title: 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 k subunits (k ≥ 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.
authors: Centro de Química-Física Molecular and Institute of Nanoscience and Nanotechnology, I.S.T, Universidade Técnica de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal.
authors: Ana M
authors: AM
authors: Melo
authors: Joana C
authors: JC
authors: Ricardo
authors: Aleksander
authors: A
authors: Fedorov
authors: Manuel
authors: M
authors: Prieto
authors: Ana
authors: A
authors: Coutinho
mesh_terms: D000682:Amyloid / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D002645:Chickens; D008081:Liposomes / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009113:Muramidase / Q000378:metabolism* / Q000648:ultrastructure; D010713:Phosphatidylcholines / Q000378:metabolism*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
chemical_list: D000682:Amyloid; D008081:Liposomes; D010713:Phosphatidylcholines; D009113:Muramidase