The evolving role of single-molecule force spectroscopy in protein biophysics

Single-molecule force spectroscopy (SMFS) techniques initially emerged as a new method to probe protein biophysics, often providing complementary insights to biochemical bulk experiments. Over time, however, advances in instrumentation and the growing recognition that mechanical forces are integral to biological function have progressively redirected its use toward exploring protein systems operating in mechanically active environments. In this review, we highlight recent applications of SMFS that shed light on how force regulates protein function, spanning diverse biological systems like cotranslational folding, protein degradation, and cellular adhesion proteins. Beyond allowing us to manipulate individual molecules, SMFS uniquely recreates the mechanical conditions under which many proteins operate, revealing mechanistic details inaccessible to traditional protein characterization methods. Looking ahead, ongoing innovation, both in instrumentation and in the integration of SMFS with complementary techniques, is bringing the field closer to mimicking physiologically relevant conditions. These developments are opening new avenues for recognizing mechanical force as a central regulator of biological systems.