Free Energy Landscape of Type III Fibronectin Domain with Identified Intermediate State and Hierarchical Symmetry

The tenth domain of type III fibronectin (FNIII_{10}) mediates cell adhesion to the extracellular matrix. Despite its structural similarity to immunoglobulin domains, FNIII_{10} exhibits unique unfolding behaviors. We employed magnetic tweezers to investigate the unfolding and folding dynamics of FNIII_{10} under physiological forces (4-50 pN). Our results showed that FNIII_{10} follows a consistent transition pathway with an intermediate state characterized by detached A and G β strands. We determined the folding free energies and all force-dependent transition rates of FNIII_{10} and found that both unfolding rates from the native state to the intermediate state and from the intermediate state to the unfolded state deviate from Bell's model. We constructed a quantitative free energy landscape with well-defined traps and barriers that exhibits a hierarchical symmetrical pattern. Our findings provide a comprehensive understanding of FNIII_{10} conformational dynamics and demonstrate how free energy landscape of multistate biomolecules can be precisely mapped, illuminating the relationship between thermal stability, intermediate states, and folding rates in protein folding.