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Abstract:

Many essential cellular processes are carried out by complex biological machines located in the cell membrane. The bacterial flagellar motor is a large membrane-spanning protein complex that functions as an ion-driven rotary motor to propel cells through liquid media. Within the motor, MotB is a component of the stator that couples ion flow to torque generation and anchors the stator to the cell wall. Here we have investigated the protein stoichiometry, dynamics and turnover of MotB with single-molecule precision in functioning bacterial flagellar motors in Escherichia coli. We monitored motor function by rotation of a tethered cell body, and simultaneously measured the number and dynamics of MotB molecules labelled with green fluorescent protein (GFP-MotB) in the motor by total internal reflection fluorescence microscopy. Counting fluorophores by the stepwise photobleaching of single GFP molecules showed that each motor contains ∼22 copies of GFP-MotB, consistent with ∼11 stators each containing two MotB molecules. We also observed a membrane pool of ∼200 GFP-MotB molecules diffusing at ∼0.008 μm²s^-1. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching showed turnover of GFP-MotB between the membrane pool and motor with a rate constant of the order of 0.04 s^-1: the dwell time of a given stator in the motor is only ∼0.5 min. This is the first direct measurement of the number and rapid turnover of protein subunits within a functioning molecular machine. © 2006 Nature Publishing Group.

Abstract:

Practical components for three-dimensional molecular nanofabrication must be simple to produce, stereopure, rigid, and adaptable. We report a family of DNA tetrahedra, less than 10 nanometers on a side, that can self-assemble in seconds with near-quantitative yield of one diastereomer. They can be connected by programmable DNA linkers. Their triangulated architecture confers structural stability; by compressing a DNA tetrahedron with an atomic force microscope, we have measured the axial compressibility of DNA and observed the buckling of the double helix under high loads.

Abstract:

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 observe 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. Transitions across energy barriers of the 26-fold symmetric LP-ring/rod flagellar bearing exhibit highly non-Poissonian kinetics spanning four orders of magnitude in time scale. At sub-millisecond timescales we observe 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 observe dynamic shifts in the preferred angular positions, indicating that the bearing’s energy landscape evolves over time.