Exchangeable GTP binding site of beta-tubulin. Identification of cysteine 12 as the major site of cross-linking by direct photoaffinity labeling.
After direct photoaffinity cross-linking of [3H]GTP to the beta-subunit of tubulin, followed by tryptic digestion and alkaline phosphatase treatment, we employed cis-diol-specific boronate gel chromatography and reversed-phase high-pressure liquid chromatography to purify a peptide containing most of the covalently bound radioactivity. The sequence of this peptide corresponded to that of residues 3-19 of beta-tubulin. Residue 10 of the peptide, which is Cys-12 in beta-tubulin, could not be identified. The fast atom bombardment mass spectrum of this peptide showed the presence of a predominant species with a molecular mass of 2022 kDa (2021 kDa for the 12C variant), which is 255 Da greater than the molecular mass of the peptide. Fast atom bombardment collision-activated decomposition mass spectrometry analysis produced fragments which are consistent with the beta(3-19) peptide but having a unit of mass of 358 at position 12. Thermolysin digestion of the tryptic peptide restricted the cross-linking site to the 9-amino acid sequence, I(L)QAGQXGNQ. The molecular mass of this peptide was 1174 kDa, which is equal to the mass of the beta(7-15) peptide containing an extra group of mass 255. To explain the molecular masses of the two labeled peptides, which are 26 atomic mass units less than expected, a mechanism of photolabeling is proposed that involves opening of the guanine ring and loss of the C-6 carbonyl function as CO2.
Shivanna
B D
BD
Department of Biochemistry, University of Kansas, Lawrence 66045-2106.
Mejillano
M R
MR
Williams
T D
TD
Himes
R H
RH
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine; D005609:Free Radicals; D006160:Guanosine Triphosphate / Q000378:metabolism*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013820:Thermolysin; D014316:Tritium; D014357:Trypsin; D014404:Tubulin / Q000302:isolation & purification / Q000378:metabolism* / Q000528:radiation effects; D014466:Ultraviolet Rays
D000345:Affinity Labels; D005609:Free Radicals; D046911:Macromolecular Substances; D010446:Peptide Fragments; D014404:Tubulin; D014316:Tritium; D006160:Guanosine Triphosphate; D014357:Trypsin; D013820:Thermolysin; D003545:Cysteine