RESUMO
We have measured fluorescence resonance energy transfer (FRET) between a fluorescent antagonist, bound to the purified detergent-solubilized serotonin type 3 receptor, and a lipophilic acceptor probe partitioned into the micelle surrounding the detergent-solubilized receptor. The experimentally observed FRET efficiency was evaluated on the basis of the characteristic dimensions of the receptor-micelle complex and the average number of acceptor molecules in such micelles. The binding site was determined to be 5.4 +/- 0.9 nm above the center of the detergent micelle. The experiments were performed below the critical micellar concentration of the detergent (C(12)E(9)) used to solubilize the receptor, under which conditions it was demonstrated that the ligand binding activity was fully preserved. This reduces considerably the fluorescence background arising from probes not associated with the receptor, allowing a precise determination of the transfer efficiency.
