Abstract
Proteins of the kinesin superfamily share a conserved motor domain, which both hydrolyses adenosine-5′-triphosphate (ATP) and binds microtubules. To determine the mechanism of action of a kinesin, it is necessary to relate the chemical cycle of ATP turnover to the mechanics of microtubule interaction. In this chapter, a number of methods are outlined by which the ATP turnover cycle of a kinesin can be analysed with a particular focus on the use of fluorescently labelled ATP and ADP analogues as a means of isolating individual steps in the cycle. By analysing the ATP turnover cycle of a kinesin, both in solution and in the presence of microtubules, the change in nucleotide state triggered upon microtubule binding can be determined. This provides information vital to understanding the coupling of the chemical and mechanical cycles that is integral to the action of members of the kinesin superfamily.
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Acknowledgements
We acknowledge with thanks Roger Goody and Aymelt Itzen for assistance in developing the protocol described in Subheading 3.2.2, and also Christopher Gell for critical reading of the manuscript. This work was supported by the Alexander Von Humboldt Foundation and the Max Planck Society.
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Friel, C.T., Bagshaw, C.R., Howard, J. (2011). Analysing the ATP Turnover Cycle of Microtubule Motors. In: Straube, A. (eds) Microtubule Dynamics. Methods in Molecular Biology, vol 777. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-252-6_13
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DOI: https://doi.org/10.1007/978-1-61779-252-6_13
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