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Advanced Insights into Bacterial Flagellar Motors and Molecular Mechanisms

This lecture overview presented by Judy Armitage delves into experimental techniques for measuring rotary molecular motors, particularly focusing on the bacterial flagellar motor and F1-ATPase. It discusses the mechanics of flagellar switch models, cooperativity mechanisms, and chemotaxis as illustrated by single-molecule experiments. Key concepts include torque, energy conservation, ATP synthesis, and the effects of varying ATP concentrations on motor speed and operation. This comprehensive exploration highlights significant findings relevant to biophysics and molecular biology.

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Advanced Insights into Bacterial Flagellar Motors and Molecular Mechanisms

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Presentation Transcript

  1. Overview of lectures • BIOLOGY was introduced by Judy Armitage’s • BIOPHYSICS - Experimental Techniques to measure rotary molecular motors Flagellar Motor F1-ATPase

  2. Single-molecule experiments on bacterial flagellar motors

  3. Stator Rotor

  4. Continuous switch model

  5. Switching F Bai, RW. Branch, D Nicolau, TPilizota, BC Steel, PK Maini, RM Berry (2010) Conformational spread as a mechanism for cooperativity in the bacterial flagellar switchScience 327:685-689

  6. Bacterial Chemotaxis

  7. 1-D Ising model of flagellar switch movie

  8. Cell body rotates at ~ 10 Hz Tethered cells Cell body Coverslip in microscope Flagellum tethered to coverslip 1 mm

  9. Frequency (Hz)

  10. Work = torque x angle Torque = d(work) / d(angle) Low Reynolds number: Torque = viscous drag coefficient x angular velocity

  11. Beads attached to the motor

  12. Finite, variable switch times

  13. Switch times distribution predicted by model

  14. … further detailed tests of model C / Co

  15. Resurrection

  16. steady-state expression One motor can contain at least 11 stators resurrection

  17. 1 mm bead 0.3 mm bead

  18. Torque-versus speed

  19. Stepping rotation

  20. Speed control for step detection using sodium-driven chimaera pmf or smf = Vm + kT/e ln (Cin/Cout)

  21. Low numbers of stators: Low-level induction of stator proteins De-energization also affects stator number

  22. Slow rotation with (probably) one stator unit Back-focal plane detection Fluorescence detection 30x slower Real speed

  23. 26 steps per revolution

  24. by energy conservation based on full energization and high loads, one proton gives a max step size of ~10 degrees. Maybe there are 2 ions per step? 34-fold model refines C-ring : 25-fold model refines M ring (& C-inner) Thomas et al 2006 Kinesin, myosin II, Myosin V, F1-ATPase: One ATP per step Step size is set by the track

  25. Single-molecule experiments on ATP-synthase FO H+ or Na+ F1 ATP ADP+ Pi 10nm

  26. Biotin-avidin link to rotating handle (actin filament, beads) F1 His-tag link to surface

  27. 0.5 micron beads Rotated by F1 Fluorescent actin filament Rotated by F1 (movie: Wolfgang Junge)

  28. High [ATP] : no wait before 90° step Medium [ATP] : ~ms before 90° step Low [ATP] : long wait before 90° step (ms) All [ATP] : ~ms before 30° step

  29. Low [ATP] : - 90° step rate-limiting- exponential distribution- single step High [ATP] : - 30° step rate-limiting- peaked distribution- double (or more) step

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