Professor Richard Berry D. Phil.

Abstract:

This chapter summarizes the current understanding on the structure and function of the bacterial flagellar motor using a combination of genetics, single molecule, and biophysical techniques, with a focus on recent results and single molecule techniques. The bacterial flagellar motor is a reversible rotary nanomachine, about 45 nm in diameter, embedded in the bacterial cell envelope. It is powered by the flux of H+ or Na+ ions across the cytoplasmic membrane driven by an electrochemical gradient. In many species, the motor switches direction stochastically, with the switching rates controlled by a network of sensory and signaling proteins. The bacterial flagellar motor was confirmed as a rotary motor in 1974 through tethered-cell experiments, the first direct observation of the function of a single molecular motor. However, due to the large size and complexity of the motor, much remains to be discovered, particularly the structural details of the torque-generating mechanism. The complex assembly pathway and requirement to anchor stators to the cell wall and locate them in an energized membrane have so far precluded the powerful in vitroreconstitution assays that have revealed so much about the other ATP-driven molecular motors in the past decade or two. Nonetheless, a great deal has been learned about the flagellar motor, including considerable recent progress in the application of single molecule techniques. This chapter summarizes the historical background and recent advances in the field. To observe the faster rotation of the motor when driving smaller loads, a variety of single molecule techniques have been used to visualize the rotating filaments of stuck or swimming cells including conventional dark field (DF), laser DF, differential interference contrast (DIC), fluorescence microscopy, back-focal-plane interferometry, and high-speed fluorescence microscopy.

Abstract:

Optical tweezers are widely used for experimental investigation of linear molecular motors. The rates and force dependence of steps in the mechanochemical cycle of linear motors have been probed giving detailed insight into motor mechanisms. With similar goals in mind for rotary molecular motors we present here an optical trapping system designed as an angle clamp to study the bacterial flagellar motor and F(1)-ATPase. The trap position was controlled by a digital signal processing board and a host computer via acousto-optic deflectors, the motor position via a three-dimensional piezoelectric stage and the motor angle using a pair of polystyrene beads as a handle for the optical trap. Bead-pair angles were detected using back focal plane interferometry with a resolution of up to 1 degrees , and controlled using a feedback algorithm with a precision of up to 2 degrees and a bandwidth of up to 1.6 kHz. Details of the optical trap, algorithm, and alignment procedures are given. Preliminary data showing angular control of F(1)-ATPase and angular and speed control of the bacterial flagellar motor are presented.