PROBING THE STRUCTURE OF THE LIGAND-BINDING CAVITY OF LIPOCALINS BY FLUORESCENCE SPECTROSCOPY

The lipocalin superfamily constitutes a phylogenetically conserved gro up of more than 40 proteins that function in the binding and transport of a variety of physiologically important ligands, Members of this fa mily subserve diverse functions as carriers of retinoids (retinol bind ing protein), odorants (odorant binding proteins), chromophores (insec ticyanin. INS), pheromones (aphrodisin) and sterols (apolipoprotein D, apoD), Despite the pivotal importance of the ligand binding function of these proteins, a suitable approach for characterizing the molecula r determinants of such binding has not been available, In studies usin g three homogeneously purified lipocalins INS, beta-lactoglobulin (BLG ) and human apoD, we find that the fluorescence reporter BIS (1,1'-bi( 4-anilino) naphthalene-5,5'-disulfonic acid) is an ideal candidate for use in rapid kinetic experiments and in fluorescence resonance energy transfer (FRET), These methods require only small amounts of reagents and yield molecular coordinates of the ligand binding cavity of lipoc alins in solution that are in remarkably close agreement to those obta ined from crystallographic work with solids, Extremely fast ligand bin ding dynamics is indicated.