Malik F, Brillinger D, Vale R D
Neuroscience Graduate Program, University of California, San Francisco 94143.
Proc Natl Acad Sci U S A. 1994 May 10;91(10):4584-8. doi: 10.1073/pnas.91.10.4584.
Kinesin is a microtubule-based motor protein that contains two identical force-generating subunits. The kinesin binding sites along the microtubule lie 8 nm apart (the dimension of the tubulin dimer), which implies that kinesin must translocate a minimum distance of 8 nm per hydrolysis cycle. Measurements of kinesin's microtubule-stimulated ATPase activity (approximately 20 ATP per sec) and velocity of transport (approximately 0.6 micron/sec), however, suggest that the net distance moved per ATP (approximately 30 nm) may be greater than one tubulin dimer under zero load conditions. To explore how kinesin translocates during its ATPase cycle, we constructed a microscope capable of tracking movement with 1-nm resolution at a bandwidth of 200 Hz and used this device to examine microtubule movement driven by a single kinesin motor. Regular stepwise movements were not observed in displacement traces of moving microtubules, although Brownian forces acting on elastic elements within the kinesin motor precluded detection of steps that were < 12 nm. Though individual steps of approximately 16 nm were occasionally observed, their infrequent occurrence suggests that kinesin rarely moves abruptly by distances of two or more tubulin subunits during its ATP hydrolysis cycle. Instead it is more likely that kinesin moves forward by the distance of only a single tubulin subunit under zero load conditions.
驱动蛋白是一种基于微管的马达蛋白，它包含两个相同的产生力的亚基。沿着微管的驱动蛋白结合位点相距8纳米（微管蛋白二聚体的尺寸），这意味着驱动蛋白每个水解循环必须至少移位8纳米。然而，对驱动蛋白的微管刺激的ATP酶活性（每秒约20个ATP）和运输速度（约0.6微米/秒）的测量表明，在零负载条件下，每个ATP移动的净距离（约30纳米）可能大于一个微管蛋白二聚体。为了探索驱动蛋白在其ATP酶循环过程中如何移位，我们构建了一台显微镜，能够在200赫兹的带宽下以1纳米的分辨率跟踪运动，并使用该设备检查由单个驱动蛋白马达驱动的微管运动。在移动微管的位移轨迹中未观察到规则的逐步运动，尽管作用于驱动蛋白马达内弹性元件的布朗力排除了检测小于12纳米的步长。尽管偶尔观察到约16纳米的单个步长，但它们的不频繁出现表明驱动蛋白在其ATP水解循环中很少突然移动两个或更多微管蛋白亚基的距离。相反，在零负载条件下，驱动蛋白更有可能仅向前移动一个微管蛋白亚基的距离。
