title: Direct observation of the binding state of the kinesin head to the microtubule.
abstract: The dimeric motor protein kinesin-1 converts chemical energy from ATP hydrolysis into mechanical work used to transport cargo along microtubules. Cargo attached to the kinesin stalk moves processively in 8-nm increments as its twin motor domains (heads) carry out an asymmetric, 'hand-over-hand' walk. The extent of individual head interactions with the microtubule during stepping, however, remains controversial. 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.
authors.affiliation: Biophysics Program, Stanford University, Stanford, California 94305, USA.
authors.forename: Nicholas R
authors.initials: NR
authors.lastname: Guydosh
authors.forename: Steven M
authors.initials: SM
authors.lastname: Block
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004331:Drosophila melanogaster; D016547:Kinesins / Q000737:chemistry* / Q000378:metabolism*; D008863:Microspheres; D008870:Microtubules / Q000378:metabolism*; D009068:Movement / Q000187:drug effects; D052898:Optical Tweezers; D011485:Protein Binding / Q000187:drug effects; D013997:Time Factors
chemical_list: D000255:Adenosine Triphosphate; D004247:DNA; D016547:Kinesins
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