STRUCTURAL AND GENETIC ASPECTS OF TUBULIN INTERACTIONS

微管是一种多亚单位的蛋白质结构 参与多种细胞功能的真核细胞。 它们主要由两种基因相关的蛋白质组成。 分子量为50,000，称为α和β微管蛋白。 从低等真核生物如酵母、真菌和 原生动物(它们的氨基酸差异可能高达30% 与哺乳动物微管蛋白的序列有很大的不同 比微管蛋白的组装、稳定性和配体结合特性 从哺乳动物的大脑中分离出来。我们的长期目标是 研究是使用遗传和生物物理技术来确定 微管的结构和功能特性 与α和β微管蛋白的氨基酸序列有关 并定义微管蛋白单体的功能结构域和 原始人。这项建议的具体目标是： 1.制备克隆的原生动物微管蛋白(非洲 锥虫)和高等真核脑(鸡)， 生物活性微管蛋白重组条件的研究 并比较组装、构象和配基 与正品锥虫体和脑微管蛋白的结合特性。 2.纯化和鉴定正品锥虫微管蛋白 其组装、稳定性和对抗有丝分裂药物的敏感性 秋水仙素和苯并咪唑类药物。 3.明确鸟嘌呤相互作用的分子机制 核苷酸和抗有丝分裂药物通过组装和 种间二聚体或基因改变的特征 (嵌合体和定点突变体)α和β微管蛋白。 4.确定锥虫或雏鸡的脑贝塔 微管蛋白(或微管蛋白的区域)在酵母中具有生物功能。 对鸟嘌呤核苷酸作用机制的认识 与微管蛋白相互作用并调节其性质可能有助于 对我们理解它在其他重要领域的作用具有重要意义 生物系统，如G蛋白、ras癌基因和 核糖体。对影响相互作用的因素的认识 含微管蛋白的抗有丝分裂药物及其相关作用 微管结构可能导致新的 用于治疗原生动物疾病的抗有丝分裂药物 比如锥虫病或者可能是恶性肿瘤。

Microtubules are multisubunit protein structures found in all eukaryotic cells which are involved in a variety of cell functions. They are composed primarily of two genetically related proteins of molecular weight 50,000 termed alpha and beta tubulin. Tubulins isolated from lower eukaryotes such as yeast, fungi, and protozoa (which may differ by as much as 30% in amino acid sequence from mammalian tubulins) exhibit greatly different assembly, stability, and ligand binding properties than tubulins isolated from mammalian brain. The long term goal of our research is to use genetic and biophysical techniques to determine how the structural and functional properties of microtubules relate to the amino acid sequence of the alpha and beta tubulins and to define functional domains of the tubulin monomers and protomers. The specific aims of this proposal are: 1. To prepare cloned tubulins of a protozoan (African trypanosome) and higher eukaryote brain (chicken), develop conditions for reconstitution of biologically active tubulin protomers, and compare the assembly, conformation, and ligand binding properties with authentic trypanosome and brain tubulin. 2. To purify and characterize authentic trypanosome tubulin in terms of its assembly, stability, and susceptibility to antimitotic agents such as colchicine and the benzimidazoles. 3. To define the molecular mechanisms of interactions of guanine nucleotides and antimitotic agents by assembly and characterization of interspecies dimers or genetically altered (chimeras and site-specific mutants) alpha and beta tubulins. 4. To determine whether the trypanosome or chick brain beta tubulins (or regions of them) are biologically functional in yeast. An understanding of the mechanisms by which guanine nucleotides interact with tubulin and modulate its properties could contribute significantly to our understanding of its role in other important biological systems such as the G proteins, ras oncogenes, and ribosome. Knowledge of the factors governing interaction of antimitotic agents with tubulin and their related effects on microtubule structure could lead to the development of new antimitotic agents useful for treatment of protozoan diseases such as trypanosomiasis or perhaps malignancies.