Localization of the nonexchangeable GTP binding site on tubulin by direct photoaffinity labeling

Tubulin is a heterodimer that binds two molecular equivalents of guanine nucleotide. One half cannot be displaced from tubulin and always exists as GTP (N site nucleotide). The other half exchanges readily with GDP or GTP from the medium. This E site GDP/GTP has been localized to the beta-tubulin subunit by many groups, and a direct photoaffinity approach using radiolabeled GTP and boronate chromatography to enrich for ribose-containing peptides demonstrated highly specific labeling of cysteine-12 of beta-tubulin. In agreement with the latter finding, we demonstrated that peptides derived from unilluminated tubulin did not bind to the boronate matrix, while peptides from irradiated tubulin did bind. We therefore replaced GDP at the exchangeable site with dGDP by successive assembly reactions with dGTP, irradiated the resulting tubulin preparation, digested the protein with cyanogen bromide, and enriched for ribose-containing peptides by boronate chromatography. The peptide(s) binding to the boronate matrix will be purified by gel electrophoresis and/or HPLC and sequenced. They presumably represent peptide(s) contributing to the N site. Their ribose moieties will be oxidized and the resulting dialdehydes tagged with radiolabeled or fluorescent markers to permit localization of the specific target amino acid(s).

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