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.
