Characterization of the Taxol Binding Site on the Microtubule

Photoaffinity labeling methods are being used to define the molecular contacts between taxol and its target protein, tubulin. Our laboratory has demonstrated previously that 3H3′-(p-azidobenzamido)taxol photolabels the N-terminal 31 amino acids of β-tubulin (Rao, S., Krauss, N. E., Heerding, J. M., Swindell, C. S., Ringel, I., Orr, G. A., and Horwitz, S. B.(1994) J. Biol. Chem. 269, 3132-3134). The interaction of a second photoaffinity analogue of taxol, 3H2-(m-azidobenzoyl)taxol, with tubulin has been investigated. This analogue specifically photolabels β-tubulin and the photolabeling is competed by both taxol and unlabeled 2-(m-azidobenzoyl)taxol indicating a common binding domain. To identify the site(s) of photoincorporation, 3H2-(m-azidobenzoyl)taxol-photolabeled β-tubulin was subjected to sequential cyanogen bromide and tryptic digestions. Radiolabeled peptides were purified by reverse phase high performance liquid chromatography, and amino acid sequencing studies identified a peptide containing amino acid residues 217-231 of β-tubulin as the major photolabeled domain. Photoaffinity labeling methods are being used to define the molecular contacts between taxol and its target protein, tubulin. Our laboratory has demonstrated previously that 3H3′-(p-azidobenzamido)taxol photolabels the N-terminal 31 amino acids of β-tubulin (Rao, S., Krauss, N. E., Heerding, J. M., Swindell, C. S., Ringel, I., Orr, G. A., and Horwitz, S. B.(1994) J. Biol. Chem. 269, 3132-3134). The interaction of a second photoaffinity analogue of taxol, 3H2-(m-azidobenzoyl)taxol, with tubulin has been investigated. This analogue specifically photolabels β-tubulin and the photolabeling is competed by both taxol and unlabeled 2-(m-azidobenzoyl)taxol indicating a common binding domain. To identify the site(s) of photoincorporation, 3H2-(m-azidobenzoyl)taxol-photolabeled β-tubulin was subjected to sequential cyanogen bromide and tryptic digestions. Radiolabeled peptides were purified by reverse phase high performance liquid chromatography, and amino acid sequencing studies identified a peptide containing amino acid residues 217-231 of β-tubulin as the major photolabeled domain.