Substeps within the 8-nm step of the ATPase cycle of single kinesin molecules

Kinesin is a molecular motor that moves processively(1-4) by regular 8-nm s teps along microtubules(5-11). The processivity of this movement is explain ed by a hand-overhand model in which the two heads of kinesin work in a coo rdinated manner. One head remains bound to the microtubule while the other steps from the alpha beta -tubulin dimer behind the attached head to the di mer in front. The overall movement is 8 nm per ATPase cycle(9-13). To inves tigate elementary processes within the 8-nm step, we have developed a new a ssay that resolves nanometre displacements of single kinesin molecules with microsecond accuracy. Our data show that the 8-nm step can be resolved int o fast and slow substeps, each corresponding to a displacement of similar t o4 nm. The substeps are most probably generated by structural changes in on e head of kinesin, leading to rectified forward thermal motions of the part ner head(14). It is also possible that the kinesin steps along the 4-nm rep eat of tubulin monomers.