Interaction of the alpha-helices of apolipophorin III with the phospholipid acyl chains in discoidal lipoprotein particles: A fluorescence quenching study

Quenching of tryptophan fluorescence by nitroxide-labeled phospholipids and nitroxide-labeled fatty acids was used to investigate the lipid-binding do mains of apolipophorin III. The location of the Trp residues relative to th e lipid bilayer was investigated in discoidal lipoprotein particles made wi th 1-palrriitoyl-2-oleoyl-sn-glycero-3-phosphocholine and five different si ngle-Trp mutants of apoLp-III. A comparison of the quenching efficiencies o f phospholipids containing nitroxide groups at the polar head, and at posit ions 5 and 16 of the sn-2 acyl chain, indicated that the protein is interac ting with the acyl chains of the phospholipid along the periphery of the bi layer of the discoidal lipoprotein. N-Bromosuccinimide readily abolished 10 0% of the fluorescence of all Trp residues in the lipid-bound state. Larger quenching rates were observed for the Trp residues in helices 1, 4, and 5 than for those located in helices 2 and 3, suggesting differences between t he interaction of these two groups of helices. However, the extent of Trp f luorescence quenching observed in lipoproteins made with any of the mutants was comparable to that reported for deeply embedded Trp residues, suggesti ng that all Trp residues interact with the phospholipid acyl chains. This s tudy provides the first experimental evidence of a massive interaction of t he alpha -helices of apoLp-III with the phospholipid acyl chains in discoid al lipoproteins. The extent of interaction deduced is consistent with the a polipoprotein adopting a highly extended conformation.