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.
