GB1 is not a two-state folder: identification and characterization of an on-pathway intermediate.

The folding pathway of the small α/β protein GB1 has been extensively studied during the past two decades using both theoretical and experimental approaches. These studies provided a consensus view that the protein folds in a two-state manner. Here, we reassessed the folding of GB1, both by experiments and simulations, and detected the presence of an on-pathway intermediate. This intermediate has eluded earlier experimental characterization and is distinct from the collapsed state previously identified using ultrarapid mixing. Failure to identify the presence of an intermediate affects some of the conclusions that have been drawn for GB1, a popular model for protein folding studies.