Expression and Purification of Functional Tubulin Domains for Crystallization Studies

这是探索性研究的一小笔助学金。微管的主要成分微管蛋白二聚体的结构的确定，对于了解微管在细胞中的组装、分解和功能具有重要的价值。此外，它将极大地提高我们对几种医学和农业上重要的化合物如何与微管结合并作用于微管的了解。以前使微管蛋白结晶的努力失败了，原因是微管蛋白的异质性和稳定性问题，以及微管蛋白倾向于组装成微管而不是结晶。为了绕过这些问题，奥克利博士建议提纯β-微管蛋白，这是形成微管蛋白二聚体的两个分子之一，并提纯一种含有重要结构域的多肽。编码分子的基因将被置于一个强大的可诱导启动子的控制下，并通过转化插入丝状真菌Nidulans中。这些分子将以高水平产生，并将通过亲和层析法使用插入在分子的羧基末端的六个组氨酸残基进行纯化。将进行GTP和药物结合研究，以确定分子是否具有天然结构，以及结构是否随着时间的推移而稳定。如果这些实验成功，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，是真核生物中普遍存在的一种结构蛋白，是我们所知的生命的基础。了解其分子结构对于了解其在自然界中的功能非常重要，并将允许使用基因工程方法使其适应可能具有商业意义的新功能(如分子开关)。微管蛋白是形状的支架，是细胞内运动的路线图，是分裂后细胞命运的决定因素；它是有丝分裂纺锤体的物质，负责将染色体分离到子细胞；它是纤毛和鞭毛的物质，负责细胞运动。微管蛋白实际上是一个基因上不同的蛋白质家族，通过翻译后修饰进一步多样化。事实证明，获得微管蛋白的结构信息是非常困难的，因为从自然来源中纯化同质蛋白质存在固有的困难。这个项目代表了一种看似合理但非常高风险的方法，以获得足够数量的均质、纯、天然、单体微管蛋白，用于结构研究。

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.