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 journalArticle

228 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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