Fluorescence studies of the beta-adrenergic receptor topology

The nature of the propranolol binding site of the beta-adrenergic receptor has been examined by utilizing the intrinsic fluorescence of propranolol as a probe. Additionally, the spatial relationship between the propranolol binding site and membrane tryptophan has been examined by utilizing I-quenching of intrinsic tryptophan fluorescence, chemical modification of membrane tryptophan, and singlet-singlet energy transfer between membrane-bound propranolol and tryptophan. Propranolol, at concentrations consistent with specific beta-receptor binding, protected approximately 42% of the membrane tryptophan fluorescence from I-quenching. Further, in the presence of propranolol, the apparent quenching constant (kq) was altered from 3.6 to 21.8 M-1. Reaction of the membrane fragments with 2-hydroxy-5-nitrobenzyl bromide (Koshland's reagent I) in the presence and absence of propranolol indicated that low concentrations of propranolol protected approximately 45% of the membrane tryptophan from the reagent. The singlet-singlet energy transfer from tryptophan to propranolol was determined by sensitized emission. The distance between these two species was found to be less than 20 A. These results have been interpreted to indicate that propranolol, when bound to the beta-adrenergic receptor, is situated such that its naphthyl moiety is inserted into a tryptophan-rich hydrophobic pocket of the receptor.