Oxford Molecular Motors

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:

The twin-arginine translocation (Tat) system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. The essential components of the Tat pathway are the membrane proteins TatA, TatB, and TatC. TatA is thought to form the protein translocating element of the Tat system. Current models for Tat transport make predictions about the oligomeric state of TatA and whether, and how, this state changes during the transport cycle. We determined the oligomeric state of TatA directly at native levels of expression in living cells by photophysical analysis of individual yellow fluorescent protein-labeled TatA complexes. TatA forms complexes exhibiting a broad range of stoichiometries with an average of approximately 25 TatA subunits per complex. Fourier analysis of the stoichiometry distribution suggests the complexes are assembled from tetramer units. Modeling the diffusion behavior of the complexes suggests that TatA protomers associate as a ring and not a bundle. Each cell contains approximately 15 mobile TatA complexes and a pool of approximately 100 TatA molecules in a more disperse state in the membrane. Dissipation of the protonmotive force that drives Tat transport has no affect on TatA complex stoichiometry. TatA complexes do not form in cells lacking TatBC, suggesting that TatBC controls the oligomeric state of TatA. Our data support the TatA polymerization model for the mechanism of Tat transport.

Abstract:

The torque generated by the power stroke of Escherichia coli F(1)-ATPase was determined as a function of the load from measurements of the velocity of the gamma-subunit obtained using a 0.25 micros time resolution and direct measurements of the drag from 45 to 91 nm gold nanorods. This result was compared to values of torque calculated using four different drag models. Although the gamma-subunit was able to rotate with a 20x increase in viscosity, the transition time decreased from 0.4 ms to 5.26 ms. The torque was measured to be 63+/-8 pN nm, independent of the load on the enzyme.