Distinguishing inchworm and hand-over-hand processive kinesin movement by neck rotation measurements

The motor enzyme kinesin makes hundreds of unidirectional 8-nanometer steps without detaching from or freely sliding along the microtubule on which it moves. We investigated the kinesin stepping mechanism by immobilizing a Drosophila kinesin derivative through the carboxyl-terminal end of the neck coiled-coil domain and measuring orientations of microtubules moved by single enzyme molecules at submicromolar adenosine triphosphate concentrations. The kinesin-mediated microtubule-surface linkage was sufficiently torsionally stiff (greater than or equal to2.0+/-0.9 x 10(-20) Newton meters per radian(2)) that stepping by the hypothesized symmetric hand-over-hand mechanism would produce 180degrees rotations of the microtubule relative to the immobilized kinesin neck. In fact, there were no rotations, a finding that is inconsistent with symmetric hand-over-hand movement, An alternative inch-worm mechanism is consistent with our experimental results.