Characterization of the taxol binding site on the microtubule: 2-(m-azidobenzoyl)taxol photolabels a peptide (amino acids 217-231) of β-tubulin

Srinivasa Rao, George A. Orr, Ashok G. Chaudhary, David G.I. Kingston, Susan Band Horwitz

Research output: Contribution to journalArticlepeer-review

229 Scopus citations

Abstract

Photoaffinity labeling methods are being used to define the molecular contacts between taxol and its target protein, tubulin. Our laboratory has demonstrated previously that [3H]3′-(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, [3H]2-(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, [3H]2-(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.

Original languageEnglish (US)
Pages (from-to)20235-20238
Number of pages4
JournalJournal of Biological Chemistry
Volume270
Issue number35
DOIs
StatePublished - Sep 1 1995

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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