Structural and Functional Characteristics of Tubulin Isoforms

针对微管蛋白同型（基因产物）和同型（翻译后变异）的结构特征的研究将继续进行。具体目标将是：(1)完成神经元特异性III类同型的结构表征，特别注意确定侧链谷氨酰化的连通性；(2)使用串联质谱法结合酶消化和特定化学修饰来测序和表征翻译后修饰，特别是多谷氨酰化和磷酸化，其他β -微管蛋白同型；(3)合成并确定秋水仙花碱类光亲和标记对微管蛋白异源二聚体结合位点定位的适用性，并对修饰后的微管蛋白进行串联质谱测序。微管是一类极其重要的细胞结构蛋白（细胞骨架），它有助于细胞的形态和功能特征：它们构成了鞭毛和纤毛结构和功能的基础；它们是与染色体运动和细胞分裂极性相关的关键细胞骨架元件；它们提供细胞内支架来维持细胞形状；由于真核细胞中的某些细胞内运输现象利用微管作为运输发生的结构轨道，它们有助于细胞功能和亚细胞动力学的方向或空间组织方面。这些研究的长期目标是了解存在于微管蛋白同工型和同工型中的结构变异的功能意义。

Research directed at structural characterization of tubulin isotypes (gene products) and isoforms (post-translational variants) will be continued. The specific objectives will be to: (1), complete the structural characterization of the neuron-specific class III isotype, with particular attention to the determination of the connectivity of the side chain glutamylation; (2), use tandem mass spectrometry in conjunction with enzymatic digestion and specific chemical modifications to sequence and characterize post-translational modification, especially polyglutamylation and phosphorylation, of other beta-tubulin isotypes; and (3), synthesize and determine the applicability of photoaffinity labels of the colchicine class for localization of the binding site on tubulin heterodimer, followed by tandem mass spectrometry sequencing of the modified tubulin. Microtubules are an extremely important class of cellular structural proteins (cytoskeleton) which contribute to the morphological and functional characteristics of the cell: they comprise the basis for structure and function of flagella and cilia; they are the key cytoskeletal elements associated with chromosomal movement and polarity of cellular divisions; they provide an intracellular scaffolding to maintain cell shape; and since certain intracellular transport phenomena in eukaryotic cells utilize microtubules as the structural track along which transport takes place, they contribute to directional or spatial organizational aspects of cellular function and subcellular dynamics. The long term goal of these studies is to understand the functional significance of the structural variations which are present in tubulin isoforms and isotypes.