Diaz J F, Andreu J M, Diakun G, Towns-Andrews E, Bordas J
Centro de Investigationces Biologicas C.S.I.C., Velazquez, Madrid, Spain.
Biophys J. 1996 May;70(5):2408-20. doi: 10.1016/S0006-3495(96)79809-0.
We have studied the self-association reactions of purified GDP-liganded tubulin into double rings and taxoid-induced microtubules, employing synchrotron time-resolved x-ray solution scattering. The experimental scattering profiles have been interpreted by reference to the known scattering profiles to 3 nm resolution and to the low-resolution structures of the tubulin dimer, tubulin double rings, and microtubules, and by comparison with oligomer models and model mixtures. The time courses of the scattering bands corresponding to the different structural features were monitored during the assembly reactions under varying biochemical conditions. GDP-tubulin essentially stays as a dimer at low Mg(2+) ion activity, in either the absence or presence of taxoid. Upon addition of the divalent cations, it associates into either double-ring aggregates or taxoid-induced microtubules by different pathways. Both processes have the formation of small linear (short protofilament-like) tubulin oligomers in common. Tubulin double-ring aggregate formation, which is shown by x-ray scattering to be favored in the GDP- versus the GTP-liganded protein, can actually block microtubule assembly. The tubulin self-association leading to double rings, as determined by sedimentation velocity, is endothermic. The formation of the double-ring aggregates from oligomers, which involves additional intermolecular contacts, is exothermic, as shown by x-ray and light scattering. Microtubule assembly can be initiated from GDP-tubulin dimers or oligomers. Under fast polymerization conditions, after a short lag time, open taxoid-induced microtubular sheets have been clearly detected (monitored by the central scattering and the maximum corresponding to the J(n) Bessel function), which slowly close into microtubules (monitored by the appearance of their characteristic J(0), J(3), and J (n) - (3) Bessel function maxima). This provides direct evidence for the bidimensional assembly of taxoid-induced microtubule polymers in solution and argues against helical growth. The rate of microtubule formation was increased by the same factors known to enhance taxoid-induced microtubule stability. The results suggest that taxoids induce the accretion of the existing Mg(2+)-induced GDP-tubulin oligomers, thus forming small bidimensional polymers that are necessary to nucleate the microtubular sheets, possibly by binding to or modifying the lateral interaction sites between tubulin dimers.
我们利用同步加速器时间分辨X射线溶液散射技术，研究了纯化的GDP结合微管蛋白自缔合形成双环以及紫杉烷诱导微管形成的反应。通过参考已知的3纳米分辨率散射曲线以及微管蛋白二聚体、微管蛋白双环和微管的低分辨率结构，并与寡聚体模型和模型混合物进行比较，对实验散射曲线进行了解释。在不同生化条件下的组装反应过程中，监测了与不同结构特征相对应的散射带的时间进程。在低Mg(2+)离子活性下，无论是否存在紫杉烷，GDP-微管蛋白基本上都保持为二聚体。加入二价阳离子后，它通过不同途径缔合形成双环聚集体或紫杉烷诱导的微管。这两个过程都有共同的小线性（短原纤维样）微管蛋白寡聚体的形成。X射线散射表明，与GTP结合蛋白相比，GDP结合蛋白更有利于形成微管蛋白双环聚集体，而这种聚集体实际上会阻碍微管组装。通过沉降速度测定，微管蛋白自缔合形成双环是吸热过程。如X射线和光散射所示，由寡聚体形成双环聚集体涉及额外的分子间接触，是放热过程。微管组装可以从GDP-微管蛋白二聚体或寡聚体开始。在快速聚合条件下，经过短暂的延迟时间后，可以清楚地检测到开放的紫杉烷诱导的微管片层（通过中心散射和对应于J(n)贝塞尔函数的最大值进行监测），这些片层会缓慢闭合形成微管（通过其特征性的J(0)、J(3)和J(n)-(3)贝塞尔函数最大值的出现进行监测）。这为溶液中紫杉烷诱导的微管聚合物的二维组装提供了直接证据，并反驳了螺旋生长的观点。已知增强紫杉烷诱导的微管稳定性的相同因素会提高微管形成的速率。结果表明，紫杉烷诱导现有Mg(2+)诱导的GDP-微管蛋白寡聚体的积累，从而形成小的二维聚合物，这些聚合物可能通过与微管蛋白二聚体之间的侧向相互作用位点结合或修饰来形成微管片层的核。
