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MINFLUX dissects the unimpeded walking of kinesin-1

Jan O. Wolff, Lukas Scheiderer, Tobias Engelhardt, Johann Engelhardt, View ORCID ProfileJessica Matthias, View ORCID ProfileStefan W. Hell
doi: https://doi.org/10.1101/2022.07.25.501426
Jan O. Wolff
1Department of Optical Nanoscopy, Max Planck Institute for Medical Research, Heidelberg, Germany
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Lukas Scheiderer
1Department of Optical Nanoscopy, Max Planck Institute for Medical Research, Heidelberg, Germany
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Tobias Engelhardt
1Department of Optical Nanoscopy, Max Planck Institute for Medical Research, Heidelberg, Germany
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Johann Engelhardt
1Department of Optical Nanoscopy, Max Planck Institute for Medical Research, Heidelberg, Germany
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Jessica Matthias
1Department of Optical Nanoscopy, Max Planck Institute for Medical Research, Heidelberg, Germany
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Stefan W. Hell
1Department of Optical Nanoscopy, Max Planck Institute for Medical Research, Heidelberg, Germany
2Department of NanoBiophotonics, Max Planck Institute for Multidisciplinary Sciences, Göttingen, Germany
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  • ORCID record for Stefan W. Hell
  • For correspondence: shell@gwdg.de
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Abstract

We report on an interferometric MINFLUX microscope that records protein movements with down to 1.7 nm precision within less than 1 ms. While such spatio-temporal resolution has so far required linking a strongly scattering 30-500 nm diameter bead to the much smaller protein, MINFLUX localization requires the detection of only down to 20 photons from an ~1-nm sized fluorophore. Harnessing this resolution, we dissect the unhindered stepping of the motor protein kinesin-1 on microtubules at up to physiological ATP concentrations. By attaching the fluorophore to different kinesin-1 sites and resolving steps and substeps of these protein constructs, we uncover a three-dimensional orientation change of the unbound kinesin head. We also find that kinesin-1 takes up ATP while only one head is bound, whereas hydrolysis of ATP occurs with both heads bound to the microtubule, resolving a long-standing conundrum of its mechanochemical cycle. Our results establish MINFLUX as a non-invasive tool for tracking protein movements and probing submillisecond structural rearrangements with nanometer resolution.

Competing Interest Statement

SWH is a co-founder of the company Abberior Instruments which commercializes MINFLUX microscopes. SWH also holds patents on the principles, embodiments and procedures of MINFLUX through the Max Planck Society. TEs current address is Abberior Instruments GmbH, Heidelberg, Germany.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-ND 4.0 International license.
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Posted July 25, 2022.
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MINFLUX dissects the unimpeded walking of kinesin-1
Jan O. Wolff, Lukas Scheiderer, Tobias Engelhardt, Johann Engelhardt, Jessica Matthias, Stefan W. Hell
bioRxiv 2022.07.25.501426; doi: https://doi.org/10.1101/2022.07.25.501426
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MINFLUX dissects the unimpeded walking of kinesin-1
Jan O. Wolff, Lukas Scheiderer, Tobias Engelhardt, Johann Engelhardt, Jessica Matthias, Stefan W. Hell
bioRxiv 2022.07.25.501426; doi: https://doi.org/10.1101/2022.07.25.501426

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