Three-color single-molecule imaging reveals conformational dynamics of dynein undergoing motility.
Three-color single-molecule imaging reveals conformational dynamics of dynein undergoing motility.
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DOI：
10.1073/pnas.2101391118
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发表时间：
2021-08-03
影响因子：
11.1
通讯作者：
Vale RD
中科院分区：
文献类型：
--
作者：
Niekamp S;Stuurman N;Zhang N;Vale RD
动力蛋白是一种二聚体马达蛋白，通过5 ' -三磷酸腺苷和热驱动的两个大环结构域（AAA环）和小微管结合结构域（mtbd）的运动沿着微管移动，两个大环结构域（AAA环）和小微管结合结构域（mtbd）被一个长线圈分开。先前的单分子研究追踪了运动过程中AAA环的位置，但没有追踪mtbd。在这里，我们通过三色纳米分辨率成像跟踪了两个mtbd和一个AAA环的相对位置。对两个mtbd的观察提供了动力蛋白如何作用于微管蛋白亚基的直接测量，同时对三个基准标记的观察揭示了运动过程中异常的灵活性和以前未知的运动构象状态。这里介绍的技术可用于探索许多其他大分子复合物的构象动力学。运动蛋白动力蛋白在微管运动过程中，其结构域发生了协调的构象变化。先前的单分子研究分析了动力蛋白同型二聚体的AAA环的运动，但没有分析沿着轨道行进的远端微管结合域（mtbd）。在这里，我们同时以纳米精度跟踪两个mtbd和一个单一动力蛋白的AAA环，因为它经历了数百个步骤，使用三色成像。我们表明，AAA环和mtbd并不总是同时步进，并且可以采取不同大小的步进。这种在AAA环和mtbd之间运动的可变性导致运动过程中意想不到的大量动力蛋白构象状态。提取构象跃迁偏差的数据，我们可以准确地在计算机上模拟动力步进。我们的研究结果表明，主要动力蛋白结构域之间的灵活性对动力蛋白的运动至关重要。
Dynein, a dimeric motor protein, moves along microtubules through adenosine 5′-triphosphate- and thermal-driven motions of two large ring domains (AAA rings) and small microtubule-binding domains (MTBDs) separated by a long coiled-coil. Previous single-molecule studies have tracked the positions of the AAA rings during motility, but not the MTBDs. Here, we tracked the relative positions of both MTBDs and one AAA ring by three-color, nanometer-resolution imaging. The observation of both MTBDs provided a direct measurement of how dynein steps on the tubulin subunits, and the simultaneous observation of three fiducial markers revealed extraordinary flexibility and previously unknown conformational states of the motor during motility. The techniques presented here can be used to explore conformational dynamics of many other macromolecular complexes. The motor protein dynein undergoes coordinated conformational changes of its domains during motility along microtubules. Previous single-molecule studies analyzed the motion of the AAA rings of the dynein homodimer, but not the distal microtubule-binding domains (MTBDs) that step along the track. Here, we simultaneously tracked with nanometer precision two MTBDs and one AAA ring of a single dynein as it underwent hundreds of steps using three-color imaging. We show that the AAA ring and the MTBDs do not always step simultaneously and can take differently sized steps. This variability in the movement between the AAA ring and MTBDs results in an unexpectedly large number of conformational states of dynein during motility. Extracting data on conformational transition biases, we could accurately model dynein stepping in silico. Our results reveal that the flexibility between major dynein domains is critical for dynein motility.
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影响因子：
16.8
作者：
通讯作者：
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DOI：
10.1126/science.1254198
发表时间：
2014-07-18
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
McKenney RJ;Huynh W;Tanenbaum ME;Bhabha G;Vale RD
通讯作者：
Vale RD
直接观察显示沿微管运动的细胞质动力蛋白电动机内的叠加和大规模柔韧性。
影响因子：
16.6
作者：
Imai H;Shima T;Sutoh K;Walker ML;Knight PJ;Kon T;Burgess SA
通讯作者：
Burgess SA
影响因子：
7.5
作者：
Gennerich A;Vale RD
通讯作者：
Vale RD
DOI：
10.1038/nrm2804
发表时间：
2009-12
期刊：
Nature reviews. Molecular cell biology
影响因子：
--
作者：
通讯作者：
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