Single-molecule experiments reveal the flexibility of a Per-ARNT-Sim domain and the kinetic partitioning in the unfolding pathway under force.

Per-ARNT-Sim (PAS) domains serve as versatile binding motifs in many signal-transduction proteins and are able to respond to a wide spectrum of chemical or physical signals. Despite their diverse functions, PAS domains share a conserved structure. It has been suggested that the structure of PAS domains is flexible and thus adaptable to many binding partners. However, direct measurement of the flexibility of PAS domains has not yet been provided. Here, we quantitatively measure the mechanical unfolding of a PAS domain, ARNT PAS-B, using single-molecule atomic force microscopy. Our force spectroscopy results indicate that the structure of ARNT PAS-B can be unraveled under mechanical forces as low as ~30 pN due to its broad potential well for the mechanical unfolding transition of ~2 nm. This allows the PAS-B domain to extend by up to 75% of its resting end-to-end distance without unfolding. Moreover, we found that the ARNT PAS-B domain unfolds in two distinct pathways via a kinetic partitioning mechanism. Sixty-seven percent of ARNT PAS-B unfolds through a simple two-state pathway, whereas the other 33% unfolds with a well-defined intermediate state in which the C-terminal β-hairpin is detached. We propose that the structural flexibility and force-induced partial unfolding of PAS-B domains may provide a unique mechanism for them to recruit diverse binding partners and lower the free-energy barrier for the formation of the binding interface.