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

Abstract

The effect of lipid association on the local environment of the two tryptophan residues of Locusta migratoria apolipophorin III (apoLp-III) has been studied. In the lipid-free state, Trp115 in helix 4 is buried in the hydrophobic interior of the helix bundle, while Trp130 is located in a loop connecting helices 4 and 5. Fluorescence spectroscopy of single Trp mutants revealed an emission maximum (lambda(max)) of 321 nm for apoLp-III-W@115 (excitation 280 nm) which red-shifted to 327 nm upon binding to dimyristoylphosphatidylcholine (DMPC). ApoLp-III-W@130 displayed a lambda(max) of 338 nm while interaction with DMPC resulted in a blue shift to 331 nm. Quenching studies with KI and acrylamide revealed decreased accessibility to Trp115 compared to Trp130, while lipid binding induced a decrease in quenching of Trp130. Aromatic circular dichroism (CD) spectra showed that Trp vibronic transitions at 278, 286, and 294 nm for lipid-free apoLp-III were caused by Trp115. Upon lipid association, aromatic extrema are reversed in sign, becoming entirely negative with both Trp residues contributing to the vibronic transitions, implying restriction in side-chain mobility of these residues. Thus, lambda(max), quencher accessibility, and aromatic CD analysis indicate that Trp115 is much less solvent-exposed than Trp130. Differences in fluorescence 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 alpha-helices, the loop region containing Trp130 comes in close contact with DMPC.