Crevel I M, Lockhart A, Cross R A
Molecular Motors Group, Marie Curie Research Institute, Oxted, Surrey, UK.
J Mol Biol. 1997 Oct 17;273(1):160-70. doi: 10.1006/jmbi.1997.1319.
The kinesin molecular motor "walks" processively along microtubules, touching down with alternate motor domains and transiently bridging between sites spaced 8 nm apart axially. To allow bridging, the coiled coil tail of kinesin would need to unzip a region immediately adjacent to the heads, and the tail region sequence at this point indeed contains potentially destabilising interruptions in the regular hydrophobic heptad repeat. We noticed that such interruptions are substantially absent from the coiled coil tails of Eg5, a slow kinesin homologue, and ncd, a reverse-directed homologue, and we wondered if this precluded their processivity. We measured the temperature dependence of kcat/K50% MTs, an index of the chemical processivity of a motor, the number of ATPs split per productive diffusional encounter of motor with microtubule. We found two-headed ncd (GSTMC5) and two-headed Eg5 (E437GST) constructs to be slightly if at all processive in solution over the range 4 degrees C to 30 degrees C. By contrast, two-headed kinesin constructs K401 and K430 were processive, and became substantially more so with increasing temperature. Arrhenius plots for the solution ATPase were linear for all three motors. Arrhenius plots for MT gliding assays were linear and essentially parallel for E437GST and GSTMC5 (Ea = 61 and 63 kJ mol-1) but for K430 the plot was biphasic, with a break at 17 degrees C, corresponding to a 30% reduction in Ea from 84 to 57 kJ mol-1. The data indicate that ncd and Eg5 are only slightly if at all processive, and suggest that this may be related to structural differences in their coiled coil neck regions.
驱动蛋白分子马达沿着微管进行持续性“行走”，交替的马达结构域与微管接触，并在轴向相距8纳米的位点之间短暂桥接。为了实现桥接，驱动蛋白的卷曲螺旋尾部需要解开紧邻头部的区域，而此时尾部区域的序列在规则的疏水七肽重复序列中确实包含潜在的不稳定中断。我们注意到，在慢速驱动蛋白同源物Eg5和反向驱动蛋白同源物ncd的卷曲螺旋尾部中基本上不存在这种中断，我们想知道这是否排除了它们的持续性。我们测量了kcat/K50% MTs的温度依赖性，kcat/K50% MTs是马达化学持续性的指标，即马达与微管每次有效扩散相遇时水解的ATP分子数。我们发现双头ncd（GSTMC5）和双头Eg5（E437GST）构建体在4摄氏度至30摄氏度的范围内在溶液中几乎没有持续性。相比之下，双头驱动蛋白构建体K401和K430具有持续性，并且随着温度升高持续性显著增强。所有三种马达的溶液ATP酶的阿累尼乌斯图都是线性的。对于E437GST和GSTMC5，MT滑行试验的阿累尼乌斯图是线性的且基本平行（活化能分别为61和63 kJ mol-1），但对于K430，该图是双相的，在17摄氏度处有一个断点，对应活化能从84 kJ mol-1降至57 kJ mol-1，降幅为30%。数据表明ncd和Eg5几乎没有持续性，这可能与它们卷曲螺旋颈部区域的结构差异有关。
