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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.
Results from a variety of single-molecule experiments have furnished insights into the mechanochemical properties of kinesin motor proteins. Individual kinesin dimers move processively, making discrete 8-nm steps at stochastic intervals, and may take a hundred or more steps before releasing from the microtubule surface. Processive motion persists even in the presence of sustained external loads up to several pN (1-3), suggesting that some portion of the kinesin dimer remains bound to the microtubule at all times. Kinesin molecules move on the microtubule surface lattice along paths parallel to the protofilaments (4, 5), interacting with one binding site per tubulin dimer (6). Finally, kinesin moves in such a way as to hydrolyze exactly one adenosine triphosphate (ATP) molecule per 8-nm step (1, 7, 8). Two broad classes of stepping pattern are consistent with the foregoing observations: hand-over-hand models, in which the two heads step alternately, exchanging leading and trailing roles with each step, and inchworm models, in which a given head remains in the lead (9, 10).
The active portion of the kinesin motor is formed from a dimer of identical heavy chains, which fold into twin heads attached to a single common stalk (11). The two globular heads, which carry enzymatic activity and bind ATP and microtubules, are joined to the stalk through short (~13 amino acids) neck linker regions, consisting of single polypeptide chains (12). The heavy chains then intertwine to form a coiled-coil dimerization domain consisting of classic heptad repeats (11). On the basis of this structure, free rotation (i.e., swiveling) of an individual head could occur, in principle, about the neck linker, but...