Direct measurements of kinesin torsional properties reveal flexible domains and occasional stalk reversals during stepping.

Kinesin is a homodimeric motor with two catalytic heads joined to a stalk via short neck linkers (NLs). We measured the torsional properties of single recombinant molecules by tracking the thermal angular motions of fluorescently labeled beads bound to the C terminus of the stalk. When kinesin heads were immobilized on microtubules (MTs) under varied nucleotide conditions, we observed bounded or unbounded angular diffusion, depending on whether one or both heads were attached to the MT. Free rotation implies that NLs act as swivels. From data on constrained diffusion, we conclude that the coiled-coil stalk domains are approximately 30-fold stiffer than its flexible "hinge" regions. Surprisingly, while tracking processive kinesin motion at low ATP concentrations, we observed occasional abrupt reversals in the directional orientations of the stalk. Our results impose constraints on kinesin walking models and suggest a role for rotational freedom in cargo transport.

On the origin of kinesin limping.

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.

Precision steering of an optical trap by electro-optic deflection.

We designed, constructed, and tested a single-beam optical trapping instrument employing twin electro-optic deflectors (EODs) to steer the trap in the specimen plane. Compared with traditional instruments based on acousto-optic deflectors (AODs), EOD-based traps offer a significant improvement in light throughput and a reduction in deflection-angle (pointing) errors. These attributes impart improved force and position resolution, making EOD-based traps a promising alternative for high-precision nanomechanical measurements of biomaterials.

Kinesin steps do not alternate in size.

Kinesin is a two-headed motor protein that transports cargo inside cells by moving stepwise on microtubules. Its exact trajectory along the microtubule is unknown: alternative pathway models predict either uniform 8-nm steps or alternating 7- and 9-nm steps. By analyzing single-molecule stepping traces from "limping" kinesin molecules, we were able to distinguish alternate fast- and slow-phase steps and thereby to calculate the step sizes associated with the motions of each of the two heads. We also compiled step distances from nonlimping kinesin molecules and compared these distributions against models predicting uniform or alternating step sizes. In both cases, we find that kinesin takes uniform 8-nm steps, a result that strongly constrains the allowed models.

Kinesin moves by an asymmetric hand-over-hand mechanism.

Kinesin is a double-headed motor protein that moves along microtubules in 8-nanometer steps. Two broad classes of model have been invoked to explain kinesin movement: hand-over-hand and inchworm. In hand-over-hand models, the heads exchange leading and trailing roles with every step, whereas no such exchange is postulated for inchworm models, where one head always leads. By measuring the stepwise motion of individual enzymes, we find that some kinesin molecules exhibit a marked alternation in the dwell times between sequential steps, causing these motors to "limp" along the microtubule. Limping implies that kinesin molecules strictly alternate between two different conformations as they step, indicative of an asymmetric, hand-over-hand mechanism.