title: Individual dimers of the mitotic kinesin motor Eg5 step processively and support substantial loads in vitro.
abstract: Eg5, a member of the kinesin superfamily of microtubule-based motors, is essential for bipolar spindle assembly and maintenance during mitosis, yet little is known about the mechanisms by which it accomplishes these tasks. Here, we used an automated optical trapping apparatus in conjunction with a novel motility assay that employed chemically modified surfaces to probe the mechanochemistry of Eg5. Individual dimers, formed by a recombinant human construct Eg5-513-5His, stepped processively along microtubules in 8-nm increments, with short run lengths averaging approximately eight steps. By varying the applied load (with a force clamp) and the ATP concentration, we found that the velocity of Eg5 was slower and less sensitive to external load than that of conventional kinesin, possibly reflecting the distinct demands of spindle assembly as compared with vesicle transport. The Eg5-513-5His velocity data were described by a minimal, three-state model where a force-dependent transition follows nucleotide binding.
authors.affiliation: Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA.
authors.forename: Megan T
authors.initials: MT
authors.lastname: Valentine
authors.forename: Polly M
authors.initials: PM
authors.lastname: Fordyce
authors.forename: Troy C
authors.initials: TC
authors.lastname: Krzysiak
authors.forename: Susan P
authors.initials: SP
authors.lastname: Gilbert
authors.forename: Steven M
authors.initials: SM
authors.lastname: Block
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001696:Biomechanical Phenomena; D002417:Cattle; D019281:Dimerization; D006639:Histidine; D006801:Humans; D016547:Kinesins / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008938:Mitosis; D020409:Molecular Motor Proteins / Q000737:chemistry* / Q000378:metabolism*; D009842:Oligopeptides; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000378:metabolism
chemical_list: C471213:His-His-His-His-His-His; C470243:KIF11 protein, human; D020409:Molecular Motor Proteins; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D006639:Histidine; D000255:Adenosine Triphosphate; D016547:Kinesins