Fluorescence spectroscopy of single tryptophan mutants of apolipophorin-III in discoidal lipoproteins of dimyristoylphosphatidylcholine.

The structure of the exchangeable apolipoprotein, apolipophorin-III from Locusta migratoria, apoLp-III, is described as a bundle of five amphipathic alpha-helices. To study the interaction of each of the helices of apoLp-III with a lipid surface, we designed five single-Trp mutants, each containing a Trp residue in a different alpha-helix. The Trp residues were located in the nonpolar domains of the amphipathic alpha-helices. The kinetics of the spontaneous interaction of the mutants with dimyristoylphosphatidylcholine (DMPC) indicated that all mutants behaved as typical exchangeable apolipoproteins. Circular dichroism in the far-UV indicated that all proteins have a high and similar helical content in the lipid-bound state. The interaction of the Trp residues with the lipid surface was investigated in recombinant lipoprotein particles made with DMPC. The properties of the Trp residues were investigated by fluorescence spectroscopy. These studies showed major changes in the spectroscopic properties of the Trp residues upon binding to lipid. These changes are observed with all single-Trp mutants, indicating that a major conformational change, which affects the properties of all helices, takes place upon binding to lipid. The position of the fluorescence maximum, the quenching efficiency of acrylamide as determined by steady-state and time-resolved fluorescence, and the fluorescence lifetimes of the single-Trp mutants suggest that helices 1, 4, and 5 interact with the nonpolar domains of the lipid. The properties of the Trp in helices 2 and 3 suggest that these helices adopt a different binding configuration than helices 1, 4, and 5. Helices 2 and 3 appear to be interacting with the polar headgroups of the phospholipids or constitute a different domain that does not interact with the lipid surface.