Novel bifunctional probe for radioisotope-free photoaffinity labeling: compact structure comprised of photospecific ligand ligation and detectable tag anchoring units.
A novel method for radioisotope-free photoaffinity labeling was developed, in which a bifunctional ligand is connected to a target protein by activation of a photoreactive group, such as an aromatic azido or 3-trifluoromethyl-3H-diazirin-3-yl group, and identification of the ligated product is achieved by anchoring of a detectable tag through the Staudinger-Bertozzi reaction with an alkyl azido moiety that survives photolysis. The chemical ground of this method was confirmed using model compounds with the bifunctional group under photoirradiation in the presence of trapping agents for reactive intermediates. The utility of the method has been demonstrated by specific labeling of the catalytic portion of human HMG-CoA reductase.
Division of Regeneration and Advanced Medical Science, Gifu University Graduate School of Medicine, Yanagido, Japan.
Takamitsu
T
Hosoya
Toshiyuki
T
Hiramatsu
Takaaki
T
Ikemoto
Masayuki
M
Nakanishi
Hiroshi
H
Aoyama
Ayako
A
Hosoya
Tomoya
T
Iwata
Kei
K
Maruyama
Makoto
M
Endo
Masaaki
M
Suzuki
D001386:Azides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006801:Humans; D006903:Hydroxymethylglutaryl CoA Reductases / Q000737:chemistry / Q000528:radiation effects; D008024:Ligands; D008027:Light; D008958:Models, Molecular; D015394:Molecular Structure; D019920:Photoaffinity Labels / Q000737:chemistry*; D010782:Photolysis; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry* / Q000528:radiation effects; D012680:Sensitivity and Specificity
D001386:Azides; D008024:Ligands; D019920:Photoaffinity Labels; D011506:Proteins; D006903:Hydroxymethylglutaryl CoA Reductases