Abstract
The ncd protein is a dimeric, ATP-powered motor that belongs to the kinesin family of microtubule motor proteins. Here we resolve single mechanochemical cycles of recombinant, dimeric, full-length ncd, using optical-tweezers-based instrumentation and a three-bead, suspended-microtubule assay. Under conditions of limiting ATP, isolated and transient microtubule-binding events exhibit exponentially distributed and ATP-concentration-dependent lifetimes. These events do not involve consecutive steps along the microtubule, quantitatively confirming that ncd is non-processive. At low loads, a single motor molecule produces ATP-triggered working strokes of about 9 nm, which occur at the ends of binding events.
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Acknowledgements
We thank S. Shoham and D. Warren for discussion of data analysis and wavelet filtering; and M. Allersma, W. Moehler and F. Gittes for help with experiments and data analysis. We also thank the Rowland Institute for Science for generous technical support. This work was supported by a predoctoral fellowship from the Whitaker Foundation (to M.J.d.), and grants from the NSF (BIR9512699 and CTS-9624907) and the NIH (1R55GM55679-01).
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Correspondence should be addressed to R. J. S. or C. F. S. Requests for materials should be addressed to R. J. S.
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deCastro, M., Fondecave, R., Clarke, L. et al. Working strokes by single molecules of the kinesin-related microtubule motor ncd. Nat Cell Biol 2, 724–729 (2000). https://doi.org/10.1038/35036357
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DOI: https://doi.org/10.1038/35036357
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