Single-Molecule AFM Force Spectroscopy — Protein Unfolding Sawtooth

An atomic force microscope cantilever tip is chemically attached to one end of a single folded, multi-domain protein, with the other end anchored to a surface. As the tip is pulled steadily away, the cantilever's tiny bend — read optically — reports the mechanical tension building in the molecule. A folded domain resists stretching until its own characteristic force threshold is reached, then abruptly unravels, releasing extra chain length and dropping the tension. Repeated over every domain, this produces the distinctive sawtooth force-extension curve used to fingerprint a protein's individual fold stabilities, one molecule at a time.