Photocrosslinking/label transfer: A key step in mapping short alpha-neurotoxin binding site on nicotinic acetylcholine receptor

Bioconjug Chem. Nov-Dec 2006;17(6):1482-91. doi: 10.1021/bc060175j.

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 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.

MeSH terms

  • Animals
  • Binding Sites
  • Cell Membrane / chemistry
  • Cell Membrane / metabolism
  • Cross-Linking Reagents / chemistry*
  • Models, Molecular
  • Molecular Structure
  • Neurotoxins / chemistry*
  • Neurotoxins / metabolism*
  • Photochemistry*
  • Protein Structure, Tertiary
  • Protein Subunits / metabolism
  • Receptors, Nicotinic / metabolism*
  • Spectrum Analysis
  • Torpedo

Substances

  • Cross-Linking Reagents
  • Neurotoxins
  • Protein Subunits
  • Receptors, Nicotinic