Motility of single-headed kinesin

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Single kinesin molecule can move a microtubule continuously. Whether the two heads of a kinesin molecule are necessary for movement or the single head is enough is yet to be established. To approach this problem, we have prepared single headed kinesin and examined in two different motility assay systems.Kinesin motor domain of 5 different lengths were expressed in E.coli : they consist of N-terminal 340 to 410 amino acids and were supplemented with a cystein residue at the C terminus ends. K340, K351 and K368 were monomers, whereas K386 and K410 existed as dimers.To examine the motility of these kinesin fragments, the C-terminal cystein residue was labeled with maleimide-biotin and tethered to a glass surface via streptavidin and biotinylated BSA. All of 5 expressed kinesin fragments moved microtubules. The velocity of double-headed species was about 0.5 mu m/sec, which is similar to that of intact kinesin. The velocity of single-headed species was about ten times lower than that of double-headed fragments.. These results suggest that the two heads of kinesin stimulate the motility, although they are not essential for the movement.To aim single molecule mechanics, we carried out a bead assay using optical tweezers. In this system, the bead binds very few numbers of kinesin fragments via biotin-avidin, and the velocity and force generated by most likely single molecule was measured from the bead displacement. The maximum velocity (0.5 mu m/sec) and the maximum force (6pN) for K351 (single-headed) was almost the same as those for K410 (double-headed). These results indicate that a single-headed kinesin is sufficient to move microtubules and generate the force at a similar level as double-headed kinesin.