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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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Nanometry of single kinesin molecules. (a) The optical trapping nanometry system (not to scale). The kinesin-coated bead captured by the optical tweezers was used as a probe to measure the kinesin displacement. (b) A typical displacement record by a single kinesin molecule at saturating ([ATPsat] = 1 mM) and limiting ([ATPlim] = 10 μM) ATP concentrations. The external load, calculated from the trap stiffness (0.05 pN nm−1), is indicated on the right.
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f1-4_11: Nanometry of single kinesin molecules. (a) The optical trapping nanometry system (not to scale). The kinesin-coated bead captured by the optical tweezers was used as a probe to measure the kinesin displacement. (b) A typical displacement record by a single kinesin molecule at saturating ([ATPsat] = 1 mM) and limiting ([ATPlim] = 10 μM) ATP concentrations. The external load, calculated from the trap stiffness (0.05 pN nm−1), is indicated on the right.

Mentions: Optical trapping nanometry was used to directly detect mechanical steps of single kinesin molecules along microtubules12,13 (Fig. 1a). Typical recordings predictably showed a distinctive 8-nm stepwise pattern in the forward and backward directions (Fig. 1b). When the ATP concentration decreased from a saturating concentration ([ATPsat] = 1 mM) to a limiting concentration ([ATPlim] = 10 μM), the velocity of the movement decreased as reported in previous studies6,9. In addition, the trapping force acted as an external load against kinesin movements. As the load increased, the stepping speed decreased and backward steps occurred more frequently.


The forward and backward stepping processes of kinesin are gated by ATP binding
Nanometry of single kinesin molecules. (a) The optical trapping nanometry system (not to scale). The kinesin-coated bead captured by the optical tweezers was used as a probe to measure the kinesin displacement. (b) A typical displacement record by a single kinesin molecule at saturating ([ATPsat] = 1 mM) and limiting ([ATPlim] = 10 μM) ATP concentrations. The external load, calculated from the trap stiffness (0.05 pN nm−1), is indicated on the right.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036606&req=5

f1-4_11: Nanometry of single kinesin molecules. (a) The optical trapping nanometry system (not to scale). The kinesin-coated bead captured by the optical tweezers was used as a probe to measure the kinesin displacement. (b) A typical displacement record by a single kinesin molecule at saturating ([ATPsat] = 1 mM) and limiting ([ATPlim] = 10 μM) ATP concentrations. The external load, calculated from the trap stiffness (0.05 pN nm−1), is indicated on the right.
Mentions: Optical trapping nanometry was used to directly detect mechanical steps of single kinesin molecules along microtubules12,13 (Fig. 1a). Typical recordings predictably showed a distinctive 8-nm stepwise pattern in the forward and backward directions (Fig. 1b). When the ATP concentration decreased from a saturating concentration ([ATPsat] = 1 mM) to a limiting concentration ([ATPlim] = 10 μM), the velocity of the movement decreased as reported in previous studies6,9. In addition, the trapping force acted as an external load against kinesin movements. As the load increased, the stepping speed decreased and backward steps occurred more frequently.

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