Wirth Jan O, Scheiderer Lukas, Engelhardt Tobias, Engelhardt Johann, Matthias Jessica, Hell Stefan W
Department of Optical Nanoscopy, Max Planck Institute for Medical Research, Heidelberg, Germany.
Department of NanoBiophotonics, Max Planck Institute for Multidisciplinary Sciences, Göttingen, Germany.
Science. 2023 Mar 10;379(6636):1004-1010. doi: 10.1126/science.ade2650. Epub 2023 Mar 9.
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.
我们介绍了一种干涉式MINFLUX显微镜，它能够以高达每毫秒1.7纳米的时空精度记录蛋白质的运动。此前，要达到这样的精度需要在蛋白质上附着不成比例的大珠子，但MINFLUX只需要检测来自一个约1纳米大小荧光团的约20个光子。因此，我们能够在接近生理浓度的腺苷-5'-三磷酸（ATP）条件下研究驱动蛋白-1在微管上的步移。我们发现无负载驱动蛋白在步移过程中其柄部和头部会发生旋转，并表明ATP在一个头部与微管结合时被摄取，而ATP水解发生在两个头部都与微管结合时。我们的结果表明，MINFLUX能够在最小干扰的情况下量化蛋白质（亚）毫秒级的构象变化。
