Dissection of the angle of single fluorophore attached to the nucleotide in corkscrewing microtubules
Dissection of the angle of single fluorophore attached to the nucleotide in corkscrewing microtubules
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DOI：
10.1016/j.bbrc.2017.01.165
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发表时间：
2017-04-08
影响因子：
3.1
通讯作者：
Nishizaka, Takayuki
中科院分区：
文献类型：
--
作者：
Fujimura, Shoko;Ito, Yuko;Nishizaka, Takayuki
在光学显微镜下直接解剖单个荧光团的角度一直是研究水溶液中蛋白质动力学的一种具有挑战性的方法。然而，对于单一底物的角度定量，由于难以构建具有单分子水平活性蛋白的实验系统，只有一篇报道（Nishizaka et al., 2014）。我们在这里展示了结合到构成微管的单个小管的单个荧光核苷酸的定向的精确估计。当固定在玻璃表面的单头动力蛋白驱动微管滑动时，微管呈现出规则螺距的螺旋状（Yajima等人，2005 & 2008）。通过三维跟踪显微镜，我们发现S8A突变体的运动蛋白也表现出精确的螺旋状，其螺距为330米，比野生型长13%。突变体的检测与散焦成像技术相结合，以可视化与螺旋微管结合的荧光核苷酸的旋转行为。值得注意的是，单个TAMRA-GTP的散焦模式周期性变化，与它的岁差运动精确相关。通过荧光团在x平面上的角度变化的时间过程，既可以估计荧光团对微管轴的取向，也可以估计分析系统定角的精度。取向以-40、- 50、- 60峰为主。为了确定它们的分子构象，通过将x射线晶体结构拟合到低温电子显微镜图上，使用原子级结构进行了严格的对接模拟。在同分异构体中，以5-或6-TAMRA标记的2'- o - eda - gdp主要被指定为可能的底物，这表明TAMRA-GTP被微管蛋白水解。(C) 2017爱思唯尔公司版权所有。
Direct dissection of the angles of single fluorophores under an optical microscope has been a challenging approach to study the dynamics of proteins in an aqueous solution. For angle quantifications of single substrates, however, there was only one report (Nishizaka et al., 2014) because of difficulties of construction of experimental systems with active proteins working at the single-molecule level. We here show precise estimation of orientation of single fluorescent nucleotides bound to single tubulins that comprise microtubule. When single -headed kinesins immobilized on a glass surface drive the sliding of microtubules, microtubules show corkscrewing with regular pitches (Yajima et al., 2005 & 2008). We found, by using a three-dimensional tracking microscope, that S8A mutant kinesin also showed precise corkscrewing with a 330-m pitch, which is 13% longer than that of the wild type. The assay with the mutant was combined with a defocused imaging technique to visualize the rotational behavior of fluorescent nucleotide bound to corkscrewing microtubule. Notably, the defocused pattern of single TAMRA-GTP periodically changed, precisely correlating to its precession movement. The time course of the change in the fluorophore angle projected to the xy-plane enabled to estimate both the fluorophore orientation against microtubule axis and the precision of angle-determination of analyses system. The orientation showed main distribution with peaks at -40, 50 and 60. To identify their molecular conformations, the rigorous docking simulations were performed using an atomic-level structure modeled by fitting x-ray crystal structures to the cryo-electron microscopy map. Among isomers, 2'-O-EDA-GDP labeled with 5- or 6-TAMRA were mainly specified as possible candidates as a substrate, which suggested the hydrolysis of TAMRA-GTP by tubulins. (C) 2017 Elsevier Inc. All rights reserved.