Kinesin Takes One 8-nm Step for Each ATP That It Hydrolyzes

Conventional kinesin is a motor protein that moves stepwise along microtubules carrying membrane-bound organelles toward the periphery of cells. The steps are of amplitude 8.1 nm, the distance between adjacent tubulin binding sites, and are powered by the hydrolysis of ATP. We have asked: how many steps does kinesin take for each molecule of ATP that it hydrolyzes? To answer this question, the motility and ATP hydrolysis of recombinant, heterotetrameric and homodimeric conventional Drosophila kinesins adsorbed to 200-nm-diameter casein-coated silica beads were assayed under identical, single-molecule conditions. Division of the speed by the maximum microtubule-activated ATPase rate gave a stoichiometry of 1.08 ± 0.09 steps for each ATP hydrolyzed at 1 mmATP. Therefore, under low loads in which the drag force ≪ 1 pN, between the and of kinesin is conventional that that steps powered by Conventional kinesin is a motor protein that moves stepwise along microtubules carrying membrane-bound organelles toward the periphery of cells. The steps are of amplitude 8.1 nm, the distance between adjacent tubulin binding sites, and are powered by the hydrolysis of ATP. We have asked: how many steps does kinesin take for each molecule of ATP that it hydrolyzes? To answer this question, the motility and ATP hydrolysis of recombinant, heterotetrameric and homodimeric conventional Drosophila kinesins adsorbed to 200-nm-diameter casein-coated silica beads were assayed under identical, single-molecule conditions. Division of the speed by the maximum microtubule-activated ATPase rate gave a stoichiometry of 1.08 ± 0.09 steps for each ATP hydrolyzed at 1 mmATP. Therefore, under low loads in which the drag force ≪ 1 pN, between the and of kinesin is conventional that that steps powered by Conventional kinesin is a protein that steps along the of a it a membrane-bound toward the periphery of a The of the steps is is the distance between binding along the and a kinesin molecule take of steps loads The steps are by the hydrolysis of kinesin is ATPase speed of ATP it a maximum of and a of and the to the by which the is to ATP this the is the stoichiometry of how many steps does kinesin take for each ATP that it hydrolyzes? is it to how motor kinesin and of that the stoichiometry is that ATP hydrolysis a in a that a the the binding is that the motor the binding in the this that at of ATP are hydrolyzed stoichiometry of to hydrolysis that to steps and to between the and a stoichiometry 1 and have to the of the of to the between and the of the are of that hydrolysis the are conventional in which the and are to a stoichiometry of to the stoichiometry of kinesin have that ATP of steps are at low ATP the the between speed and ATP low ATP that ATP is to a were the speed the a of the ATP is by at low ATP that between steps are were the are a stoichiometry of 1 the that the binding of ATP molecule in a the that a the this the stoichiometry of kinesin by rate of ATP hydrolysis to rate of in which a kinesin molecule is along a of the speed of by the ATPase the of the motor Division of the by the 8.1 the of the stoichiometry 1 kinesin ATPase have in motility have the to a to a in for Drosophila kinesin to low for a kinesin motor protein of between speed and ATPase that binding of kinesin to a have the speed this have the ATP hydrolysis rate of kinesin under to to the kinesin to 200-nm-diameter silica low the motility is to the stoichiometry is the stoichiometry of the motility and ATP hydrolysis of recombinant, heterotetrameric and homodimeric conventional Drosophila kinesin adsorbed to 200-nm-diameter casein-coated silica beads were assayed under identical, single-molecule conditions. kinesin are in ± ± ± ± ± ± ± ± ± ± ± ± ± ± and ATPase of kinesin to silica beads at kinesin and The stoichiometry by the distance ATP by the and by to for the of the ATPase The were by the the in motility to in the of and in the ATPase in a of the beads along microtubules adsorbed to the of the by the of the the of the beads for The distance each of a along a to at the of the were a the of beads have of are to a kinesin The of to that by the the to in the of are to for kinesin to the at that that each kinesin have a of and speed of beads were the kinesin the rate of to microtubules a of the is a of of a of the the in The by the distance by the of speed of the beads a of the rate at which the beads to the microtubules the of The rate of at the that the of a binding to a a at a of is that of a that a of the adsorbed are the under that the adsorbed to beads to speed at which beads along microtubules of the in and The speed ± ± for this at rate of ATP hydrolysis by kinesin to the 200-nm-diameter beads a The that of the beads kinesin to kinesin The hydrolysis rate the and the were the the motility is in and the hydrolysis rate