Mechanochemical mechanism of kinesin processive movement studied with single molecule techniques

Kinesin is an essential motor enzyme responsible for many cellular transport processes, such as membrane and protein transport, mitosis, and meiosis. Abnormalities in kinesin functions may be linked to amyotrophic lateral sclerosis and Alzheimer's disease. Single kinesin molecules hydrolyze ATP to drive plus-end-directed transport along the microtubule for many micrometers without detaching, and such movement can sustain external load of ∼7pN. These features undoubtedly help to serve kinesin's long-distance organelle transport in vivo . My studies aim to understand the mechanochemical (conversion of chemical energy into mechanical work) mechanism of kinesin movement along microtubules by using various single molecule techniques. First, by attaching a 100nm polystyrene beads to the end of the coiled-coil neck of the kinesin and observing the stochastic stepping behavior of kinesin under low ATP concentrations, we showed that kinesin hydrolyzes a single ATP molecule for each 8nm step—a distance equal to the microtubule protofilament lattice spacing. One-to-one coupling of mechanical steps to chemical reactions establishes a simple framework for further mechanochemical studies. Next, two alternative mechanisms for kinesin processive movement were tested. In a symmetric hand-over-hand mechanism, every step is hypothesized to be accompanied by 180° rotation of the dimeric enzyme around the axis of its coiled-coil neck domain because the heads are identical and equivalent in their functions. In an inchworm mechanism, the head domains do not swap positions so identical heads function differently throughout the cycles. Three different single molecule experiments were independently carried out to show that 8nm stepping occurred without detectable neck rotation. This is inconsistent with symmetric hand-over-hand mechanism and supporting inchworm mechanism. Finally, to study the intermediate steps during 8nm/1ATP mechanochemical cycle, a slowly hydrolysable ATP analog—ATPγS was used in place of ATP to drive kinesin movement. 8nm steps were observed but not separated into two 4nm steps. This suggests that the ATP binding triggers full 8nm forward movement of the kinesin neck. Combining all the results, a detailed inchworm type mechanism is proposed to explain kinesin processive movement along microtubules.