One-headed kinesin derivatives move by a nonprocessive, low-duty ratio mechanism unlike that of two-headed kinesin.
A single molecule of the "two-headed" motor enzyme kinesin can move along a microtubule continuously for many enzymatic turnovers (processive movement), and the velocity produced by one kinesin molecule is the same as that produced by many kinesin molecules (high duty ratio). We studied the microtubule movement driven at 1 mM ATP by biotinated N-terminal fragments of Drosophila kinesin heavy chain attached to streptavidin-coated coverslips at various surface densities. K448-BIO has velocity at a high density of vmax = 750 nm s-1 and is dimeric (hence two-headed); K365-BIO (vmax = 200 nm s-1) and K340-BIO (vmax = 90 nm s-1) are monomeric. Escape of microtubules from the surface was prevented by methylcellulose so that continuous trajectories of microtubules not continuously attached to motor molecules could be recorded by video microscopy. The component of instantaneous velocity parallel to the microtubule axis (v) was analyzed in trajectories with a mean velocity 0.4-0.7 times vmax. In K448-BIO trajectories, the distribution of v was bimodal with peaks near 0 and 750 nm s-1. Temporal autocorrelation analysis of v detected lengthy episodes of high-velocity movement consistent with isolated processive microtubule runs driven at vmax by single K448-BIO dimers. K365-BIO and K340-BIO trajectories had unimodal distributions of v and autocorrelation times much shorter than those for K448-BIO. Therefore the monomeric motors have duty ratio < 55% (i.e., no forward movement is generated for at least 45% of the enzymatic cycle time) or processivity below the detection limit of approximately 300 turnovers even in methylcellulose. Continuous movement at maximal velocity thus requires more than one kinesin head.
Young
E C
EC
Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254, USA.
Mahtani
H K
HK
Gelles
J
J
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002417:Cattle; D004330:Drosophila / Q000201:enzymology / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D066298:In Vitro Techniques; D016547:Kinesins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008747:Methylcellulose; D008870:Microtubules / Q000378:metabolism; D009068:Movement; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D010446:Peptide Fragments; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D008747:Methylcellulose; D016547:Kinesins