to ± molecule ± The hydrolysis rate at a tubulin of ± ± were kinesin kinesin ATPase rate of heterotetrameric kinesin and homodimeric kinesin adsorbed to 200-nm-diameter silica are the to the are that have the ATPase a a of of kinesin To a are each to the a kinesin molecule to the and the ATP hydrolysis molecule a of of kinesin are to beads at kinesin the ATP hydrolysis at tubulin of ± 1 to the rate at a of ± 1 that kinesin a by that the ATPase rate is the of ATP hydrolysis the of ATP and the of the hydrolysis To this of the ATP hydrolyzed the of To the of a low of the hydrolysis assayed and ATPase in the of the speed ± to ± The ATP hydrolysis rate at in the of beads ± to ± that the the kinesin hydrolysis rate in by the rate at which kinesin moves along a and by for The of kinesin and stoichiometry were for this the to and the speed of kinesin adsorbed to a silica by maximum hydrolysis rate of ± and ± for the and ± for the the to by to the of in the ATPase in the motility for this and by the of 8.1 stoichiometry of 1.08 ± 0.09 hydrolyzed the by the of The is the in the the ATPase the kinesin the and the of in the motility and ATPase the to of the stoichiometry the of the of the kinesin this the of kinesin the of that ATP by each kinesin molecule We that kinesin to the silica beads the of of the kinesin adsorbed to the beads a for binding of the adsorbed were by microtubules to Therefore, the of kinesin to the beads is a of the to and to to is that the of is the of the motility is the of the that the beads along the that kinesin between and a the of of this were the the that of a the ATPase rate the ATPase rate of the and the stoichiometry a of that the of in a is the were have beads binding to the microtubules for this of beads to the the were a that the of the to the microtubules at tubulin the are to beads in this is to Drosophila kinesin in to that the for the ATPase is to that for at tubulin the are to the the of of a the speed of of silica beads under the of kinesin by the ATP hydrolysis rate of the beads under the have that kinesin 1.08 ± 0.09 ± steps for each molecule of ATP that it is a stoichiometry of were of kinesin ATP molecule kinesin steps is ATP of at ATP that are of steps the were Therefore, the that the is ATP hydrolysis in a along a the low in the between motility and ATP hydrolysis is a stoichiometry of 1.08 ± 0.09 that at of the hydrolysis a 1.08 0.09 The in is the drag force a at 1 is in which is of the maximum force of that a kinesin molecule the at is stoichiometry of 1 is the and that a between the and of a motor and of that the in the that steps powered by ATP Conventional kinesin is a protein that steps along the of a it a membrane-bound toward the periphery of a The of the steps is is the distance between binding along the and a kinesin molecule take of steps loads The steps are by the hydrolysis of kinesin is ATPase speed of ATP it a maximum of and a of and the to the by which the is to ATP this the is the stoichiometry of how many steps does kinesin take for each ATP that it hydrolyzes? is it to how motor kinesin and of that the stoichiometry is that ATP hydrolysis a in a that a the the binding is that the motor the binding in the this that at of ATP are hydrolyzed stoichiometry of to hydrolysis that to steps and to between the and a stoichiometry 1 and have to the of the of to the between and the of the are of that hydrolysis the are conventional in which the and are to a stoichiometry of to the stoichiometry of kinesin have that ATP of steps are at low ATP the the between speed and ATP low ATP that ATP is to a were the speed the a of the ATP is by at low ATP that between steps are were the are a stoichiometry of 1 the that the binding of ATP molecule in a the that a the this the stoichiometry of kinesin by rate of ATP hydrolysis to rate of in which a kinesin molecule is along a of the speed of by the ATPase the of the motor Division of the by the 8.1 the of the stoichiometry 1 kinesin ATPase have in motility have the to a to a in for Drosophila kinesin to low for a kinesin motor protein of between speed and ATPase that binding of kinesin to a have the speed this have the ATP hydrolysis rate of kinesin under to to the kinesin to 200-nm-diameter silica low the motility is to the stoichiometry is the stoichiometry of the motility and ATP hydrolysis of recombinant, heterotetrameric and homodimeric conventional Drosophila kinesin adsorbed to 200-nm-diameter casein-coated silica beads were assayed under identical, single-molecule conditions. kinesin are in ± ± ± ± ± ± ± ± ± ± ± ± ± ± and ATPase of kinesin to silica beads at kinesin and The stoichiometry by the distance ATP by the and by to for the of the ATPase The were by the the in motility to in the of and in the ATPase in a of the beads along microtubules adsorbed to the of the by the of the the of the beads for The distance each of a along a to