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Nat Chem Biol. 2016 Apr;12(4):290-7. doi: 10.1038/nchembio.2028. Epub 2016 Feb 29.

Direct observation of intermediate states during the stepping motion of kinesin-1.

Author information

1
Department of Applied Physics, School of Engineering, The University of Tokyo, Tokyo, Japan.
2
Okazaki Institute for Integrative Bioscience, Institute for Molecular Science, National Institutes of Natural Sciences, Aichi, Japan.
3
Department of Functional Molecular Science, School of Physical Sciences, The Graduate University for Advanced Studies (SOKENDAI), Kanagawa, Japan.
4
Quantum-Phase Electronics Center, School of Engineering, The University of Tokyo, Tokyo, Japan.
5
Department of Applied Chemistry, School of Engineering, The University of Tokyo, Tokyo, Japan.

Abstract

The dimeric motor protein kinesin-1 walks along microtubules by alternatingly hydrolyzing ATP and moving two motor domains ('heads'). Nanometer-precision single-molecule studies demonstrated that kinesin takes regular 8-nm steps upon hydrolysis of each ATP; however, the intermediate states between steps have not been directly visualized. Here, we employed high-temporal resolution dark-field microscopy to directly visualize the binding and unbinding of kinesin heads to or from microtubules during processive movement. Our observations revealed that upon unbinding from microtubules, the labeled heads were displaced rightward and underwent tethered diffusive movement. Structural and kinetic analyses of wild-type and mutant kinesins with altered neck linker lengths provided evidence that rebinding of the unbound head to the rear-binding site is prohibited by a tension increase in the neck linker and that ATP hydrolysis by the leading head is suppressed when both heads are bound to the microtubule, thereby explaining how the two heads coordinate to move in a hand-over-hand manner.

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PMID:
26928936
DOI:
10.1038/nchembio.2028
[Indexed for MEDLINE]

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