Extract Motive Energy from Single-Molecule Trajectories.

Single-molecule trajectories from nonequilibrium unfolding experiments are widely used to recover a biomolecule's intrinsic free-energy profile. Trajectories of molecular motors from similar single-molecule experiments may be mapped to biased diffusion over an inclined free-energy profile. Such an effective potential is not a static equilibrium property anymore, and how it can benefit molecular motor study is unclear. Here, we introduce a method to deduce this effective potential from motor trajectories with realistic temporal-spatial resolution and find that the potential yields a motor's stall force─a quantity that not only characterizes a motor's force-generating capacity but also largely determines its energy efficiency. Interestingly, this potential allows the extraction of a motor's stall force from trajectories recorded at a single resisting force or even zero force, as verified with trajectories from two molecular motor models and also experimental trajectories from a real artificial motor. This finding drastically reduces the difficulty of stall force measurement, making it accessible even to force-incapable optical tracking experiments (commonly regarded as irrelevant to stall force determination). This study further provides a method for experimentally measuring a second-law-decreed least energy price for submicroscopic directionality─a previously elusive but thermodynamically important quantity pertinent to efficient energy conversion of molecular motors.