Detection and Structure Determination of an Equilibrium Unfolding Intermediate of Rd-apocytochrome b562: Native Fold with Non-native Hydrophobic Interactions

The absence of detectable kinetic and equilibrium folding intermediates by optical probes is commonly taken to indicate that protein folding is a two-state process. However, for some small proteins with apparent two-state behavior, unfolding intermediates have been identified in native-state hydrogen exchange or kinetic unfolding experiments monitored by nuclear magnetic resonance. Rd-apocytochrome b562, a four-helix bundle, is one such protein. Here, we found another unfolding intermediate for Rd-apocytochrome b562. It is based on a cooperative transition of 15N chemical shifts of amide protons as a function of urea concentrations before the global unfolding. We have solved the high-resolution structure of the protein at 2.8 M urea, which is after this cooperative transition but before the global unfolding. All four helices remained intact, but a number of hydrophobic core residues repacked. This intermediate provides a possible structural interpretation for the kinetic unfolding intermediates observed using nuclear magnetic resonance methods for several proteins and has important implications for theoretical studies of protein folding.