Photocrosslinking/label transfer: A key step in mapping short alpha-neurotoxin binding site on nicotinic acetylcholine receptor.
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 alpha-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 alpha subunit (59%), followed by the gamma subunit (28%), with the delta 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 alpha subunit followed by Edman degradation of the purified peptides showed that alphaTyr190 and alphaTyr198 were the most labeled residues, with a less important labeling on alphaCys192. We believe that the novel photoactivatable probe will be of great use to identify key residues of ligands interacting with macromolecules.
Kessler
Pascal
P
CEA, DSV, DIEP, Gif-sur-Yvette, F-91191, France. pascal.kessler@cea.fr
Thai
Robert
R
Beau
Fabrice
F
Tarride
Jean-Luc
JL
Ménez
André
A
D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry*; D008958:Models, Molecular; D015394:Molecular Structure; D009498:Neurotoxins / Q000737:chemistry* / Q000378:metabolism*; D010777:Photochemistry; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000378:metabolism*; D013057:Spectrum Analysis; D014101:Torpedo
D003432:Cross-Linking Reagents; D009498:Neurotoxins; D021122:Protein Subunits; D011978:Receptors, Nicotinic