C-terminus of a long alpha-neurotoxin is highly mobile when bound to the nicotinic acetylcholine receptor: a time-resolved fluorescence anisotropy approach.

To better understand how alpha-neurotoxins interact with the acetylcholine receptor, four fluorescein isothiocyanate derivatives of the siamemsis alpha-cobratoxin were prepared (conjugated to the epsilon-amino group in Lys(23), Lys(35), Lys(49), or Lys(69)) and the time-resolved fluorescence anisotropy of each conjugate was measured free in solution and bound to the Torpedo acetylcholine receptor. All the conjugated reporter groups displayed a high and comparable level of mobility free in solution. When receptor bound, on the other hand, significant differences in the conformational dynamics of the reporter groups were observed with the C-terminal Lys(69) derivative displaying by far the greatest mobility strongly suggesting that the C-terminal domain of the bound neurotoxin is highly mobile and does not participate in the toxin-nAChR binding surface. Additionally, this study demonstrates the utility of time-resolved fluorescence anisotropy to characterize the interaction of heteroproteins.