STRUCTURE AND ASSEMBLY OF CYTOSKELETAL FILAMENTS

我们的主要目标是了解“动态”的机制 不稳定性”，这是一种公认的现象， 缓慢伸长和快速缩短阶段之间的循环。 可能 最重要的悬而未决的问题是“灾难”的机制， 从伸长到缩短的突然转变。 在青睐 当前模型的灾难被认为是“GTP帽”的丢失， 当存在时，其稳定MT的末端并有利于伸长。 帽损失暴露了GDP微管蛋白亚基的非常不稳定的核心， 然后迅速分解。 因此，灾难是由 GTP帽的动态，其中涉及至少三个随机 GTP微管蛋白亚基的结合和解离反应 MT的结束和GTP的水解。 目前有 猜测，但很少有确切的数据，对上限的大小， GTP水解的速率，以及帽损失的机制。 我们在下一个资助期的首要任务是延长我们的学习时间 动态不稳定性的视频显微镜的单个微管。 我们 最近完成了一项详尽的研究， E. D实验室三文鱼 这项研究测量了所有 动态不稳定的阶段，但仅限于一个单一的缓冲区。 我们 现在建议扩大这些研究，以探讨重要的缓冲区 条件和配体。 正在进行的研究已经表明， 镁对快速起酥油反应具有显著影响， 在7mM Mg下解聚速率高达每秒8，000个亚基。 钙，pH值和核苷酸，所有潜在的生理调节剂 重要性，将在该系统中进行调查，以确定 动态不稳定的阶段，它们发挥其作用。 我们建议补充这些研究个别微管 使用急冷流和停流技术测量GTP水解 以及在低至5毫秒的时间间隔处的电容损失。 我们最近的实验， 在10和1秒的时间间隔，已经为 上限，但需要更快的技术来解决机制 凯普动力公司 急冷流动研究不仅应确定 水解的速率，而且在帽内的位置， 发生水解。 解聚的停流分析 还应确定上限的大小，由不同的方法。 通过 我们希望在一系列组装条件下重复这些实验， 以确定帽动态和突变的完整机制。 对微管蛋白生物化学的最终理解将需要 X射线晶体学中的原子结构 我们处在一个极好的 尝试结晶。 第一步是 确定保持微管蛋白作为单一蛋白质溶液的条件 两人同时发出满足的呻吟声。 微管蛋白的正常联系：MT和环的形成。 我们建议 确定必要的生化条件， 结晶尝试。

Our major objective is to understand the mechanisms of "dynamic instability", the well established phenomenon in which microtubules cycle between phases of slow elongation and rapid shortening. Probably the most important unsolved question is the mechanism of "catastrophe," the abrupt transition from elongation to shortening. In the favored current model catastrophe is thought to be the loss of a "GTP cap," which when present, stabilizes the end of the MT and favors elongation. Cap loss exposes the very labile core of GDP tubulin subunits, which then rapidly disassembles. Catastrophe is therefore determined by the dynamics of the GTP cap, which involves at least three stochastic reactions" association and dissociation of GTP tubulin subunits from the end of the MT, and hydrolysis of GTP. At present there are speculations, but little definitive data, on the size of the cap, the rate of GTP hydrolysis, and the mechanism of cap loss. Our first priority in the next grant period will be to extend our study of dynamic instability by video microscopy of single microtubules. We have recently completed an exhaustive study in collaboration with the laboratory of E.D. Salmon. This study measured the kinetics for all phases of dynamic instability, but was limited to a single buffer. We now propose to extend these studies to explore important buffer conditions and ligands. Ongoing studies have already demonstrated that magnesium has a dramatic effect on the rapid shortening reaction, with depolymerization rates up to 8,000 subunits per sec at 7 mM Mg. Calcium, pH, and nucleotides, all potential regulators of physiological importance, will be investigated in this system to determine at which phases(s) of dynamic instability they exert their effects. We propose to complement these studies of individual microtubules by using quench-flow and stopped-flow techniques to measure GTP hydrolysis and cap loss at time intervals down to 5 msec. Our recent experiments, at time intervals of 10 and 1 s, have set upper bounds for the size of the cap, but much faster techniques are needed to resolve the mechanisms of cap dynamics. The quench-flow studies should determine not only the rate of hydrolysis but also the location within the cap at which hydrolysis takes place. The stopped-flow analysis of depolymerization should also determine the size of the cap, by a different approach. By repeating these experiments over a range of assembly conditions we hope to determine the complete mechanism of cap dynamics and catastrophe. The ultimate understanding of tubulin biochemistry will require an atomic structure, from x-ray crystallography. We are in an excellent position to attempt crystallization. The first step will be to determine conditions that keep tubulin as a solution of single protein molecules, stable for weeks, and at the same time poisoned for the two normal associations of tubulin: MT and ring formation. We propose to determine the necessary biochemical conditions and initiate crystallization attempts.