RETINOL AND RETINOIC ACID BIND TO A SURFACE CLEFT IN BOVINE BETA-LACTOGLOBULIN - A METHOD OF BINDING-SITE DETERMINATION USING FLUORESCENCE RESONANCE ENERGY-TRANSFER

Two potential ligand binding sites in the lipocalin beta-lactoglobulin have been postulated for small hydrophobic molecules such as retinol or retinoic acid. An agreement on one of the two alternatives, an inte rior cavity or a surface cleft, however, has not been achieved. In ord er to discriminate between these two possibilities, we measured the ef ficiency of fluorescence resonance energy transfer between the two int rinsic Trp-residues of beta-lactoglobulin and the ligands retinol, ret inoic acid and bis-ANS. Using the crystallographic coordinates of beta -lactoglobulin, this efficiency could be accurately computed fur both the interior cavity and the surface cleft as ligand binding sites. For the surface cleft, the theoretical value was found to be in excellent agreement with the measured value, whereas for the interior cavity an y reasonable agreement would require a dramatic ligand-induced conform ational change that can be ruled out due to the protein's known struct ural stability. Our conclusion that these ligands bind to the surface pocket rather than the interior cavity was further confirmed by compet itive binding studies. (C) 1998 Elsevier Science B.V. All rights reser ved.