KINETICS OF FORCE GENERATION BY SINGLE KINESIN MOLECULES ACTIVATED BYLASER PHOTOLYSIS OF CAGED-ATP

To relate transients of force by single kinesin molecules with the ele mentary steps of the ATPase cycle, we measured the time to force gener ation by kinesin after photorelease of ATP from caged ATP. Kinesin-coa ted beads were trapped by an infrared laser and brought onto microtubu les fixed to a coverslip, Tension was applied to a kinesin-microtubule rigor complex using the optical trap, and ATP was released by flash p hotolysis of caged ATP with a UV laser, Kinesin started to generate fo rce and move stepwise with a step size of 8 nm at average times of 31, 45, and 79 ms after photorelease of 450, 90, and 18 mu M ATP, respect ively, The kinetics of force generation were consistent with a two-ste p reaction: ATP binding, with an apparent second-order rate constant o f 0.7 mu M-1.s (-1), followed by force generation at 45 s(-1) per kine sin molecule. The transient rate of force generation was close to the rate of the ATPase cycle in solution, suggesting that the rate-limitin g step of ATPase cycle is involved with the force generation.