Heterogeneity and multi-scale dynamics in the molecular bearing of the bacterial flagellum

The bacterial flagellum is a protein-based rotary machine that drives bacterial motility. It comprises the bacterial flagellar motor (BFM), consisting of a stator which is anchored to the cell wall and a rotor in the cytoplasmic membrane, linked via the flagellar rod to the extracellular hook and filament. We previously showed that active rotation of the flagellum was dominated by discrete steps of 1/26th of a revolution. This corresponds to the 26-fold symmetry more recently observed in the structure of the flagellar LP-ring, a bearing that anchors the flagellum in the cell envelope. Here we observed passive rotational diffusion of six individual Escherichia Coli flagella lacking torque-generating units, via polarization microscopy of single gold nanorods attached to the hook, sampled at 250 kHz. The 26-fold bearing symmetry dominated rotation, creating barriers that impeded free diffusion. We resolved transitions across these barriers that exhibited highly non-Poissonian kinetics spanning four orders of magnitude in time scale. From the average time scales we estimated barrier heights between 12 and 14 kBT, just low enough to be overcome by a single torque-generating stator unit. At sub-millisecond timescales we observed anomalous ultra-slow diffusion typically associated with 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 bearing’s potential energy landscape evolves over time. These findings raise the question of whether heterogeneous dynamics are a universal feature of large protein machines