Fluorescence Detection of Lipid-Induced Oligomeric Intermediates Involved in Lysozyme “Amyloid-Like” Fiber Formation Driven by Anionic Membranes

Abstract

Recent findings implicate that “amyloid-like” fiber formation by several non-amyloidogenic proteins/peptides can be triggered by negatively charged lipid membranes. In order to elucidate the factors that govern the formation of these structures, the interaction of lysozyme with phosphatidylserine-containing lipid vesicles was studied by steady-state and time-resolved fluorescence measurements. Three consecutive stages in the interaction of Alexa488-fluorescently labeled lysozyme (Lz-A488) with acidic lipid vesicles were identified in ensemble average measurements. The variation of the mean fluorescence lifetime of Lz-A488 as a function of the surface coverage of the liposomes was quantitatively described by a cooperative partition model that assumes that monomeric lysozyme molecules partition into the bilayer surface and reversibly assemble into oligomers with <i>k</i> subunits (<i>k</i> ≥ 6). The global fit to the experimental data covering a wide range of experimental conditions was performed by taking into account electrostatic effects by means of the Gouy–Chapman theory using a single self-consistent pair of parameters (aggregation constant and stoichiometry). The lipid–protein supramolecular assemblies formed at a low lipid/protein molar ratio were further characterized by fluorescence lifetime imaging microscopy at the single-fiber level, which reported that quenched oligomers are the predominant species in these structures