Expression and Purification of Functional Tubulin Domains for Crystallization Studies

这是一个探索性研究的小补助金。 的 微管蛋白二聚体的结构测定，主要 微管的组成部分，将是非常有价值的理解 微管是如何在细胞中组装、分解和发挥作用的。 在 此外，它应该大大提高我们的知识如何几个 医学和农业上重要的化合物结合到 在微管上。 先前对微管蛋白结晶的努力， 失败的原因是微管蛋白的异质性和稳定性 由于微管蛋白倾向于组装成 微管而不是结晶。 为了规避这些 奥克利博士提出了纯化β-微管蛋白的方法， 形成微管蛋白二聚体的分子，并纯化多肽 含有β-微管蛋白的重要结构域。 的 编码这些分子的基因将被置于一个 强大的诱导型启动子，并通过转化插入到 丝状真菌，构巢曲霉。 这些分子将 在高水平产生，并将通过亲和纯化 使用六个组氨酸残基插入在 分子的羧基末端。 GTP和药物结合研究 将进行，以确定分子是否具有其天然的 结构是否稳定，以及结构是否随时间而稳定。 如果这些 如果实验成功，纯化的分子将 随后用于结构研究。 %%% 微管蛋白是微管的单体成分，是一种结构性的蛋白质。 在真核世界中普遍存在的蛋白质，是生命的基础 了解它的分子结构 对于理解其在自然界中的功能至关重要， 允许使用基因工程方法来适应新的 可能具有商业利益的功能（例如分子 开关）。 微管蛋白是形状的脚手架， 细胞内运动性，一个决定裂解后细胞命运的因素; 它是有丝分裂纺锤体的物质，负责染色体 分离成子细胞，纤毛和鞭毛的材料， 负责细胞运动。 微管蛋白实际上是一个家族 基因上不同的蛋白质，这是进一步多样化， 翻译后修饰。 事实证明， 获得微管蛋白的结构信息， 从天然来源纯化同质蛋白质所固有的。 该项目代表了一种看似合理但风险非常高的方法， 获得足够量的均匀的、纯的、天然的、 用于结构研究的单体微管蛋白。

This is a Small Grant for Exploratory Research. The determination of the structure of the tubulin dimer, the major component of microtubules, would be of great value in understanding how microtubules assemble, disassemble and function in cells. In addition, it should greatly improve our knowledge of how several medically and agriculturally important compounds bind to and act upon microtubules. Previous efforts to crystallize tubulin have failed because of problems with tubulin heterogeneity and stability and because of the tendency of tubulin to assemble into microtubules rather than crystallize. To circumvent these problems, Dr. Oakley proposes to purify beta-tubulin, one of two molecules that form the tubulin dimer, and to purify a polypeptide containing an important structural domain of beta-tubulin. The genes encoding the molecules will be placed under the control of a powerful inducible promoter and inserted by transformation in the filamentous fungus, Aspergillus nidulans. The molecules will be produced at high levels and will be purified by affinity chromatography using six histidine residues inserted at the carboxyl termini of the molecules. GTP and drug binding studies will be performed to determine if the molecules have their native structure and if the structure is stable over time. If these experiments are successful, the purified molecules will subsequently be used for structural studies. %%% Tubulin, the monomeric component of microtubules, is a structural protein ubiquitous in the eukaryotic world and fundamental to life as we know it. Understanding its molecular structure would be important for understanding its functions in nature, and would allow genetic engineering approaches to be used to adapt it for new functions of possible commercial interest (such as molecular switches). Tubulin is a scaffolding for shape, a road map for intracellular motility, a determinant of post-cleavage cell fate; it is the stuff of the mitotic spindle, responsible for chromosome segregation to daughter cells, and the stuff of cilia and flagella, responsible for cellular locomotion. Tubulin is actually a family of genetically distinct proteins, which are further diversified by post-translational modifications. It has proven very difficult to get structural information on tubulin, because of the difficulties inherent in purifying a homogeneous protein from natural sources. This project represents a plausible yet very high-risk approach to obtaining sufficient quantities of homogeneous, pure, native, monomeric tubulin for structural studies.