Spann U, Renner W, Mandelkow E M, Bordas J, Mandelkow E
Biochemistry. 1987 Feb 24;26(4):1123-32. doi: 10.1021/bi00378a021.
This paper describes a time-resolved X-ray scattering study of microtubule assembly by synchrotron radiation. The method is complementary to light scattering but allows a better distinction between oligomeric and polymeric assembly states. With an improved rapid temperature jump device, it is shown that temperature-induced microtubule assembly is preceded by prenucleation and nucleation events involving oligomers of tubulin, in analogy with earlier results from near-equilibrium temperature scans. In general, the two phases closely overlap, but in certain conditions they can be observed separately. The prenucleation events seen by X-rays can be described as a rapid temperature-dependent equilibrium, with ring oligomers dissociating into smaller oligomers and subunits at elevated temperature. Different solution conditions affect mainly the time lag between the prenucleation and nucleation phases; this in turn determines the apparent magnitude of the prenucleation steps. By contrast, the temperature dependence of the equilibrium between the prenucleation oligomers shows little influence on solution conditions. The results suggest that the ring-forming and tubule-forming assembly modes of tubulin are governed by different interactions between subunits, although they may be based on a pool of similar intermediates.
本文描述了一项利用同步辐射对微管组装进行时间分辨X射线散射研究。该方法是对光散射的补充，但能更好地区分寡聚体和聚合物组装状态。利用改进的快速温度跳跃装置，结果表明，温度诱导的微管组装之前存在涉及微管蛋白寡聚体的预成核和成核事件，这与早期近平衡温度扫描的结果类似。一般来说，这两个阶段紧密重叠，但在某些条件下可以分别观察到。X射线观察到的预成核事件可描述为一种快速的温度依赖性平衡，在升高的温度下，环状寡聚体解离成较小的寡聚体和亚基。不同的溶液条件主要影响预成核和成核阶段之间的时间间隔；这反过来又决定了预成核步骤的表观大小。相比之下，预成核寡聚体之间平衡的温度依赖性对溶液条件影响较小。结果表明，微管蛋白的成环和成管组装模式受亚基之间不同相互作用的支配，尽管它们可能基于一组相似的中间体。
