Single-molecule observation of multi-scale dynamic heterogeneity in the molecular bearing of the bacterial flagellum

The bacterial flagellar motor (BFM) is a protein-based rotary machine that drives bacterial motility. It comprises a stator anchored to the cell wall and a rotor in the cytoplasmic membrane, linked via the flagellar rod to the extracellular hook and filament. We describe high-speed polarization microscopy of gold nanorods attached to the hook, allowing unprecedented continuous recording of single BFMs with a few degrees resolution at 250 kHz for tens of minutes. The angular distribution of actively rotating motors exhibited multiple periodicities that are consistent with structure of the bearing between the rod and LP-ring. We observed passive diffusional rotation of this bearing, revealing complex dynamics at odds with the expected classical Ornstein-Uhlenbeck process in a smooth potential. Resolved transitions between the main 26-fold bearing states exhibited highly non-Poissonian kinetics spanning four orders of magnitude in timescale. Using the average transition times, we estimated the interaction barrier in the bearing to be close to 13 kBT, suggesting that the previously estimated torque generated by a single stator unit is just sufficient to overcome this barrier. At sub-millisecond timescales we observed anomalous ultra-slow diffusion typically associated with multi-scale disordered systems, despite the ordered crystalline atomic structure of the bearing revealed by cryo-Electron Microscopy. Over longer periods, we observed dynamic shifts in the preferred angular positions, indicating that the motor's potential energy landscape evolves over time. We speculate that these newly discovered rich dynamics may be a universal feature of protein machines.