TUBULIN FOLDING AND MICROTUBULE DYNAMICS

微管蛋白折叠和微管动力学

• 批准号: 2695187
• 负责人: ROBLEY C WILLIAMS-JR
• 金额: $ 24.39万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2695187
• 关键词: Escherichia coli; SDS polyacrylamide gel electrophoresis; bioenergetics; centrifugation; chemical stability; cytoskeleton; gel filtration chromatography; microtubule associated protein; microtubules; molecular chaperones; polymerization; posttranslational modifications; protein biosynthesis; protein folding; protein structure; protein structure function; radionuclides; recombinant proteins; scintillation cameras; tubulin

Microtubules, polymers of alphabeta-tubulin and its associated proteins, are essential organizers of the eukaryotic cytoplasm. They are dynamic structures: within a time span short in comparison to the cell cycle, they repeatedly dissociate into, and then reassemble themselves from, their constituent proteins. This behavior, subject to regulation by a variety of known factors, affects cellular differentiation, intracellular transport and determination of cell shape. Its mechanism requires modulation of the affinities of the microtubule's molecules for each other and of the rates at which they associate and dissociate. The long-term goal of this continuing project has been to measure and interpret these affinities and rates and thus to contribute to understanding how tubulin participates in formation and remodeling of the dynamic cytoskeleton. It is now proposed to extend the project's scope to study the folding of tubulin's individual polypeptide chains, both to provide a technical means to obtain active tubulin from recombinant sources and to begin to learn how folding, and possibly refolding, may influence microtubular structure and dynamics. Recent discoveries have shown that a molecular chaperone, CCT, with its cofactors, facilitates and governs tubulin's folding. It is proposed to mobilize this new knowledge to generate homogeneous and well-defined functional tubulin, and to study its assembly into microtubules. By making use of denatured recombinant alpha-and beta-chains produced in E. coli, and by using the chaperone system to fold them in vitro, we intend: (1) to understand the kinetics of the in vitro folding reaction and to use the information to increase the yield of active folded protein, (2) to study folding of post-translationally modified tubulins to probe the influence of modifications on folding and to learn if the CCT system can serve a refolding function as well as folding newly synthesized chains; (3) to initiate study of the dynamics of assembly and disassembly of microtubules which incorporate newly folded tubulins. The results should help to bring together the molecular biological and functional studies of this indispensable part of the cytoskeleton and thus contribute to eventual understanding of its cellular functions, both in health and in disease.

微管、α-β-微管蛋白聚合物及其相关蛋白， 是真核细胞质的重要组织者。它们是动态的 结构：在与细胞周期相比较短的时间跨度内， 它们反复地分离，然后重新组合起来， 它们的组成蛋白。这一行为，受到 各种已知因素，影响细胞分化， 细胞内转运和细胞形状的确定。其作用机制 需要调节微管分子的亲和力 以及它们相互关联和分离的速率。这个 这一持续项目的长期目标一直是衡量和 解释这些亲和力和比率，从而有助于 了解微管蛋白如何参与血管形成和重塑 动态细胞骨架。现在提议延长该项目的 研究微管蛋白的单个多肽链的折叠， 两者都提供了一种技术手段，从 重组资源，并开始学习如何折叠，并可能 折叠，可能会影响微管的结构和动力学。近期 发现表明，一种分子伴侣CCT及其 辅助因子，促进和管理微管蛋白的折叠。有建议说 为了调动这一新知识，以产生同质和定义明确的 功能微管蛋白，并研究其组装成微管的情况。通过 利用产生的变性重组α-链和β-链 通过使用伴侣系统在体外折叠它们，我们 目的：(1)了解体外折叠反应的动力学 并利用这些信息来提高活性折叠的产率 蛋白质，(2)研究翻译后修饰微管蛋白的折叠 探索修饰对折叠的影响，并了解是否 CCT系统除了具有新的折叠功能外，还可以提供重折叠功能 合成链；(3)启动组装动力学研究 以及包含新折叠的微管蛋白的微管的拆卸。 这一结果应该有助于将分子生物学和 细胞骨架中这一不可或缺的部分的功能研究 从而有助于最终了解其细胞功能， 无论是在健康上还是在疾病上。