experimental studies of other native proteins at elevated temperature and the chemical denaturant equivalent of T(m).