Fluorescence energy transfer between cobra alpha-toxin molecules bound to the acetylcholine receptor.
An approach was developed with steady state fluorescence energy transfer measurements to examine the spatial relationship between the two alpha-toxins bound to the acetylcholine receptor. By taking advantage of the slow dissociation rates of alpha-toxins (Naja naja siamensis 3) from the receptor and of the equal probability with which alpha-toxins bind to the two alpha-toxin-binding sites, we derived an equation which allows prediction of a "true" efficiency of transfer based on the relationship between fractional site occupancy and the observed transfer efficiency ascertained from donor quenching. Using this approach, we examined the efficiency of energy transfer between two fluorescently labeled alpha-toxins, N epsilon-fluorescein isothiocyanate lysine 23 alpha-toxin and monolabeled tetramethylrhodamine isothiocyanate alpha-toxin bound to the receptor from the Torpedo californica electric organ. Significantly greater (32 versus 14%) energy transfer was observed with the membrane-associated than with the solubilized receptor, suggesting that transfer between fluorophores on separate receptor molecules is greater than that occurring intramolecularly between the two sites on the receptor. The magnitude of the distances calculated from the intrareceptor energy transfer efficiency combined with the considerable inter-receptor energy transfer indicate that the fluorophores would reside on the outer perimeter of the receptor molecule rather than near the central axis perpendicular to the plane of the membrane.
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003039:Cobra Neurotoxin Proteins / Q000378:metabolism*; D004546:Elapid Venoms / Q000378:metabolism*; D004557:Electric Organ / Q000378:metabolism*; D004735:Energy Transfer; D005456:Fluorescent Dyes; D007700:Kinetics; D008433:Mathematics; D011950:Receptors, Cholinergic / Q000302:isolation & purification / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014101:Torpedo
D003039:Cobra Neurotoxin Proteins; D004546:Elapid Venoms; D005456:Fluorescent Dyes; D011950:Receptors, Cholinergic; C025154:alpha-cobratoxin