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Overview of lectures

Overview of lectures. BIOLOGY was introduced by Judy Armitage’s BIOPHYSICS - Experimental Techniques to measure rotary molecular motors Flagellar Motor F1-ATPase. Single-molecule experiments on bacterial flagellar motors. Stator. Rotor. Continuous switch model. Switching.

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Overview of lectures

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  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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