Single-Molecule Force Spectroscopy Reveals the Mechanical Role of p47 in Protein Stabilization

Mechanical force plays a pivotal role in regulating protein folding, trafficking, and processing in cells. While canonical chaperones are well-known to govern force-responsive conformational transitions, the capacity of accessory cofactors to directly modulate substrate mechanics remains poorly understood. Here, we uncover a noncanonical mechanical role for p47, a cofactor of the AAA⁺ ATPase p97, showing that it independently enhances the mechanical stability of talin, a mechanosensitive protein with well-defined two-state folding. Single-molecule magnetic tweezers reveal that p47 shifts talin's half-point force (of folding probability) from 8.4 pN to 16.6 pN and reduces the distance to the transition state from 8 to 0.93 nm, indicating a transition to a mechanically compact and rigid conformation. p47 also increases both unfolding and refolding forces and boosts mechanical work output to ∼199.5 zJ, underscoring its ability to stabilize folded states under load. These findings position p47 as a force-responsive cofactor capable of modulating protein energy landscapes, revealing new principles in mechanoregulated proteostasis and expanding the functional repertoire of accessory proteins.