Fluorescence resonance energy transfer: FRET studies of ligand binding to cell surface receptors

We describe a simple optical system employing fluorescence resonance energy transfer (FRET) to identify potential binding domains on the macrophage sc avenger receptor for the ligand maleylated bovine serum albumin (mal-BSA). Using a plasma membrane vesicle system, we placed donor probes on the ligan d and acceptor probes in the membrane to determine the distance of bound li gand from the cell surface. Two donors and three accepters were employed. T ransfer between ligand covalently modified with multiple dansyl molecules a nd hexadecanoylaminoeosin in the membrane yielded a distance of 46.5 +/- 7. 5 Angstrom; transfer from the same type of donors to octade-cylrhodamine B in the membrane gave a distance of 58.5 +/- 3.0 Angstrom. No transfer was o bserved between ligand mono-labeled with fluorescein and 1,1'-dioctadecyl-3 ,3,3',3' -tetramethylindocarbocyanineperchlorate in the membrane. This sugg ests that the orientation of mal-BSA bound to the receptor places the fluor escein probe too far from the lipid surface to experience energy transfer. The distance information identifies a potential location for the binding si te, which can be compared to structural information about the receptor and used to extract a binding sequence.