CONFORMATION & RECOGNITION IN MICROTUBLE DYNAMICS

结构

• 批准号: 9175796
• 负责人: Luke W Rice
• 金额: $ 32.07万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9175796
• 关键词: Address; Adopted; Affect; Behavior; Binding; Biochemical; Biochemistry; Cells; Chromosome Segregation; Coupling; Cryoelectron Microscopy; Defect; Engineering; Equilibrium; Frequencies; Genetic Materials; Goals; Growth; Guanosine Triphosphate Phosphohydrolases; In Vitro; Kinetics; Laboratories; Link; Malignant Neoplasms; Measurement; Measures; Mediating; Methods; Microtubule Polymerization; Microtubules; Molecular; Molecular Conformation; Mutagenesis; Mutation; Negative Staining; Paclitaxel; Pharmaceutical Preparations; Polymerase; Polymers; Positioning Attribute; Property; Protein Engineering; Proteins; Reagent; Regulation; Role; Site; Structure; Surface; Testing; Time; Tubulin; Variant; Vinca Alkaloids; Work; Yeasts; base; beta Tubulin; design; genetic regulatory protein; insight; mutant; novel; polymerization; reconstitution; research study; residence; response; segregation; self assembly; single molecule

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. It is increasingly appreciated that the polymerizing αβ-tubulin subunits adopt distinct conformations as part of the GTPase-dependent polymerization dynamics, and that regulatory proteins selectively recognize subsets of these conformations to promote elongation, shrinking, or catastrophe. However, integrating these structural and biochemical findings into a mechanistic understanding of MT dynamics and regulation remains a central challenge. In the prior project period, we pioneered a powerful approach based on structure-inspired site- directed αβ-tubulin mutants. Using these methods, we advanced the understanding of the regulatory MT polymerase Stu2p by discovering that two simple concepts – selective binding to a MT-incompatible conformation of αβ-tubulin and tethering-based concentration of reactants – could explain the catalytic action of the polymerase. We also advanced the understanding of MT dynamics, discovering by studying buried mutations in β-tubulin that a tunable allosteric response to GDP in the lattice dictates the frequency of MT catastrophe and the rate of post-catastrophe shrinking. Our laboratory is now uniquely positioned to answer fundamental questions about MT dynamics and regulation. In Aim 1 we will use a combination of protein engineering and in vitro reconstitution, including single molecule experiments, to answer most of the remaining questions about the mechanism of MT polymerases: the molecular origin of processivity, what determines the degree of polymerase saturation on the MT end, and how maximal polymerase activity depends on the number and type of TOG domains. This will result in a state-of-the-art understanding of a MT regulatory protein that integrates structure and biochemistry with bulk and single-molecule kinetic results In Aim 2 we capitalize on our finding that α:E255A, a surface mutation at the site of GTPase activity, causes a `straightening defect'. We will use mutagenesis, measurements of MT dynamics, negative stain and cryo electron microscopy, and other approaches to discover the mechanism of assembly-dependent αβ-tubilin straightening, identify allosteric coupling within the heterodimer, and show how they contribute to MT dynamics and the structure of αβ-tubulin assemblies. In Aim 3 we will use a stable of new reagents to determine αβ-tubulin structures without binding partners or bound to a TOG domain. This work will answer longstanding questions about conformation(s) of unpolymerized αβ-tubulin, will reveal whether allosteric mutations change it, and it will clarify what αβ-tubulin conformations can be recognized by TOG domains. Our approach is distinctive and promises to deliver previously unobtainable insight into fundamental mechanisms of MT dynamics and regulation.

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。是 人们越来越认识到，聚合的αβ-微管蛋白亚基采用不同的构象作为结构的一部分， GTP酶依赖的聚合动力学，以及调节蛋白选择性地识别 这些构象，以促进伸长，收缩，或灾难。然而，整合这些结构 和生物化学的研究结果转化为对MT动力学和调节机制的理解仍然是一个核心问题。 挑战.在上一个项目期间，我们开创了一种基于结构灵感的强大方法- 定向αβ-微管蛋白突变体。利用这些方法，我们进一步了解了调节MT 聚合酶Stu 2 p通过发现两个简单的概念-选择性结合到MT不相容的 αβ-微管蛋白的构象和基于反应物浓度的束缚可以解释催化作用 的聚合酶。通过对隐伏区的研究发现了大地电磁动力学的新认识 β-微管蛋白中的突变使得晶格中对GDP的可调变构反应决定了MT的频率 灾难和灾难后萎缩的速度。我们的实验室现在处于独特的位置， 关于MT动力学和调节的基本问题。在目标1中，我们将使用蛋白质的组合 工程和体外重建，包括单分子实验，以回答大多数问题。 关于MT聚合酶机制的剩余问题：持续合成能力的分子起源， 决定了MT末端聚合酶的饱和度，以及最大的聚合酶活性 取决于TOG域的数量和类型。这将导致对MT的最新理解 一种将结构和生物化学与整体和单分子动力学结果相结合的调节蛋白， 目的2：我们利用我们的发现，α：E255 A，一个在GT3活性位点的表面突变，导致了一个新的突变。 “矫直缺陷”。我们将使用诱变，MT动力学测量，负染色和冷冻， 电子显微镜和其他方法来发现组装依赖性αβ-微管蛋白的机制 拉直，识别异二聚体内的变构偶联，并显示它们如何对MT做出贡献 动力学和αβ-微管蛋白组装体的结构。在目标3中，我们将使用稳定的新试剂， 确定没有结合配偶体或与TOG结构域结合的αβ-微管蛋白结构。这项工作将回答 关于未聚合的αβ-微管蛋白构象的长期问题，将揭示变构是否 突变改变了它，这将阐明TOG结构域可以识别哪些αβ-微管蛋白构象。 我们的方法是独特的，并承诺提供以前无法获得的洞察基本 MT动态和调节机制。