Asenjo Ana B, Sosa Hernando
Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Proc Natl Acad Sci U S A. 2009 Apr 7;106(14):5657-62. doi: 10.1073/pnas.0808355106. Epub 2009 Mar 25.
Kinesin1 is a motor protein that uses the energy from ATP hydrolysis to move intracellular cargoes along microtubules. It contains 2 identical motor domains, or heads, that coordinate their mechano-chemical cycles to move processively along microtubules. The molecular mechanism of coordination between head domains remains unclear, partly because of the lack of structural information on critical intermediates of the kinesin1 mechano-chemical cycle. A point of controversy has been whether before ATP binding, in the so called ATP-waiting state, 1 or 2 motor domains are bound to the microtubule. To address this issue, here we use ensemble and single molecule fluorescence polarization microscopy (FPM) to determine the mobility and orientation of the kinesin1 heads at different ATP concentrations and in heterodimeric constructs with microtubule binding impaired in 1 head. We found evidence for a mobile head during the ATP-waiting state. We incorporate our results into a model for kinesin translocation that accounts well for many reported experimental results.
驱动蛋白1是一种马达蛋白，它利用ATP水解产生的能量沿着微管移动细胞内的货物。它包含2个相同的马达结构域，即头部，它们协调其机械化学循环以沿着微管持续移动。头部结构域之间协调的分子机制仍不清楚，部分原因是缺乏驱动蛋白1机械化学循环关键中间体的结构信息。一个有争议的问题是，在ATP结合之前，即在所谓的ATP等待状态下，是1个还是2个马达结构域与微管结合。为了解决这个问题，我们在这里使用整体和单分子荧光偏振显微镜（FPM）来确定在不同ATP浓度下以及在一个头部微管结合受损的异二聚体构建体中驱动蛋白1头部的迁移率和取向。我们发现了ATP等待状态下存在一个可移动头部的证据。我们将我们的结果纳入一个驱动蛋白易位模型，该模型很好地解释了许多已报道的实验结果。
