PT  - JOURNAL ARTICLE
AU  - Jan O. Wolff
AU  - Lukas Scheiderer
AU  - Tobias Engelhardt
AU  - Johann Engelhardt
AU  - Jessica Matthias
AU  - Stefan W. Hell
TI  - MINFLUX dissects the unimpeded walking of kinesin-1
AID  - 10.1101/2022.07.25.501426
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.07.25.501426
4099  - http://biorxiv.org/content/early/2022/07/25/2022.07.25.501426.short
4100  - http://biorxiv.org/content/early/2022/07/25/2022.07.25.501426.full
AB  - 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 StatementSWH 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.