Studies of Conformational Changes in Tubulin

这个项目的最终目标是了解微管蛋白中与核苷酸水解相关的结构变化，并导致微管的动态性质。动态不稳定性是微管的一个基本属性，它是有丝分裂和其他细胞功能发展所必需的。GTP在β-微管蛋白中的水解会导致二聚体的构象变化，从而削弱侧向相互作用，并诱导原丝卷曲，从而导致微管解聚。我们将比较代表蛋白质高能态和低能态的两种不同构象中微管蛋白的结构：在直的原丝中，gdp-微管蛋白分子被聚合物限制在“类GTP”状态。 晶格，以及弯曲的原丝中的构象，其中GDP-微管蛋白处于不受约束的松弛状态。我们通过锌诱导的薄片的电子结晶学获得了约束状态下微管蛋白的原子结构，以及完整微管的8埃分辨率模型。因此，本项目致力于通过低温电子显微镜和图像重建获得GDP-微管蛋白的低能结构。我们已经能够获得一种形式的GDP-微管蛋白，在这种形式中，弯曲的原丝填充到一个易于螺旋重建的结晶管中。我们的最终目标是重建这些管子 在高于10埃的分辨率下，单个阿尔法螺旋可以被可视化。然后，微管蛋白的晶体结构将被用来模拟这种不受约束的构象，并测试不同的假设，将核苷酸位置与微管蛋白上的聚合表面联系起来。 许多细胞因子通过稳定微管结构或诱导其解体来调节动态不稳定性。此外，许多具有抗癌特性的抗有丝分裂药物通过与微管蛋白结合并破坏正常的微管动力学来发挥作用。在这两种情况下，配基都可以被认为是作用于微管蛋白，以增强、修饰或消除GTP水解的影响。因此，对GTP水解引发的构象变化的详细了解对于我们理解这些过程是至关重要的。在这个项目中，我们还计划通过研究隐孢霉素1对微管蛋白结构的影响来直接解决抗有丝分裂药物的影响。

This project has as its final goal to understand the structural changes in tubulin related to nucleotide hydrolysis and resulting in the dynamic nature of microtubules. Dynamic instability is an essential property of microtubule required for the progression of mitosis and other cellular functions. GTP hydrolysis in beta-tubulin causes a conformational change in the dimer that weakens lateral interaction and induces the curling of protofilaments that results in microtubule depolymerization. We will compare the structures of tubulin in two different conformations representing the high and low energy states of the protein: the conformation in a straight protofilament where the GDP-tubulin molecule is constrained in a "GTP-like" state by the polymer lattice, and the conformation in curved protofilaments where GDP-tubulin is in an unconstrained, relaxed state. We have an atomic structure of tubulin in the constrained state obtained by electron crystallography of zinc-induced sheets, as well as an 8 Angstrom resolution model of the intact microtubule. Thus the present project is concerned with obtaining the low energy structure of GDP-tubulin by cryo-electron microscopy and image reconstruction. We have been able to obtain a form of GDP-tubulin in which curved protofilaments pack into a crystalline tube amenable to helical reconstruction. Our final goal is to obtain a reconstruction of these tubes at better than 10 Angstrom resolution where individual alpha helices can be visualized. The crystal structure of tubulin will then be used to model this unconstrained conformation and to test different hypotheses linking the nucleotide site with polymerization surfaces on tubulin. A number of cellular factors regulate dynamic instability by either stabilizing microtubule structure or inducing its disassembly. In addition, a variety of antimitotic agents with anticancer properties function by binding to tubulin and disrupting normal microtubule dynamics. In both cases the ligand can be thought as operating on tubulin to enhance, modify or eliminate the effect of GTP hydrolysis. A detailed knowledge of the conformational changes triggered by GTP hydrolysis is thus essential for our understanding of these processes. Within this project we also plan to address the effect of antimitotic agents directly by investigating the effect of cryptophycin 1 on tubulin structure.