CONFORMATION & RECOGNITION IN MICROTUBLE DYNAMICS

微管（MT）是染色体分离和细胞内增殖所必需的动态聚合物。 组织，并且是抗癌化学治疗剂如紫杉醇和紫菀生物碱的直接靶标。的 MT的动力学特性对其功能至关重要，它们来自于结构和生物化学特性， 单个微管蛋白亚基的性质以及它们如何在MT晶格内相互作用。越来越 我们认识到，微管蛋白亚基采用不同的构象作为GTP酶依赖性蛋白的一部分， 聚合动力学，并且调节蛋白选择性地识别这些构象的子集， 控制MT伸长、稳定性和切换。这项研究的长期目标是建立一个结构性的 了解变构和微管蛋白构象周期如何决定MT动力学，以及 调节因子控制MT动力学的机制。在以前的项目期间，我们开创了一个 基于结构启发的定点αβ-微管蛋白突变体的强大方法。在本提案中， 通过三个具体目标，我们将在这些主题的基础上提供独特的、基本的新见解 MT动力学的物理起源和调节机制。我们会用生化，重组， 和建模以定义XMAP 215家族聚合酶活性的一般生化机制， 持续性我们将通过结构揭示改变MT动力学的“变构”突变如何影响 微管蛋白构象在人类和酵母MT，我们将提供新的构象循环突变体，以扩大 我们对MT动力学变构的理解。最后，我们将确定生化和结构设计 CLASP TOG与微管蛋白的相互作用如何抑制灾难并促进救援的基本原理。 这项工作将提供关于αβ-微管蛋白构象的新信息，以及“变构”突变如何影响αβ-微管蛋白的构象。 可以扰乱MT动力学和微管蛋白构象。这项工作也将扩大我们对如何 不同的TOG域实现不同的监管结果，对基础的影响 微管动力学机制。

Microtubules (MTs) are essential dynamic polymers required for chromosome segregation and intracellular organization, and are the direct targets of anti-cancer chemotherapeutics like taxol and the Vinca alkaloids. The dynamic properties of MTs are central to their function, and they derive from the structural and biochemical properties of individual tubulin subunits and how they interact within the MT lattice. It is increasingly appreciated that tubulin subunits adopt distinct conformations as part of the GTPase-dependent polymerization dynamics, and that regulatory proteins selectively recognize subsets of these conformations to control MT elongation, stability, and switching. The long-term goal of this research is to build a structural understanding of how allostery and the tubulin conformation cycle dictate MT dynamics, and of the mechanisms by which regulatory factors control MT dynamics. In prior project periods, we pioneered a powerful approach based on structure-inspired site-directed αβ-tubulin mutants. In the present proposal, through three specific aims, we will build on these themes to provide unique and fundamental new insights into the physical origins and regulatory mechanism of MT dynamics. We will use biochemistry, reconstitution, and modeling to define general biochemical mechanisms for XMAP215-family polymerase activity and processivity. We will reveal through structures how an `allosteric' mutation that alters MT dynamics affects tubulin conformation in human and yeast MTs, and we will provide new conformation cycle mutants to expand our understanding of allostery in MT dynamics. Finally, we will identify biochemical and structural design principles underlying how CLASP TOG interactions with tubulin suppress catastrophe and promote rescue. This work will provide new information about the conformation(s) of αβ-tubulin and how `allosteric' mutations can perturb MT dynamics and tubulin conformation. The work will also expand our understanding of how different TOG domains achieve different regulatory outcomes, with implications for the underlying mechanisms of microtubule dynamics.