1. MotorMax ForceMax SpeedMax Power ROTARY ROTARY Flagellar motor (8 units, E. coli) 2400 pN nm 95 pN 300 Hz 35  m/s 2000 pN nm @ 150 Hz 1.9 x 10 6 pN nm /s (Vibrio)1700 Hz 220  m/s (single unit)300 pN nm 12 pN 300 Hz 35  m/s 250 pN nm @ 150 Hz 2.4 x 10 5 pN nm /s F 1 -ATPase40 pN nm 40 pN 150 Hz 0.9  m/s 20 pN nm @ 75 Hz 9 x 10 3 pN nm /s LINEARLINEAR Myosin (single molecule in muscle or in vitro) 6 pN 10  m/s (speed of array, each molecule mostly detached) 2 pN @ 10 /s x 20 nm 400 pN nm /s Kinesin5 pN 1  m/s2.5 pN @ 0.5  m /s 1.25 x 10 3 pN nm /s RNA polymerase 20 pN 0.01  m/s ~200 pN nm /s

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

3. Single-molecule experiments on bacterial flagellar motors

5. Rotor Stator

8. Continuous switch model

10. 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 switch Science 327:685-689

11. Bacterial Chemotaxis

12. 1-D Ising model of flagellar switch movie

13. Tethered cells Cell body rotates at ~ 10 Hz 1  m Flagellum tethered to coverslip Cell body Coverslip in microscope

14. Frequency (Hz)

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

16. Beads attached to the motor

17. Finite, variable switch times

18. Switch times distribution predicted by model

19. … further detailed tests of model C / C o

20. Resurrection

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

22. 1  m bead 0.3  m bead

23. Torque-versus speed

24. Stepping rotation

25. Speed control for step detection using sodium-driven chimaera pmf or smf = V m + kT/e ln (C in /C out )

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

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

28. 26 steps per revolution

29. Kinesin, myosin II, Myosin V, F1-ATPase: Step size is set by the track 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 One ATP per step

30. ATP ADP+ P i 10nm F1F1 FOFO H + or Na + Single-molecule experiments on ATP-synthase

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

33. 0.5 micron beads Rotated by F1 Fluorescent actin filament Rotated by F1 (movie: Wolfgang Junge)

35. 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

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