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The forward and backward stepping processes of kinesin are gated by ATP binding

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ABSTRACT

The kinesin motor converts the chemical energy from ATP turnover into mechanical work, which produces successive 8-nm steps in the forward and backward direction along a microtubule. A key problem for kinesin mechanochemistry is explaining how ATP turnover is coordinated with mechanical work. We investigated this by measuring the ATP dependent properties of kinesin forward and backward steps using optical trapping nanometry. The results showed that the rate for both forward and backward steps are ATP-dependent, indicating that ATP binding to kinesin triggers both forward and backward steps. This suggests that ATP turnover in kinesin is not rigidly coupled to total mechanical work at high load.

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Kinetic analysis of kinesin steps. (a) Kinetic pathway of kinesin steps. Forward steps, backward steps, or detachments occur by passing through a sequence of load-independent transitions (state 1 to state 2) and a load-dependent transition (state 2 to forward step, backward step or detachment). (b) Load-dependence of rate constants (mean±s.e.m) for the forward steps (kf, blue), backward steps (kb, red) and detachments (kd, green) at different [ATP]s. The lines are best fits to equation (1). All kinetic parameters determined are listed in Table 1. The dotted lines in the right ([ATPlim]) are the data from the left ([ATPsat]) and are included for comparison.
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f3-4_11: Kinetic analysis of kinesin steps. (a) Kinetic pathway of kinesin steps. Forward steps, backward steps, or detachments occur by passing through a sequence of load-independent transitions (state 1 to state 2) and a load-dependent transition (state 2 to forward step, backward step or detachment). (b) Load-dependence of rate constants (mean±s.e.m) for the forward steps (kf, blue), backward steps (kb, red) and detachments (kd, green) at different [ATP]s. The lines are best fits to equation (1). All kinetic parameters determined are listed in Table 1. The dotted lines in the right ([ATPlim]) are the data from the left ([ATPsat]) and are included for comparison.

Mentions: We further analyzed the directionality of stepping by examining the propagation of forward steps, backward steps and detachments as a function of load and [ATP] (Fig. 2c). The proportion of forward steps exponentially decreased from almost 1 at zero load to 0.5 at about 7–8 pN, while both backward steps and detachments increased. We obtained similar results for all ATP concentrations studied. Attributing the load-dependence of stepping direction to the load dependent transition14 (Fig. 3a), the rate constants for forward steps (kf), backward steps (kb) and detachments (kd) can be given by: kζ = pζ/τm / (ζ=f, b, d), where pζ are the proportion of forward steps (f), backward steps (b) and detachments (d), and τm is the time constant of the load-dependent mechanical transition.


The forward and backward stepping processes of kinesin are gated by ATP binding
Kinetic analysis of kinesin steps. (a) Kinetic pathway of kinesin steps. Forward steps, backward steps, or detachments occur by passing through a sequence of load-independent transitions (state 1 to state 2) and a load-dependent transition (state 2 to forward step, backward step or detachment). (b) Load-dependence of rate constants (mean±s.e.m) for the forward steps (kf, blue), backward steps (kb, red) and detachments (kd, green) at different [ATP]s. The lines are best fits to equation (1). All kinetic parameters determined are listed in Table 1. The dotted lines in the right ([ATPlim]) are the data from the left ([ATPsat]) and are included for comparison.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036606&req=5

f3-4_11: Kinetic analysis of kinesin steps. (a) Kinetic pathway of kinesin steps. Forward steps, backward steps, or detachments occur by passing through a sequence of load-independent transitions (state 1 to state 2) and a load-dependent transition (state 2 to forward step, backward step or detachment). (b) Load-dependence of rate constants (mean±s.e.m) for the forward steps (kf, blue), backward steps (kb, red) and detachments (kd, green) at different [ATP]s. The lines are best fits to equation (1). All kinetic parameters determined are listed in Table 1. The dotted lines in the right ([ATPlim]) are the data from the left ([ATPsat]) and are included for comparison.
Mentions: We further analyzed the directionality of stepping by examining the propagation of forward steps, backward steps and detachments as a function of load and [ATP] (Fig. 2c). The proportion of forward steps exponentially decreased from almost 1 at zero load to 0.5 at about 7–8 pN, while both backward steps and detachments increased. We obtained similar results for all ATP concentrations studied. Attributing the load-dependence of stepping direction to the load dependent transition14 (Fig. 3a), the rate constants for forward steps (kf), backward steps (kb) and detachments (kd) can be given by: kζ = pζ/τm / (ζ=f, b, d), where pζ are the proportion of forward steps (f), backward steps (b) and detachments (d), and τm is the time constant of the load-dependent mechanical transition.

View Article: PubMed Central - PubMed

ABSTRACT

The kinesin motor converts the chemical energy from ATP turnover into mechanical work, which produces successive 8-nm steps in the forward and backward direction along a microtubule. A key problem for kinesin mechanochemistry is explaining how ATP turnover is coordinated with mechanical work. We investigated this by measuring the ATP dependent properties of kinesin forward and backward steps using optical trapping nanometry. The results showed that the rate for both forward and backward steps are ATP-dependent, indicating that ATP binding to kinesin triggers both forward and backward steps. This suggests that ATP turnover in kinesin is not rigidly coupled to total mechanical work at high load.

No MeSH data available.


Related in: MedlinePlus