Direct observation of the binding state of the kinesin head to the microtubule.
Direct observation of the binding state of the kinesin head to the microtubule.
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二聚体运动驱动蛋白-1 将 ATP 水解产生的化学能耦合到用于沿着微管运输货物的机械功中。附着在驱动蛋白柄上的货物随着其双运动域（头）进行不对称的手拉手行走，以 8 nm 的增量持续移动。然而，在行走过程中个体头部与微管相互作用的程度仍然存在争议。一个主要的实验限制是缺乏监测运动过程中单个头部与微管的附着的方法，因此需要间接方法。我们开发了一种单分子测定法，可以直接报告行走驱动蛋白分子中的头部结合，并表明在低 ATP 浓度的步骤之间只有一个头部与微管结合。一个珠子通过短 DNA 系链连接到两个驱动蛋白头之一，并用于通过光陷阱施加快速交替的阻碍和辅助负载。珠子向前和向后位移之间随时间变化的差异在步进过程中在两个离散值之间交替，对应于链接头采用束缚或非束缚状态时的那些间隔。只有当 ATP 与其配对头结合后，连接的头才能重新结合微管。
The dimeric motor kinesin-1 couples chemical energy from ATP hydrolysis into mechanical work used to transport cargo along microtubules. Cargo attached to the kinesin stalk moves processively in 8-nm increments as its twin motor domains (heads) carry out an asymmetric, hand-over-hand walk. The extent of individual head interactions with the microtubule during stepping, however, remains controversial. A major experimental limitation has been the lack of a means to monitor the attachment of individual heads to the microtubule during movement, necessitating indirect approaches. We developed a single-molecule assay that can directly report head binding in a walking kinesin molecule, and show that only a single head is bound to the microtubule between steps at low ATP concentrations. A bead was linked to one of the two kinesin heads via a short DNA tether and used to apply rapidly alternating hindering and assisting loads with an optical trap. The time-dependent difference between forward and rearward displacements of the bead alternated between two discrete values during stepping, corresponding to those intervals when the linked head adopted bound or unbound states. The linked head could only rebind the microtubule once ATP had become bound to its partner head.
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DOI：
10.1073/pnas.0436709100
发表时间：
2003-03-04
影响因子：
11.1
作者：
Block, SM;Asbury, CL;Lang, MJ
通讯作者：
Lang, MJ
影响因子：
64.8
作者：
Kerssemakers, Jacob W. J.;Munteanu, E. Laura;Dogterom, Marileen
通讯作者：
Dogterom, Marileen
影响因子：
11.4
作者：
Kikkawa, Masahide;Hirokawa, Nobutaka
通讯作者：
Hirokawa, Nobutaka
影响因子：
56.9
作者：
Asbury, CL;Fehr, AN;Block, SM
通讯作者：
Block, SM
DOI：
10.1073/pnas.91.15.6865
发表时间：
1994-07-19
影响因子：
11.1
作者：
HACKNEY, DD
通讯作者：
HACKNEY, DD