What kinesin does at roadblocks: the coordination mechanism for molecular walking
What kinesin does at roadblocks: the coordination mechanism for molecular walking
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DOI：
10.1038/sj.emboj.7600042
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发表时间：
2004-01-14
期刊：
影响因子：
11.4
通讯作者：
Cross, RA
中科院分区：
文献类型：
--
作者：
Crevel, IMTC;Nyitrai, M;Cross, RA
可以通过引入路障来防止引导头附着来测试驱动蛋白中进行性步进协调的竞争模型。我们使用 T93N（一种不可逆结合的突变单体）作为路障，并使用微闪光解结合停流测量了有或没有 T93N 路障的核苷酸诱导的驱动蛋白单体或二聚体的脱离率。对照核苷酸诱导的单体(rK340) ATP 解结合时间为73.6 s(-1)，ADP 解结合时间为40.5 s(-1)。对照 ADP 诱导的二聚体 (rK430) 解结合时间为 18.6 s(-1)。添加 20 mM Pi 会减慢单体和二聚体的解离速度。路障就位后，二聚体的前导头附着被阻止，并且 ATP 诱导的后头解结合时间为 42 s(-1)。这比无阻碍的 rK430 二聚体的步进速率 (50 - 70 s(-1)) 慢不到两倍，表明在行走过程中，引线头附着至多仅引起轻微（小于两倍）的步道头分离加速。正如我们所讨论的，这意味着协调模型具有非常快（>2000 s(-1)）ATP 诱导的引线头附着，随后从引线头释放较慢的、应变敏感的 ADP。
Competing models for the coordination of processive stepping in kinesin can be tested by introducing a roadblock to prevent lead head attachment. We used T93N, an irreversibly binding mutant monomer, as a roadblock, and measured the rates of nucleotide-induced detachment of kinesin monomers or dimers with and without the T93N roadblock using microflash photolysis combined with stopped flow. Control nucleotide-induced monomer (rK340) unbinding was 73.6 s(-1) for ATP and 40.5 s(-1) for ADP. Control ADP-induced dimer (rK430) unbinding was 18.6 s(-1). Added 20 mM Pi slowed both monomer and dimer unbinding. With the roadblock in place, lead head attachment of dimers is prevented and ATP-induced trail head unbinding was then 42 s(-1). This is less than two-fold slower than the stepping rate of unimpeded rK430 dimers (50 - 70 s(-1)), indicating that during walking, lead head attachment induces at most only a slight ( less than twofold) acceleration of trail head detachment. As we discuss, this implies a coordination model having very fast (>2000 s(-1)) ATP-induced attachment of the lead head, followed by slower, strain-sensitive ADP release from the lead head.