DISTANCES BETWEEN THE PACLITAXEL, COLCHICINE, AND EXCHANGEABLE GTP-BINDING SITES ON TUBULIN

Distances between the paclitaxel, colchicine, and exchangeable GTP bin ding sites on tubulin polymers have been probed using fluorescence spe ctroscopy. Techniques for measuring fluorescence resonance energy tran sfer (FRET) between fluorescent or chromophoric ligands for each bindi ng site were employed, 2-Debenzoyl-2-(m-aminobenzoyl)paclitaxel (2-AB- PT) was the fluorophore ligand for the paclitaxel binding site, thioco lchicine, 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 dis tance between the colchicine and paclitaxel binding sites was determin ed with two different acceptor ligands in the colchicine site. An aver age distance distribution of 17 Angstrom was found in both cases. Ener gy transfer between 2-AB-PT bound to the paclitaxel site and TNP-GTP ( acceptor) bound to the exchangeable GTP site was observed in the polym er. The average distance distribution between the fluorophores was 16. 0 Angstrom, but the half-width of the distribution was large (17.9 Ang strom), which indicates that energy transfer between more than one don or-acceptor pair occurred in the system. One interpretation of this re sult is that 2-AB-PT serves as an energy transfer donor for two GTP si tes, one contained on the same subunit and one on an adjacent protofil ament. No FRET was observed between ligands bound to the colchicine an d exchangeable GTP sites, indicating that the result of colchicine bin ding on the GTP region of beta-tubulin is a long range, allosteric eff ect. The results from these experiments are interpreted in terms of kn own structural features of microtubules.