Direct photoaffinity labeling of cysteine 211 or a nearby amino acid residue of beta-tubulin by guanosine 5'-diphosphate bound in the exchangeable site.
Tubulin with [8-14C]GDP bound in the exchangeable site was exposed to ultraviolet light, and radiolabel was cross-linked to two peptide regions of the beta-subunit. Following enrichment for peptides cross-linked to guanosine by boronate chromatography, we confirmed that the cysteine 12 residue was the major site of cross-linking. However, significant radiolabel was also incorporated into a peptide containing amino acid residues 206 through 224. Although every amino acid in this peptide except cysteine 211 was identified by sequential Edman degradation, implying that this was the amino acid residue cross-linked to guanosine, radiolabel at C-8 was usually lost during peptide processing (probably during chromatography at pH 10). Consequently, the radiolabeled amino acid could not be unambiguously identified.
Bai
R
R
Science Applications International Corporation-Frederick, NCI-Frederick Cancer Research and Development Center, National Institutes of Health, Frederick, Maryland 21702, USA.
Ewell
J B
JB
Nguyen
N Y
NY
Hamel
E
E
D000595:Amino Acid Sequence; D001665:Binding Sites; D002853:Chromatography, Liquid; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D008969:Molecular Sequence Data; D019920:Photoaffinity Labels; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism*
D019920:Photoaffinity Labels; D014404:Tubulin; D006153:Guanosine Diphosphate; D003545:Cysteine