Photocrosslinking/Label Transfer: A Key Step in Mapping Short α-Neurotoxin Binding Site on Nicotinic Acetylcholine Receptor

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Abstract
We developed a novel radioactive short bifunctional photoprobe, which could be coupled through a cleavable bond to an engineered cysteinyl residue on an analogue of a nicotinic acetylcholine receptor-specific α-neurotoxin. This cysteine was put on the tip of loop II in place of Arg33, a major residue for the interaction with the receptor. To facilitate the purification of the nAChR labeled subunits, we tagged the ligand with a desthiobiotin moiety. After irradiation of the photosensitive toxin−nAChR complex, gel electrophoresis showed that most of the radioactivity was attached to the α subunit (59%), followed by the γ subunit (28%), with the δ subunit (13%) being less labeled. On a preparative scale, the labeled subunits were purified on streptavidin beads before separation on SDS-PAGE. “In-gel” CNBr cleavage of the labeled α subunit followed by Edman degradation of the purified peptides showed that αTyr190 and αTyr198 were the most labeled residues, with a less important labeling on αCys192. We believe that the novel photoactivatable probe will be of great use to identify key residues of ligands interacting with macromolecules.

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