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

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

Membrane Order Is a Key Regulator of Divalent Cation-Induced Clustering of PI(3,5)P₂ and PI(4,5)P₂

Although the evidence for the presence of functionally important nanosized phosphorylated phosphoinositide (PIP)-rich domains within cellular membranes has accumulated, very limited information is available regarding the structural determinants for compartmentalization of these phospholipids. Here, we used a combination of fluorescence spectroscopy and microscopy techniques to characterize differences in divalent cation-induced clustering of PI­(4,5)­P₂ and PI­(3,5)­P₂. Through these methodologies we were able to detect differences in divalent cation-induced clustering efficiency and cluster size. Ca²⁺-induced PI­(4,5)­P₂ clusters are shown to be significantly larger than the ones observed for PI­(3,5)­P₂. Clustering of PI­(4,5)­P₂ is also detected at physiological concentrations of Mg²⁺, suggesting that in cellular membranes, these molecules are constitutively driven to clustering by the high intracellular concentration of divalent cations. Importantly, it is shown that lipid membrane order is a key factor in the regulation of clustering for both PIP isoforms, with a major impact on cluster sizes. Clustered PI­(4,5)­P₂ and PI­(3,5)­P₂ are observed to present considerably higher affinity for more ordered lipid phases than the monomeric species or than PI(4)­P, possibly reflecting a more general tendency of clustered lipids for insertion into ordered domains. These results support a model for the description of the lateral organization of PIPs in cellular membranes, where both divalent cation interaction and membrane order are key modulators defining the lateral organization of these lipids