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.
Johnson
David A
DA
Division of Biomedical Sciences, University of California, Riverside, CA 92521-0121, USA. david.johnson@ucr.edu
D000818:Animals; D017815:Elapidae; D016650:Fluorescein-5-isothiocyanate; D005454:Fluorescence Polarization; D009068:Movement; D009498:Neurotoxins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000378:metabolism*; D013997:Time Factors; D014101:Torpedo
D009498:Neurotoxins; D011978:Receptors, Nicotinic; D016650:Fluorescein-5-isothiocyanate