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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

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Eg5 is interesting because:

- Member of the kinesin-5 family.

- Speculated to move nonprocessively, unlike regular Kinesin.

- Also speculated to move exactly like kinesin (i.e. walk with ~8 nm steps)

- Human Eg5 is not easily purified from cells.

Clearly we need single molecule biophysics!

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Experimental setup

Notes: Recombinant human Eg5-513-5His from E.coliPLL-PEG-biotin = biotinylated poly-(L-lysine)-graft-polyethylene polymersVideo tracking, data sampling at 2 kHz; Fixed position optical trap.

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How many dimers does it take to move a bead?

How many dimers does it take to move a bead?

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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 mMe: Load = –4 pN and c(APT) = 31 µMf: Load = +4 pN and c(APT) = 2 mMNotes: 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)

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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.

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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 steady-state solution kinetics is caused by frequent detachments from microtubles.

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Velocity vs. ATP concentration/force

Michaelis-Menten kinetics gives us:

Looks fine, but does velocity really increase with positive force?

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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.

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Another point of interest

Stall force:

No load force greater than –5 pN was recorded. because Eg5-513-5His 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.

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Speculations

Eg5:

Dissociates at high loads

Walks roughly 8.3 steps

V ~ 100 nm s-1

Kinesin:

Slows down at high loads

Walks >100 steps

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).