One of the characteristic properties of tubulin, a 100 kdal protein comprising the central core of microtubules, is its ability to bind colchicine, an alkaloid isolated from the Autumn crocus. Colchicine binding to tubulin results in substoichiometric inhibition of tubulin polymerization and subsequent disruption of microtubule-mediated processes. The ubiquity and specificity of colchicine binding activity in higher eukaryotes implicates the colchicine binding site as having a possible role in the in vivo regulation of microtubule assembly. In order to confirm or deny the validity of this proposition, the interactions between tubulin and ligands binding to the colchicine site must first be understood. The location and sequence of the colchicine site on tubulin will be found using several novel photoaffinity labeling derivatives of colchicine in which the photolabile groups are situated very close to the interior of the binding site. This approach is innovative in that it is designed to identify peptides in the individual subdomains of the binding site. The unique spectroscopic properties of colchicine bound to tubulin have led the PI to propose that ligands bound to tubulin are engaged in a pi-stacking interaction with a tryptophan in the binding site. Associations of this type are known to occur between single-stranded DNA binding proteins and nucleic acids but have not yet been observed between a small molecule and a protein. Dr. Hastie will use the method of optically detected magnetic resonance (ODMR) to directly examine the effect of ligand binding on tubulin tryptophan(s). By employing heavy atom derivatives of the ligands, she intends to determine which aromatic ring of the ligand is stacking with the tryptophan. Kinetic and equilibrium parameters of selected ligands binding to tubulin will be determined in order to test a unified mechanism that explains how ligands which are structurally diverse bind to the protein at a single site. This information coupled with the photoaffinity labeling and ODMR results, will be used to construct a low resolution map of the receptor site.