at the of the were a the of beads have of are to a kinesin The of to that by the the to in the of are to for kinesin to the at that that each kinesin have a of rate at which the beads to the microtubules the of The rate of at the that the of a binding to a a at a of is that of a that a of the adsorbed are the under that the adsorbed to beads to speed at which beads along microtubules of the in and The speed ± ± for this at rate of ATP hydrolysis by kinesin to the 200-nm-diameter beads a The that of the beads kinesin to kinesin The hydrolysis rate the and the were the the motility is in and the hydrolysis rate to ± molecule ± The hydrolysis rate at a tubulin of ± ± were kinesin kinesin ATPase rate of heterotetrameric kinesin and homodimeric kinesin adsorbed to 200-nm-diameter silica are the to the are that have the ATPase a a of of kinesin To a are each to the a kinesin molecule to the and the ATP hydrolysis molecule a of of kinesin are to beads at kinesin the ATP hydrolysis at tubulin of ± 1 to the rate at a of ± 1 that kinesin a by that the ATPase rate is the of ATP hydrolysis the of ATP and the of the hydrolysis To this of the ATP hydrolyzed the of To the of a low of the hydrolysis assayed and ATPase in the of the speed ± to ± The ATP hydrolysis rate at in the of beads ± to ± that the the kinesin hydrolysis rate in by the rate at which kinesin moves along a and by for The of kinesin and stoichiometry were for this the to and the speed of kinesin adsorbed to a silica by maximum hydrolysis rate of ± and ± for the and ± for the the to by to the of in the ATPase in the motility for this and by the of 8.1 stoichiometry of 1.08 ± 0.09 hydrolyzed the by the of The is the in the the ATPase the kinesin the and the of in the motility and ATPase the to of the stoichiometry the of the of the kinesin this the of kinesin the of that ATP by each kinesin molecule We that kinesin to the silica beads the of of the kinesin adsorbed to the beads a for binding of the adsorbed were by microtubules to Therefore, the of kinesin to the beads is a of the to and to to is that the of is the of the motility is the of the that the beads along the that kinesin between and a the of of this were the the that of a the ATPase rate the ATPase rate of the and the stoichiometry a of that the of in a is the were have beads binding to the microtubules for this of beads to the the were a that the of the to the microtubules at tubulin the are to beads in this is to Drosophila kinesin in to that the for the ATPase is to that for at tubulin the are to the the of of a To the stoichiometry of the motility and ATP hydrolysis of recombinant, heterotetrameric and homodimeric conventional Drosophila kinesin adsorbed to 200-nm-diameter casein-coated silica beads were assayed under identical, single-molecule conditions. kinesin are in and ATPase of kinesin to silica beads at kinesin and The stoichiometry by the distance ATP by the and by to for the of the ATPase The were by the the in motility to in the of and in the ATPase of the beads along microtubules adsorbed to the of the by the of the the of the beads for The distance each of a along a to at the of the were a the of beads have of are to a kinesin The of to that by the the to in the of are to for kinesin to the at that that each kinesin have a of rate at which the beads to the microtubules the of The rate of at the that the of a binding to a a at a of is that of a that a of the adsorbed are the under that the adsorbed to beads to speed at which beads along microtubules of the in and The speed ± ± for this at rate of ATP hydrolysis by kinesin to the 200-nm-diameter beads a The that of the beads kinesin to kinesin The hydrolysis rate the and the were the the motility is in and the hydrolysis rate to ± molecule ± The hydrolysis rate at a tubulin of ± ± were kinesin kinesin are that have the ATPase a a of of kinesin To a are each to the a kinesin molecule to the and the ATP hydrolysis molecule a of of kinesin are to beads at kinesin the ATP hydrolysis at tubulin of ± 1 to the rate at a of ± 1 that kinesin a by that the ATPase rate is the of ATP hydrolysis the of ATP and the of the hydrolysis To this of the ATP hydrolyzed the of To the of a low of the hydrolysis assayed and ATPase in the of the speed ± to ± The ATP hydrolysis rate at in the of beads ± to ± that the the kinesin hydrolysis rate in by the rate at which kinesin moves along a and by for The of kinesin and stoichiometry were for this the to and the speed of kinesin adsorbed to a silica by maximum hydrolysis rate of ± and ± for the and ± for the the to by to the of in the ATPase in the motility for this and by the of 8.1 stoichiometry of 1.08 ± 0.09 hydrolyzed the by the of The is the in the the ATPase the kinesin the and the of in the motility and ATPase the to of the stoichiometry the of the of the kinesin this the of kinesin the of that ATP by each kinesin