Backtracking due to residual structure in the unfolded state changes the folding of the third fibronectin type III domain from tenascin-C.

Residual structure in the unfolded state of a protein may play a crucial role in folding and stability. In the present study, using an all (heavy)-atom structure based model and replica exchange molecular dynamics simulations, we explored the folding landscape of the third fibronectin type III domain from tenascin-C (TNfn3). Specifically, both the wild type (WT) and a variant with two additional amino acids, Gly-Leu (GL), at the C-terminus (WT(+GL)) were studied. We found that, although both domains of TNfn3 are topologically frustrated, the early formation of the native contacts from the C-terminal end of WT(+GL) causes more "backtracking" than in the WT. As a result, the WT exhibits a two-state folding behavior with a broad transition-state ensemble, whereas the WT(+GL) folds through a metastable intermediate state. Furthermore, our study confirmed that the core of both proteins is conformationally heterogeneous and noncompact, and folds late mainly due to backtracking of the part of the core. Finally, in agreement with the previous experimental studies, our results clearly demonstrated distinct thermodynamic behavior of the two proteins with WT(+GL) being more stable.