Materials and Methods Som Text Figs. S1 to S3 References and Notes Kinesin Moves by an Asymmetric Hand-over-hand Mechanism-猫眼课题宝

DOI：

发表时间：

期刊：

影响因子：

通讯作者：

中科院分区：

其他

文献类型：

作者：

W J Lee;J. D. Lee;V. Kravchenko;R. Ulevitch;P. Brey;C. Asbury;Adrian N. Fehr;S. Block

28. 我们感谢实验室的同事对手稿提出的批评意见，W.-J。感谢 Lee 赠送的 GNBP1 试剂，J.-M. Ubeda 提出了双过度表达实验，M.-C. Lacombe 寻求专家技术帮助。得到法国国家科学研究中心 (CNRS) 和国家教育研究与技术部以及美国国立卫生研究院 (NIH) 的支持。对 GNBP1 osi (e03371) 库存的请求应发送给 Exelixis。驱动蛋白是一种双头运动蛋白，以 8 纳米的步长沿着微管移动。两大类模型被用来解释驱动蛋白运动：交接手模型和尺蠖模型。在交接手模型中，头部每一步都会交换引导和尾随角色，而对于尺蠖模型则不假设这种交换，因为尺蠖模型中总是有一个头部在引导。通过测量单个酶的逐步运动，我们发现一些驱动蛋白分子在连续步骤之间的停留时间表现出明显的交替，导致这些马达沿着微管“跛行”。跛行意味着驱动蛋白分子在行走时严格地在两种不同构象之间交替，表明不对称的切换机制。各种单分子实验的结果提供了对驱动蛋白运动蛋白的机械化学特性的深入了解。各个驱动蛋白二聚体连续移动，以随机间隔进行离散的 8 nm 步长，并且在从微管表面释放之前可能需要一百或更多步。即使存在高达几个 pN (1-3) 的持续外部负载，过程运动也会持续存在，这表明驱动蛋白二聚体的某些部分始终与微管结合。驱动蛋白分子在微管表面晶格上沿着与原丝平行的路径移动 (4, 5)，与每个微管蛋白二聚体的一个结合位点相互作用 (6)。最后，驱动蛋白的移动方式是每 8 纳米步长恰好水解一个三磷酸腺苷 (ATP) 分子 (1, 7, 8)。两大类迈步模式与上述观察结果一致：切换手模型，其中两个头交替迈步，每一步交换前导和尾随角色；以及尺蠖模型，其中给定的头保持在前导位置 (9, 10)。驱动蛋白马达的活性部分由相同重链的二聚体形成，它们折叠成连接到单个公共茎上的双头（11）。两个球状头具有酶活性并结合 ATP 和微管，通过短颈（ϳ13 个氨基酸）与茎相连……

28. We thank our colleagues in the laboratory for critical comments on the manuscript, W.-J. Lee for the kind gift of GNBP1 reagents, J.-M. Ubeda for suggesting the double-overexpression experiment, and M.-C. Lacombe for expert technical help. Supported by CNRS and the Ministère de l'Education Nationale de la Re-cherche et de la Technologie and by NIH. Requests for the GNBP1 osi (e03371) stock should be addressed to Exelixis. Kinesin is a double-headed motor protein that moves along microtubules in 8-nano-meter steps. Two broad classes of model have been invoked to explain kinesin movement: handover hand and inchworm. In handover hand models, the heads exchange leading and trailing roles with every step, whereas no such exchange is postulated for inchworm models, where one head always leads. By measuring the stepwise motion of individual enzymes, we find that some kinesin molecules exhibit a marked alternation in the dwell times between sequential steps, causing these motors to " limp " along the microtubule. Limping implies that kinesin molecules strictly alternate between two different conformations as they step, indicative of an asymmetric, handover hand mechanism. Results from a variety of single-molecule experiments have furnished insights into the mechanochemical properties of kinesin motor proteins. Individual kinesin dimers move pro-cessively, making discrete 8-nm steps at sto-chastic intervals, and may take a hundred or more steps before releasing from the micro-tubule surface. Processive motion persists even in the presence of sustained external loads up to several pN (1–3), suggesting that some portion of the kinesin dimer remains bound to the microtubule at all times. Kinesin molecules move on the microtubule surface lattice along paths parallel to the protofila-ments (4, 5), interacting with one binding site per tubulin dimer (6). Finally, kinesin moves in such a way as to hydrolyze exactly one adenosine triphosphate (ATP) molecule per 8-nm step (1, 7, 8). Two broad classes of stepping pattern are consistent with the foregoing observations: handover hand models, in which the two heads step alternately, exchanging leading and trailing roles with each step, and inchworm models, in which a given head remains in the lead (9, 10). The active portion of the kinesin motor is formed from a dimer of identical heavy chains, which fold into twin heads attached to a single common stalk (11). The two globular heads, which carry enzymatic activity and bind ATP and microtubules, are joined to the stalk through short (ϳ13 amino acids) neck …

Materials and Methods Som Text Figs. S1 to S3 References and Notes Kinesin Moves by an Asymmetric Hand-over-hand Mechanism

文献摘要

被引文献

相似文献