molecule We that kinesin to the silica beads the of of the kinesin adsorbed to the beads a for binding of the adsorbed were by microtubules to Therefore, the of kinesin to the beads is a of the to and to to is that the of is the of the motility is the of the that the beads along the that kinesin between and a the of of this were the the that of a the ATPase rate the ATPase rate of the and the stoichiometry a of that the of in a is the were have beads binding to the microtubules for this of beads to the the were a that the of the to the microtubules at tubulin the are to beads in this is to Drosophila kinesin in to that the for the ATPase is to that for at tubulin the are to the the of of a of the beads along microtubules adsorbed to the of the by the of the the of the beads for The distance each of a along a to at the of the were a the of beads have of are to a kinesin The of to that by the the to in the of are to for kinesin to the at that that each kinesin have a of rate at which the beads to the microtubules the of The rate of at the that the of a binding to a a at a of is that of a that a of the adsorbed are the under that the adsorbed to beads to speed at which beads along microtubules of the in and The speed ± ± for this at rate of ATP hydrolysis by kinesin to the 200-nm-diameter beads a The that of the beads kinesin to kinesin The hydrolysis rate the and the were the the motility is in and the hydrolysis rate to ± molecule ± The hydrolysis rate at a tubulin of ± ± were kinesin kinesin are that have the ATPase a a of of kinesin To a are each to the a kinesin molecule to the and the ATP hydrolysis molecule a of of kinesin are to beads at kinesin the ATP hydrolysis at tubulin of ± 1 to the rate at a of ± 1 that kinesin a by that the ATPase rate is the of ATP hydrolysis the of ATP and the of the hydrolysis To this of the ATP hydrolyzed the of To the of a low of the hydrolysis assayed and ATPase in the of the speed ± to ± The ATP hydrolysis rate at in the of beads ± to ± that the the kinesin hydrolysis rate in by the rate at which kinesin moves along a and by for The of kinesin and stoichiometry were for this the to and the speed of kinesin adsorbed to a silica by maximum hydrolysis rate of ± and ± for the and ± for the the to by to the of in the ATPase in the motility for this and by the of 8.1 stoichiometry of 1.08 ± 0.09 hydrolyzed the by the of The is the in the the ATPase the kinesin the and the of in the motility and ATPase the to of the stoichiometry the of the of the kinesin this the of kinesin the of that ATP by each kinesin molecule We that kinesin to the silica beads the of of the kinesin adsorbed to the beads a for binding of the adsorbed were by microtubules to Therefore, the of kinesin to the beads is a of the to and to to is that the of is the of the motility is the of the that the beads along the that kinesin between and a the of of this were the the that of a the ATPase rate the ATPase rate of the and the stoichiometry a of that the of in a is the were have beads binding to the microtubules for this of beads to the the were a that the of the to the microtubules at tubulin the are to beads in this is to Drosophila kinesin in to that the for the ATPase is to that for at tubulin the are to the the of of a of the beads along microtubules adsorbed to the of the by the of the the of the beads for The distance each of a along a to at the of the were a the of beads have of are to a kinesin The of to that by the the to in the of are to for kinesin to the at that that each kinesin have a of rate at which the beads to the microtubules the of The rate of at the that the of a binding to a a at a of is that of a that a of the adsorbed are the under that the adsorbed to beads to speed at which beads along microtubules of the in and The speed ± ± for this at The of the beads along microtubules adsorbed to the of the by the of the the of the beads for The distance each of a along a to at the of the were a the of beads have of are to a kinesin The of to that by the the to in the of are to for kinesin to the at that that each kinesin have a of The rate at which the beads to the microtubules the of The rate of at the that the of a binding to a a at a of is that of a that a of the adsorbed are the under that the adsorbed to beads to The speed at which beads along microtubules of the in and The speed ± ± for this at ATPase rate of ATP hydrolysis by kinesin to the 200-nm-diameter beads a The that of the beads kinesin to kinesin The hydrolysis rate the and the were the the motility is in and the hydrolysis rate to ± molecule ± The hydrolysis rate at a tubulin of ± ± were kinesin kinesin are that have the ATPase a a of of kinesin To a are each to the a kinesin molecule to the and the ATP hydrolysis molecule a of of kinesin are to beads at kinesin the ATP hydrolysis at tubulin of ± 1 to the rate at a of ± 1 that kinesin a by that the