Distances between the paclitaxel, colchicine, and exchangeable GTP binding sites on tubulin

Distances between the paclitaxel, colchicine, and exchangeable GTP binding sites on tubulin polymers have been probed using fluorescence spectroscopy. Techniques for measuring fluorescence resonance energy transfer (FRET) between fluorescent or chromophoric ligands for each binding site were employed, 2-Debenzoyl-2-(m-aminobenzoyl)paclitaxel (2-AB-PT) was the fluorophore ligand for the paclitaxel binding site, thiocolchicine, allocolchicine, and MDL 27048 were probes for the colchicine site, and 2'(or 3')-O-(trinitrophenyl)guanosine 5'-triphosphate (TNP-GTP) was the fluorophore ligand for the exchangeable GTP site. The distance between the colchicine and paclitaxel binding sites was determined with two different acceptor ligands in the colchicine site. An average distance distribution of 17 Angstrom was found in both cases. Energy transfer between 2-AB-PT bound to the paclitaxel site and TNP-GTP (acceptor) bound to the exchangeable GTP site was observed in the polymer. The average distance distribution between the fluorophores was 16.0 Angstrom, but the half-width of the distribution was large (17.9 Angstrom), which indicates that energy transfer between more than one donor-acceptor pair occurred in the system. One interpretation of this result is that 2-AB-PT serves as an energy transfer donor for two GTP sites, one contained on the same subunit and one on an adjacent protofilament. No FRET was observed between ligands bound to the colchicine and exchangeable GTP sites, indicating that the result of colchicine binding on the GTP region of beta-tubulin is a long range, allosteric effect. The results from these experiments are interpreted in terms of known structural features of microtubules.