MINFLUX dissects the unimpeded walking of kinesin-1

• 成果类型: Article
• 署名作者: Wolff, Jan O.; Scheiderer, Lukas; Engelhardt, Tobias; Engelhardt, Johann; Matthias, Jessica; Hell, Stefan W.
• 署名单位: Max Planck Society
• 刊物名称: SCIENCE
• ISSN/ISSBN: 0036-9703
• DOI: 10.1126/science.ade2650
• 发表日期: 2023-03-10
• 页码: 1004-1010
• 关键词: hand-over-hand; tracking; microtubules; nucleotide; molecules; MOVEMENT; waits; cycle; head; atp

摘要

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.