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bioRxiv · 10.1101/2022.07.25.501426

MINFLUX dissects the unimpeded walking of kinesin-1

Wolff, J. O.·Scheiderer, L.·Engelhardt, T.·Engelhardt, J.·Matthias, J.·Hell, S. W.
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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.

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Wolff, J. O., Scheiderer, L., Engelhardt, T., Engelhardt, J., Matthias, J., Hell, S. W.. 2022-07-25. MINFLUX dissects the unimpeded walking of kinesin-1. https://doi.org/10.1101/2022.07.25.501426

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