Lipid binding of the exchangeable apolipoprotein apolipophorin III inducesmajor changes in fluorescence properties of tryptophans 115 and 130

The effect of lipid association on the local environment of the two tryptop han residues of Locusta migratoria apolipophorin III (apoLp-III) has been s tudied. In the lipid-free state, Trp115 in helix 4 is buried in the hydroph obic interior of the helix bundle, while Trp130 is located in a loop connec ting helices 4 and 5. Fluorescence spectroscopy of single Trp mutants revea led an emission maximum (lambda(max)) of 321 nm for apoLp-III-W@115 (excita tion 280 nm) which red-shifted to 327 nm upon binding to dimyristoylphospha tidylcholine (DMPC). ApoLp-III-W @ 130 displayed a lambda(max) of 338 nm wh ile interaction with DMPC resulted in a blue shift to 331 nm. Quenching stu dies with KI and acrylamide revealed decreased accessibility to Trp115 comp ared to Trp130, while lipid binding induced a decrease in quenching of Trp1 30. Aromatic circular dichroism (CD) spectra showed that Trp vibronic trans itions at 278, 286, and 294 nm for lipid-free apoLp-III were caused by Trp1 15. Upon lipid association, aromatic extrema are reversed in sign, becoming entirely negative with both Trp residues contributing to the vibronic tran sitions, implying restriction in side-chain mobility of these residues. Thu s, lambda(max), quencher accessibility, and aromatic CD analysis indicate t hat Trp115 is much less solvent-exposed than Trp130. Differences in fluores cence properties of these residues are minimized in the lipid-bound state, a result of relocation of Trp115 and Trp130 into the lipid milieu. Thus, in addition to the hydrophobic faces of apoLp-III amphipathic cr-helices, the loop region containing Trp130 comes in close contact with DMPC.