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

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.


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Dwell time and step directionality. (a) Histogram of dwell times between steps at different [ATP]s. Inset, an expansion of the histogram around a shorter time range. The curves are one (light blue) or two (pink) rate-limiting transition scheme fits. (b) Load-dependence of the time constants (mean±s.e.m) obtained from the dwell time distribution for forward steps (blue), backward steps (red) and detachments (green). The time constants obtained for the slow decay phase of the histogram (τm, closed symbols) and for the fast rise phase (τc, open symbols) were plotted. (c) Load dependence of the proportion (mean±s.e.m) of forward steps (pf, blue), backward steps (pb, red) and detachments (pd, green). The curves are calculated as: pf= kf/(kf+ kb+ kd), pb= kb/(kf+ kb+ kd) and pd= kd/(kf+ kb+ kd) using the kinetic parameters summarized in Table 1.
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f2-4_11: Dwell time and step directionality. (a) Histogram of dwell times between steps at different [ATP]s. Inset, an expansion of the histogram around a shorter time range. The curves are one (light blue) or two (pink) rate-limiting transition scheme fits. (b) Load-dependence of the time constants (mean±s.e.m) obtained from the dwell time distribution for forward steps (blue), backward steps (red) and detachments (green). The time constants obtained for the slow decay phase of the histogram (τm, closed symbols) and for the fast rise phase (τc, open symbols) were plotted. (c) Load dependence of the proportion (mean±s.e.m) of forward steps (pf, blue), backward steps (pb, red) and detachments (pd, green). The curves are calculated as: pf= kf/(kf+ kb+ kd), pb= kb/(kf+ kb+ kd) and pd= kd/(kf+ kb+ kd) using the kinetic parameters summarized in Table 1.

Mentions: To examine the ATP-dependent kinetics of the kinesin steps, the distribution of dwell time between steps was analyzed. The distribution showed an exponential decay with the rise phase occurring over a short time range (Fig. 2a). The distributions was fitted significantly better to a two rate-limiting transition scheme (χ2(98)=90.1, P>0.1, at 10 μM ATP and 5–7 pN; for example, see Fig. 2a) than a one rate-limiting transition scheme (χ2(98)=170.3, P<0.005, for the same data set). Similar results were obtained from the dwell-time distributions for all loads and ATP concentrations. We obtained time constants corresponding to two transitions from the fitting. The results showed that one time constant increased with load while the other remained constant (Fig. 2b). This indicates that one of the two rate-limiting transitions is a load-dependent mechanical transition and the other is a load-independent biochemical transition14,15. In addition, a decrease in the ATP concentration did not affect the load-independent transition but did make the load-dependent transition slower. This indicates that the load-dependent transition originates in the ATP binding reaction. Furthermore, these time constants were unchanged even if the analyzed data was limited to just one of forward steps, backward steps or detachments (Fig. 2b), indicating that these three events occured in parallel.


The forward and backward stepping processes of kinesin are gated by ATP binding
Dwell time and step directionality. (a) Histogram of dwell times between steps at different [ATP]s. Inset, an expansion of the histogram around a shorter time range. The curves are one (light blue) or two (pink) rate-limiting transition scheme fits. (b) Load-dependence of the time constants (mean±s.e.m) obtained from the dwell time distribution for forward steps (blue), backward steps (red) and detachments (green). The time constants obtained for the slow decay phase of the histogram (τm, closed symbols) and for the fast rise phase (τc, open symbols) were plotted. (c) Load dependence of the proportion (mean±s.e.m) of forward steps (pf, blue), backward steps (pb, red) and detachments (pd, green). The curves are calculated as: pf= kf/(kf+ kb+ kd), pb= kb/(kf+ kb+ kd) and pd= kd/(kf+ kb+ kd) using the kinetic parameters summarized in Table 1.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036606&req=5

f2-4_11: Dwell time and step directionality. (a) Histogram of dwell times between steps at different [ATP]s. Inset, an expansion of the histogram around a shorter time range. The curves are one (light blue) or two (pink) rate-limiting transition scheme fits. (b) Load-dependence of the time constants (mean±s.e.m) obtained from the dwell time distribution for forward steps (blue), backward steps (red) and detachments (green). The time constants obtained for the slow decay phase of the histogram (τm, closed symbols) and for the fast rise phase (τc, open symbols) were plotted. (c) Load dependence of the proportion (mean±s.e.m) of forward steps (pf, blue), backward steps (pb, red) and detachments (pd, green). The curves are calculated as: pf= kf/(kf+ kb+ kd), pb= kb/(kf+ kb+ kd) and pd= kd/(kf+ kb+ kd) using the kinetic parameters summarized in Table 1.
Mentions: To examine the ATP-dependent kinetics of the kinesin steps, the distribution of dwell time between steps was analyzed. The distribution showed an exponential decay with the rise phase occurring over a short time range (Fig. 2a). The distributions was fitted significantly better to a two rate-limiting transition scheme (χ2(98)=90.1, P>0.1, at 10 μM ATP and 5–7 pN; for example, see Fig. 2a) than a one rate-limiting transition scheme (χ2(98)=170.3, P<0.005, for the same data set). Similar results were obtained from the dwell-time distributions for all loads and ATP concentrations. We obtained time constants corresponding to two transitions from the fitting. The results showed that one time constant increased with load while the other remained constant (Fig. 2b). This indicates that one of the two rate-limiting transitions is a load-dependent mechanical transition and the other is a load-independent biochemical transition14,15. In addition, a decrease in the ATP concentration did not affect the load-independent transition but did make the load-dependent transition slower. This indicates that the load-dependent transition originates in the ATP binding reaction. Furthermore, these time constants were unchanged even if the analyzed data was limited to just one of forward steps, backward steps or detachments (Fig. 2b), indicating that these three events occured in parallel.

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