On the Origin of Kinesin Limping

Abstract

Kinesin is a dimeric motor with twin catalytic heads joined to a common stalk. Kinesin molecules move processively along microtubules in a hand-over-hand walk, with the two heads advancing alternately. Recombinant kinesin constructs with short stalks have been found to "limp", i.e., exhibit alternation in the dwell times of successive steps. Limping behavior implies that the molecular rearrangements underlying even- and odd-numbered steps must differ, but the mechanism by which such rearrangements lead to limping remains unsolved. Here, we used an optical force clamp to measure individual, recombinant dimers and test candidate explanations for limping. Introducing a covalent cross-link into the stalk region near the heads had no effect on limping, ruling out possible stalk misregistration during coiled-coil formation as a cause. Limping was equally unaffected by mutations that produced 50-fold changes in stalk stiffness, ruling out models where limping arises from an asymmetry in torsional strain. However, limping was enhanced by perturbations that increased the vertical component of load on the motor, including increases in bead size or net load, and decreases in the stalk length. These results suggest that kinesin heads take different vertical trajectories during alternate steps, and that the rates for these motions are differentially sensitive to load.

Regulation of a heterodimeric kinesin-2 through an unprocessive motor domain that is turned processive by its partner

“…Limping has in fact been observed also for conventional, homodimeric kinesin-1. The degree of limping was strongly affected by the magnitude of the vertical force acting on the motor and was influenced, e.g., by the length of the stalk (37). In our assays, motors are unspecifically attached to beads.…”

Motor proteins and molecular motors: how to operate machines at the nanoscale

“…Optical-trap spectroscopy is one of the most powerful single-molecule experimental methods that have been widely applied for studying dynamic properties of different classes of molecular motors [24,25,30,33–38,42,44,48,52,57,60,63,64,66]. This approach uses a laser beam to monitor the displacements of a bead to which a single motor protein molecule is chemically attached [5,22,25,30,85,136,137].…”

Strain-Dependent Kinetic Properties of KIF3A and KIF3C Tune the Mechanochemistry of the KIF3AC Heterodimer

“…Previous work in which dimers were constructed with non-identical motors showed the alternating stepping rates expected from the alternating head kinetics (36). Even homodimeric kinesins have been reported to limp with alternating long and short dwell times before each step (37,38). Thus, it was surprising that we did not observe alternating dwell times with KIF3AC.…”