ATPase rate is the of ATP hydrolysis the of ATP and the of the hydrolysis To this of the ATP hydrolyzed the of To the of a low of the hydrolysis assayed and ATPase in the of the speed ± to ± The ATP hydrolysis rate at in the of beads ± to ± that the the kinesin hydrolysis rate in by the rate at which kinesin moves along a and by for The of kinesin and stoichiometry were for this the to The rate of ATP hydrolysis by kinesin to the 200-nm-diameter beads a The that of the beads kinesin to kinesin The hydrolysis rate the and the were the the motility is in and the hydrolysis rate to ± molecule ± The hydrolysis rate at a tubulin of ± ± were kinesin kinesin are that have the ATPase a a of of kinesin To a are each to the a kinesin molecule to the and the ATP hydrolysis molecule a of of kinesin are to beads at kinesin the ATP hydrolysis at tubulin of ± 1 to the rate at a of ± 1 that kinesin a by that the ATPase rate is the of ATP hydrolysis the of ATP and the of the hydrolysis To this of the ATP hydrolyzed the of To the of a low of the hydrolysis assayed and ATPase in the of the speed ± to ± The ATP hydrolysis rate at in the of beads ± to ± that the the kinesin hydrolysis rate in by the rate at which kinesin moves along a and by for The of kinesin and stoichiometry were for this the to and the speed of kinesin adsorbed to a silica by maximum hydrolysis rate of ± and ± for the and ± for the the to by to the of in the ATPase in the motility for this and by the of 8.1 stoichiometry of 1.08 ± 0.09 hydrolyzed the by the of The is the in the the ATPase the kinesin the and the of in the motility and ATPase the to of the stoichiometry the of the of the kinesin this the of kinesin the of that ATP by each kinesin molecule We that kinesin to the silica beads the of of the kinesin adsorbed to the beads a for binding of the adsorbed were by microtubules to Therefore, the of kinesin to the beads is a of the to and to to is that the of is the of the motility is the of the that the beads along the that kinesin between and a the of of this were the the that of a the ATPase rate the ATPase rate of the and the stoichiometry a of that the of in a is the were have beads binding to the microtubules for this of beads to the the were a that the of the to the microtubules at tubulin the are to beads in this is to Drosophila kinesin in to that the for the ATPase is to that for at tubulin the are to the the of of a the speed of kinesin adsorbed to a silica by maximum hydrolysis rate of ± and ± for the and ± for the the to by to the of in the ATPase in the motility for this and by the of 8.1 stoichiometry of 1.08 ± 0.09 hydrolyzed the by the of The is the in the the ATPase the kinesin the and the of in the motility and ATPase the to of the stoichiometry the of the of the kinesin this the of kinesin the of that ATP by each kinesin molecule We that kinesin to the silica beads the of of the kinesin adsorbed to the beads a for binding of the adsorbed were by microtubules to Therefore, the of kinesin to the beads is a of the to and to to is that the of is the of the motility is the of the that the beads along the that kinesin between and a the of of this were the the that of a the ATPase rate the ATPase rate of the and the stoichiometry a of that the of in a is the were have beads binding to the microtubules for this of beads to the the were a that the of the to the microtubules at tubulin the are to beads in this is to Drosophila kinesin in to that the for the ATPase is to that for at tubulin the are to the the of of a the speed of of silica beads under the of kinesin by the ATP hydrolysis rate of the beads under the have that kinesin 1.08 ± 0.09 ± steps for each molecule of ATP that it is a stoichiometry of were of kinesin ATP molecule kinesin steps is ATP of at ATP that are of steps the were Therefore, the that the is ATP hydrolysis in a along a the low in the between motility and ATP hydrolysis is a stoichiometry of 1.08 ± 0.09 that at of the hydrolysis a 1.08 0.09 The in is the drag force a at 1 is in which is of the maximum force of that a kinesin molecule the at is stoichiometry of 1 is the and that a between the and of a motor and of that the in the that steps powered by ATP the speed of of silica beads under the of kinesin by the ATP hydrolysis rate of the beads under the have that kinesin 1.08 ± 0.09 ± steps for each molecule of ATP that it is a stoichiometry of were of kinesin ATP molecule kinesin steps is ATP of at ATP that are of steps the were Therefore, the that the is ATP hydrolysis in a along a the low in the between motility and ATP hydrolysis is a stoichiometry of 1.08 ± 0.09 that at of the hydrolysis a 1.08 0.09 The in is the drag force a at 1 is in which is of the maximum force of that a kinesin molecule the at is stoichiometry of 1 is the and that a between the and of a motor and of that the in the that steps powered by ATP We and for of the