Direct photoaffinity labeling of cysteine-295 of α-tubulin by guanosine 5'-triphosphate bound in the nonexchangeable site

Ruoli Bai, Kevin Choe, John B. Ewell, Nga Y. Nguyen, Ernest Hamel

Research output: Contribution to journalArticle

11 Scopus citations

Abstract

The αβ-tubulin heterodimer has two high affinity guanosine 5- triphosphate binding sites, so that purified tubulin usually contains two molecules of bound guanosine nucleotide. Half this nucleotide is freely exchangeable with exogenous guanine nucleotide, and its binding site has been readily localized to the β-subunit. The remaining nonexchangeable guanosine 5'-triphosphate can only be released from tubulin by denaturing the protein. We replaced the exchangeable site nucleotide of tubulin with 2'- deoxyguanosine 5'-diphosphate, exposed the resulting tubulin to ultraviolet light, degraded the protein, and isolated ribose-containing peptide derived from the nonexchangeable site. A large cyanogen bromide peptide was recovered, and its further degradation with endoproteinase Glu-C established that cysteine-295 of α-tubulin was the major reactive amino acid cross- linked to guanosine by ultraviolet irradiation.

Original languageEnglish (US)
Pages (from-to)9894-9897
Number of pages4
JournalJournal of Biological Chemistry
Volume273
Issue number16
DOIs
StatePublished - Apr 17 1998

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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