MINFLUX dissects the unimpeded walking of kinesin-1

We introduce an interferometric MINFLUX microscope that records protein movements with up to 1.7 nanometer per millisecond spatiotemporal precision. Such precision has previously required attaching disproportionately large beads to the protein, but MINFLUX requires the detection of only about 20 photons from an approximately 1-nanometer-sized fluorophore. Therefore, we were able to study the stepping of the motor protein kinesin-1 on microtubules at up to physiological adenosine-5 ' triphosphate (ATP) concentrations. We uncovered rotations of the stalk and the heads of load-free kinesin during stepping and showed that ATP is taken up with a single head bound to the microtubule and that ATP hydrolysis occurs when both heads are bound. Our results show that MINFLUX quantifies (sub)millisecond conformational changes of proteins with minimal disturbance.

Automated summary

We introduce an interferometric MINFLUX microscope that can record protein movements with up to 1.7 nanometer per millisecond spatiotemporal precision. It only requires the detection of about 20 photons from a 1-nanometer-sized fluorophore, eliminating the need for attaching large beads to the protein. With this microscope, we were able to study the stepping of kinesin-1 on microtubules at physiological ATP concentrations, revealing rotations of the stalk and heads of the protein during stepping.