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ADP-induced rocking of the kinesin motor domain revealed by single-molecule fluorescence polarization microscopy

Nature Structural Biology volume 8, pages 540–544 (2001)Cite this article

Abstract

Kinesin is an ATP-driven molecular motor protein that moves processively along microtubules. Despite considerable research, the detailed mechanism of kinesin motion remains elusive. We applied an enhanced suite of single- and multiple-molecule fluorescence polarization microscopy assays to report the orientation and mobility of kinesin molecules bound to microtubules as a function of nucleotide state. In the presence of analogs of ATP, ADP-Pi or in the absence of nucleotide, the kinesin head maintains a rigid orientation. In the presence of ADP, the motor domain of kinesin, still bound to the microtubule, adopts a previously undescribed, highly mobile state. This state may be general to the chemomechanical cycle of motor proteins; in the case of kinesin, the transition from a highly mobile to a rigid state after ADP release may contribute to the generation of the 8 nm step.

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Figure 1: Probe location and orientation.
Figure 2: Fluorescence polarization measurements of axonemes decorated with KMC2–BSR.
Figure 3: Fluorescence polarization from single molecules.
Figure 4: Proposed structural states of kinesin.

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Acknowledgements

We thank R. Sakowicz for axoneme preparations, advice and discussions; R. Dickson, R. Vale and D. Pierce for their advice in the initial phases of this project; L. Gross, H. Deng and L. Siconolfi-Baez for mass spectrometry analysis; A. Asenjo for biochemical assays, and S. Brasselet and B. Lounis for helpful discussions and experimental suggestions. L.S.B. Goldstein is an investigator of the Howard Hughes Medical Institute. This project was supported by NSF grants.

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Author notes

  1. Hernando Sosa

    Present address: Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York, 10461, USA

  2. Erwin J.G. Peterman

    Present address: Division of Physics and Astronomy, Vrije Universiteit, Amsterdam, The Netherlands

Authors and Affiliations

  1. Department of Cellular and Molecular Medicine and Howard Hughes Medical Institute, University of California San Diego, La Jolla, 92093-0683, California, USA

    Hernando Sosa & Lawrence S.B. Goldstein

  2. Department of Chemistry, Stanford University, Stanford, 94305-5080, California, USA

    Erwin J.G. Peterman & W.E. Moerner

  3. Department of Pharmacology, University of California San Diego, La Jolla, 92093-0683, California, USA

    Lawrence S.B. Goldstein

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  1. Hernando Sosa
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  3. W.E. Moerner
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  4. Lawrence S.B. Goldstein
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Correspondence to Hernando Sosa.

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Sosa, H., Peterman, E., Moerner, W. et al. ADP-induced rocking of the kinesin motor domain revealed by single-molecule fluorescence polarization microscopy. Nat Struct Mol Biol 8, 540–544 (2001). https://doi.org/10.1038/88611

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