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

The Journal of Biological Chemistry
R BaiE Hamel

Abstract

The alphabeta-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 beta-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 alpha-tubulin was the major reactive amino acid cross-linked to guanosine by ultraviolet irradiation.

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Citations

Dec 26, 2001·Proceedings of the National Academy of Sciences of the United States of America·M J DayelR D Mullins
Apr 8, 2015·Chemical Biology & Drug Design·Shanthi NagarajanAe Nim Pae
Jul 19, 2002·Biophysical Journal·Ozlem KeskinDavid G Covell
Apr 28, 2009·Plant Physiology and Biochemistry : PPB·Bon-Sung KooMoon-Young Yoon

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