R
O
T
A
R
Y
Motor
Max Force
Max Speed
Max Power
Flagellar motor
(8 units, E. coli)
2400 pN nm
95 pN
300 Hz
35 mm/s
2000 pN nm @ 150 Hz
1.9 x 106 pN nm /s
(Vibrio)
(single unit)
F1 -ATPase
L
I
N
E
A
R
Myosin
1700 Hz
220 mm/s
300 pN nm
12 pN
300 Hz
35 mm/s
250 pN nm @ 150 Hz
2.4 x 105 pN nm /s
40 pN nm
40 pN
150 Hz
0.9 mm/s
20 pN nm @ 75 Hz
9 x 103 pN nm /s
6 pN
10 mm/s
2 pN @ 10 /s x 20 nm
400 pN nm /s
(single molecule in
muscle or in vitro)
(speed of array, each
molecule mostly
detached)
Kinesin
5 pN
1 mm/s
2.5 pN @ 0.5 mm /s
1.25 x 103 pN nm /s
RNA
polymerase
20 pN
0.01 mm/s
~200 pN nm /s
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
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
Bacterial Chemotaxis
1-D Ising model of flagellar switch
movie
Tethered cells
Cell body
Coverslip in microscope
Cell body rotates
at ~ 10 Hz
1 mm
Flagellum tethered
to coverslip
Frequency (Hz)
Work = torque x angle
Torque = d(work) / d(angle)
Low Reynolds number:
Torque = viscous drag coefficient x angular velocity
Beads attached to the motor
Finite, variable switch times
Switch times distribution predicted by model
… further detailed tests of model
C / Co
Resurrection
One motor can contain at least 11 stators
resurrection
steady-state expression
1 mm bead
0.3 mm bead
Torque-versus speed
Stepping rotation
Speed control for step detection using sodium-driven chimaera
pmf or smf = Vm + kT/e ln (Cin/Cout)
Low numbers of stators:
Low-level induction of
stator proteins
De-energization also
affects stator number
Slow rotation with (probably) one stator unit
Back-focal plane detection
Real speed
Fluorescence detection
30x slower
26 steps per revolution
Kinesin, myosin II, Myosin V, F1-ATPase:
One ATP per step
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
Single-molecule experiments
on ATP-synthase
FO
H+ or Na+
F1
ATP
ADP+ Pi
10nm
Biotin-avidin link to
rotating handle
(actin filament, beads)
F1
His-tag link to surface
0.5 micron beads
Rotated by F1
Fluorescent actin filament
Rotated by F1
(movie: Wolfgang Junge)
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
Low [ATP] :
- 90° step rate-limiting
- exponential distribution
- single step
High [ATP] :
- 30° step rate-limiting
- peaked distribution
- double (or more) step