A cysteine-linkable, short cleavable photoprobe with dual functionality to explore protein-protein interfaces.
We developed a bifunctional photoprobe with dual functionality, that can be specifically tethered to cysteinyl residues of peptides and proteins through a short cleavable disulfide bond. Thus, an aryldiazonium moiety is positioned at approximately 8.5 A from the modified cysteinyl alpha-carbon, leading to one of the shortest cleavable linkages. In a sodium azide-containing buffer, the aryldiazonium moiety is transformed into an aryl azide. Therefore, with one bifunctional photoprobe two types of photogenerated species can be obtained: a hydrophilic and positively charged arylcation or a hydrophobic nitrene. We coupled the aryldiazonium probe, in a site-directed manner, to a nicotinic acetylcholine receptor competitive antagonist, obtained by chemical engineering of an analogue of a snake alpha-neurotoxin. In this molecule, Arg33, which is known to interact with the receptor, was replaced by a cysteine residue, where the photoprobe could be attached. Under inactinic light, this novel photosensitive snake toxin behaved as a reversible ligand on the Torpedo acetylcholine receptor. However, when irradiated at 391 nm, it generated a highly reactive arylcation which labeled mostly the receptor alpha-subunit, confirming the location of the tip of the second toxic loop near this receptor subunit. Finally, we showed that reduction of the disulfide bond, linking the ligand to the photocoupled receptor, allowed introduction of radioactivity on the labeled residue(s), opening the way to further characterization and avoiding the synthesis of a radioactive bifunctional photoprobe.
Teixeira-Clerc
Fatima
F
CEA/Saclay, Département d'Ingénierie et d'Etudes des Protéines, 91191 Gif-sur-Yvette, France.
Michalet
Sophie
S
Ménez
André
A
Kessler
Pascal
P
D000818:Animals; D001665:Binding Sites / Q000502:physiology; D003545:Cysteine / Q000138:chemical synthesis / Q000378:metabolism*; D009498:Neurotoxins / Q000138:chemical synthesis / Q000378:metabolism; D018733:Nicotinic Antagonists / Q000378:metabolism; D019920:Photoaffinity Labels / Q000138:chemical synthesis / Q000378:metabolism*; D010777:Photochemistry; D011485:Protein Binding / Q000502:physiology; D025941:Protein Interaction Mapping / Q000379:methods*; D014101:Torpedo
D009498:Neurotoxins; D018733:Nicotinic Antagonists; D019920:Photoaffinity Labels; D003545:Cysteine