Fluorescence detection of lipid-induced oligomeric intermediates involved in lysozyme "amyloid-like" fiber formation driven by anionic membranes

J Phys Chem B. 2013 Mar 14;117(10):2906-17. doi: 10.1021/jp310396v. Epub 2013 Feb 1.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amyloid / metabolism*
  • Amyloid / ultrastructure
  • Animals
  • Chickens
  • Liposomes / metabolism*
  • Microscopy, Confocal
  • Microscopy, Fluorescence
  • Models, Biological
  • Muramidase / metabolism*
  • Muramidase / ultrastructure
  • Phosphatidylcholines / metabolism*
  • Protein Binding
  • Spectrometry, Fluorescence

Substances

  • Amyloid
  • Liposomes
  • Phosphatidylcholines
  • Muramidase