Single kinesin molecules studied with a molecular force clamp

Kinesin is a two-headed, ATP-driven motor protein that moves processively a long microtubules in discrete steps of 8 nm, probably by advancing each of its heads alternately in sequence(1-4). Molecular details of how the chemic al energy stored in ATP is coupled to mechanical displacement remain obscur e. To shed light on this question, a force clamp was constructed, based on a feedback-driven optical trap capable of maintaining constant loads on sin gle kinesin motors(5). The instrument provides unprecedented resolution of molecular motion and permits mechanochemical studies under controlled exter nal loads. Analysis of records of kinesin motion under variable ATP concent rations and loads revealed several new features. First, kinesin stepping ap pears to be tightly coupled to ATP hydrolysis over a wide range of forces, with a single hydrolysis per 8-nm mechanical advance. Second, the kinesin s tall force depends on the ATP concentration. Third, increased loads reduce the maximum velocity as expected, but also raise the apparent Michaelis-Men ten constant. The kinesin cycle therefore contains at least one load-depend ent transition affecting the rate at which ATP molecules bind and subsequen tly commit to hydrolysis, It is likely that at least one other load-depende nt rate exists, affecting turnover number. Together, these findings will ne cessitate revisions to our understanding of how kinesin motors function.