Exploring Fluorescence Lifetime and Homo-FRET Measurements to Monitor Lysozyme Oligomerization in Anionic Lipid Membranes: Relation to “Amyloid-Like” Fibril Formation

Abstract

Negatively-charged lipid membranes have been suggested to trigger “amyloid-like” fibril formation by several non-amyloidogenic proteins, e.g. lysozyme [1]. We aimed to elucidate the factors that govern the formation of these “amyloid-like” fibrils and to characterize their structural and dynamical properties. Lysozyme was labeled with Alexa 488 (A488-Lz) and its interaction with POPC LUVs containing 20 and 30 mol% of POPS was studied using both steady-state and time-resolved fluorescence techniques. The variation of the mean fluorescence lifetime of A488-Lz as a function of the surface coverage of the liposomes was quantitatively described by a three-state model that assumes that monomeric lysozyme molecules partition into the bilayer surface and reversibly assemble into oligomers with N subunits (N ≥ 6) (cooperative partition model). The global fit was done using the partition coefficients previously determined for A488-Lz by fluorescence correlation spectroscopy (FCS) [2] and by taking into account electrostatic effects by means of the Gouy-Chapman theory. To better evaluate the oligomerization state of membrane-bound lysozyme, the steady-state fluorescence anisotropy of A488-Lz was also measured for two different fluorophore labeling. The extent of energy migration between A488-Lz (decrease in fluorescence anisotropy) was adequately described only for N= 6 ± 1 when the binomial distribution of fluorescently-labeled monomers among the oligomers was considered. Finally, the lipid-protein supramolecular complexes formed at a low lipid/protein molar ratio [1] were characterized by fluorescence lifetime imaging microscopy (FLIM). The average fluorescence lifetime of A488-Lz had a uniform spatial distribution on these structures, being much shorter than the values measured for free and membrane-bound monomeric A488-Lz, reporting the aggregated state of lysozyme.[1] Zhao et al. 2004 Biochemistry 43: 10302[2] Melo et al. 2011 Biochim. Biophys. Acta 1808: 2559