Transcript Dias nummer 1

Individual dimers of the mitotic kinesin motor Eg5 step
processively and support substantial loads in vitro
Megan T. Valentine, Polly M. Fordyce, Troy C. Krzysiak, Susan P. Gilbert and
Steven M. Block, 2006, Nature Cell Biology 8: 470-476
Jeg hedder Anders
University of Copenhagen
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Eg5 is interesting because:
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Member of the kinesin-5 family.
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Speculated to move nonprocessively, unlike regular
Kinesin.
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Also speculated to move exactly like kinesin (i.e.
walk with ~8 nm steps)
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Human Eg5 is not easily purified from cells.
Clearly we need single molecule biophysics!
University of Copenhagen
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Experimental setup
Notes: Recombinant human Eg5-513-5His from E.coli
PLL-PEG-biotin = biotinylated poly-(L-lysine)-graft-polyethylene polymers
Video tracking, data sampling at 2 kHz; Fixed position optical trap.
University of Copenhagen
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How many dimers does it take to move a bead?
How many dimers does it take to
move a bead?
University of Copenhagen
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Steps vs. time
a, b, d: Load = –4 pN and c(APT) = 8 µM
c:
Load = –4 pN and c(APT) = 2 mM
e:
Load = –4 pN and c(APT) = 31 µM
f:
Load = +4 pN and c(APT) = 2 mM
Notes: c(ATP) in human cells is in the range of 1-10 mM says Wikipedia.
Step size is identical to that of kinesin (8.1 ± 0.1 nm)
University of Copenhagen
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Average run length
Average run length of
Eg5-513-5His:
67± 7 nm or 8.3 steps
Average run length of regular
kinesin is more than 100 steps
per run.
The authors discard the first
three bins to obtain a good
exponential fit.
Eg5-513-5His dissociates
easily compared to kinesin.
University of Copenhagen
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Sammenligning med ensemble værdier
Steady-state solution kinetics:
KM
= 8 ± 2 µM (for Eg5-513)
kcat = 0.48 ± 0.02 s-1 (for Eg5-513)
kcat = 0.45 ± 0.04 s-1 (for Eg5-513-5His)
Single-molecule velocities at zero force:
KM
= 10 ± 2 µM (for Eg5-513-5His)
kcat = 11.9 ± 0.2 s-1 (for Eg5-513-5His)
Possible explanation: The kcat underestimation in steadystate solution kinetics is caused by frequent detachments from
microtubles.
University of Copenhagen
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Velocity vs. ATP concentration/force
Michaelis-Menten kinetics gives us:
Looks fine, but does velocity really increase with positive force?
University of Copenhagen
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Rate limiting steps/mechanisms
a: F = –4 pN; APT concentration is clearly rate limiting at low
concentrations.
b: At saturated ATP concentrations, the load force acts slightly
rate limiting.
 At least two rate limiting steps/mechanisms are involved.
University of Copenhagen
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Another point of interest
Stall force:
No load force greater than –5 pN was recorded. because Eg5513-5His is dissociates from microtubles at this force. –7 pN
has been recorded in another experiment.
It’s like a strong car with too little traction.
University of Copenhagen
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Speculations
Eg5:
Kinesin:
Dissociates at high loads
Slows down at high loads
Walks roughly 8.3 steps
Walks >100 steps
V ~ 100 nm s-1
V ~ 6-8 times faster
The authors have lots of speculations on why these characteristics
may be practical in vivo, but very few references (one).
University of Copenhagen
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