Hidden Intermediate State and Second Pathway Determining Folding and Unfolding Dynamics of GB1 Protein at Low Forces

Atomic force microscopy experiments found that GB1, a typical two-state model protein used for study of folding and unfolding dynamics, can sustain forces of more than 100 pN, but its response to low forces still remains unclear. Using ultrastable magnetic tweezers, we discovered that GB1 has an unexpected nonmonotonic force-dependent unfolding rate at 5-160 pN, from which a free energy landscape with two main barriers and a hidden intermediate state was constructed. A model combining two separate models by Dudko et al. with two pathways between the native state and this intermediate state is proposed to rebuild the unfolding dynamics over the full experimental force range. One candidate of this transient intermediate state is the theoretically proposed molten globule state with a loosely collapsed conformation, which might exist universally in the folding and unfolding processes of two-state proteins.