Force-dependent mechanical unfolding pathways of GFP

We characterize the force-dependent unfolding pathways and intermediate configurations of the green fluorescence protein (GFP) using novel atomistic simulations based on potential energy surface exploration. By using this approach, we are able to unfold GFP to significantly longer end-to-end distances, i.e. 40 nm, as compared to that seen in previous atomistic simulation studies. We find that there are four intermediate states between 5 and 40 nm end-to-end distance, where the intermediate configurations and unfolding pathways are strongly force-dependent. We additionally calculate the force-dependent lifetime of the 14 nm αβ1 intermediate, and demonstrate that it obeys Bell's formula.