CHARACTERIZATION OF THE TAXOL BINDING-SITE ON THE MICROTUBULE - 2-(M-AZIDOBENZOYL)TAXOL PHOTOLABELS A PEPTIDE (AMINO-ACIDS 217-231) OF BETA-TUBULIN

Photoaffinity labeling methods are being used to define the molecular contacts between taxol and its target protein, tubulin. Our laboratory has demonstrated previously that [H-3]3'-(p-azidobenzamido)taxol photolabels the N-terminal 31 amino acids of beta-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, [H-3]2-(m-azidobenzoyl)taxol, with tubulin has been investigated. This analogue specifically photolabels beta-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, [H-3]2-(m-azidobenzoyl)taxol-photolabeled beta-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 beta-tubulin as the major photolabeled domain.