Verbrugge Sander, Lansky Zdenek, Peterman Erwin J G
Department of Physics and Astronomy and Laser Centre, VU University, de Boelelaan 1081, 1081 HV, Amsterdam, The Netherlands.
Proc Natl Acad Sci U S A. 2009 Oct 20;106(42):17741-6. doi: 10.1073/pnas.0905177106. Epub 2009 Oct 1.
The motor protein Kinesin-1 drives intracellular transport along microtubules, with each of its two motor domains taking 16-nm steps in a hand-over-hand fashion. The way in which a single-motor domain moves during a step is unknown. Here, we use Förster resonance energy transfer (FRET) between fluorescent labels on both motor domains of a single kinesin. This approach allows us to resolve the relative distance between the motor domains and their relative orientation, on the submillisecond timescale, during processive stepping. We observe transitions between high and low FRET values for certain kinesin constructs, depending on the location of the labels. These results reveal that, during a step, a kinesin motor domain dwells in a well-defined intermediate position for approximately 3 ms.
驱动蛋白-1这种运动蛋白沿着微管推动细胞内运输，其两个运动结构域中的每一个都以交替的方式迈出16纳米的步长。单个运动结构域在一步过程中的移动方式尚不清楚。在这里，我们利用单个驱动蛋白两个运动结构域上荧光标记之间的荧光共振能量转移（FRET）。这种方法使我们能够在亚毫秒时间尺度上解析运动结构域之间的相对距离及其相对取向，在持续步移过程中。我们观察到某些驱动蛋白构建体的FRET值在高和低之间转换，这取决于标记的位置。这些结果表明，在一步过程中，驱动蛋白运动结构域会在一个明确的中间位置停留约3毫秒。
