User:Steven J. Koch/Notebook/Kochlab/2010/09/04/Curr Bio (2010) Shastry, Hancock neck linker kinesin1,2

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Shastry, S., & Hancock, W. O. (2010). Neck linker length determines the degree of processivity in kinesin-1 and kinesin-2 motors. Current biology : CB, 20(10), 939-43.

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  • 80 mM PIPES to minimize electrostatic effects on processivity. Processivity measured via TIRF and GFP constructs.
  • Shortening the neck linker of the kinesin-1 construct abolished processivity (as if the evolved neck linker is optimal
  • They created a kinesin-2/kinesin-1 hybrid that has the head and neck linker of kinesin-2, and the coiled-coil region of kinesin-1. Thus, any charge or structural effects of the coiled-coil should be the same between the kinesin-1 and the kinesin-1/2 hybrid.
  • The kinesin-2 construct is about half as fast as kinesin-1, and 2 or 3 times less processive.
  • Systematic length changes of neck linkers
    • There is a proline in the kinesin-2 neck linker that makes the neck linker less compliant than would be expected from a random coil
      • This is supported by less processivity when deleting two neck linker aa
      • This is supported by their molecular dynamics simulation of force-extension curves
      • Deleting this proline eliminates this effect
    • The proline-less kinesin-2 construct and the kinesin-1 construct have remarkably similar processivity when their neck linkers are adapted to be the same (either by shortening kinesin-2 or lengthening kinesin-1). Their speeds remain different, however.
    • Thus, neck linker length appears to be the dominant player in processivity when charge is not an issue (high ionic strength/ no artificially introduced lysines)
    • Stochastic modeling supports notion that (a) front head gating of ATP binding (inhibited by high strain in their interpretation) and (b) rear head gating (acceleration of head unbinding by strain) are both necessary to explain the results.
      • They model front-head gating as lowering the ATP on rate. I'll have to discuss with Larry, but I think we model it as increased ATP off rate (because it cannot bind the switch and behaves like ATP).
  • Neck linker charge
    • Adding + charges to neck linker or lowering the ionic strength increase the processivity. Yildiz et al. (Yildiz, A., Tomishige, M., Gennerich, A., & Vale, R. D. (2008). Intramolecular strain coordinates kinesin stepping behavior along microtubules. Cell, 134(6), 1030-41. doi: 10.1016/j.cell.2008.07.018.) use low ionic strength compared to the more typical 80 mM PIPES. Hancock et al. blame this for the discrepancy with Yildiz, and I think I agree.

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