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Direct observation of the binding state of the kinesin head to the microtubule


The dimeric motor protein kinesin-1 converts chemical energy from ATP hydrolysis into mechanical work used to transport cargo along microtubules1,2. Cargo attached to the kinesin stalk moves processively in 8-nm increments3 as its twin motor domains (heads) carry out an asymmetric, ‘hand-over-hand’ walk4,5,6,7. The extent of individual head interactions with the microtubule during stepping, however, remains controversial4,8,9,10,11,12,13,14. A major experimental limitation has been the lack of a means to monitor the attachment of an individual head to the microtubule during movement, necessitating indirect approaches. Here we report the development of a single-molecule assay that can directly report head binding in a walking kinesin molecule, and show that only a single head is bound to the microtubule between steps at low ATP concentrations. A bead was linked to one of the two kinesin heads by means of a short DNA tether and used to apply rapidly alternating hindering and assisting loads with an optical trap. The time-dependent difference between forwards and backwards displacements of the bead alternated between two discrete values during stepping, corresponding to those intervals when the linked head adopted a bound or an unbound state. The linked head could only rebind the microtubule once ATP had become bound to its partner head.

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Figure 1: Single-molecule observations of kinesin head motion.
Figure 2: Motion of a bead linked to one of the kinesin heads under alternating loads.
Figure 3: Histograms of bead displacements under alternating load.
Figure 4: A model for strain-based mechanochemical gating.

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We thank A. Dunn, B. Choi and W. Hwang for advice on labelling kinesin; S. Gilbert for advice on expressing kinesin; and B. Gutiérrez-Medina, C. Sindelar, C. Perez and K. Frieda for comments on the manuscript. This work was supported by grant GM51453 from the US National Institutes of Health.

Author Contributions N.R.G. designed the project, expressed and labelled the protein, and collected and analysed data. N.R.G. and S.M.B. discussed the data and co-wrote the manuscript.

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Correspondence to Steven M. Block.

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Guydosh, N., Block, S. Direct observation of the binding state of the kinesin head to the microtubule. Nature 461, 125–128 (2009).

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