Herein, we describe a straightforward, easy method for generating stable lipid bilayer vesicle nanoparticles and show their usefulness for efficient in vitro tracking of lipid intermembrane transfer activity. Bilayer model membrane discs, i.e., bicelles, are initially produced and then rapidly diluted, a process that transforms them into stable uniform-sized, unilamellar lipid bilayer vesicles as confirmed by cryo-EM. The resulting "donor" vesicles contain minor amounts of fluorescently labeled "substrate" lipids for specific lipid transfer proteins (LTPs), whereas "acceptor" vesicles do not. Upon donor and acceptor vesicle incubation with LTPs, fluorophore-labeled lipid departure from donor vesicles is continuously tracked in real time via Förster resonance energy transfer. FRET and various other means for measuring in vitro intermembrane lipid transfer have been detailed previously (Kenoth R, Brown RE, Kamlekar RK, Methods Mol Biol 1949:237-256 (2019)). The novelty of the methodology presented here lies in the use of bicelle dilution to easily generate stable donor and acceptor nanoparticles needed for tracking lipid transfer activity. The approach is readily adjustable for assessing lipid transfer involving (i) various lipid types by specific LTPs, (ii) crude or highly purified protein sample preparations, and (iii) no protein, i.e., spontaneous, and should facilitate the development of high throughput, plate reader-type LTP screening assays.
Keywords: Amphitropic protein; Bicelle dilution; CPTP; Cryo-EM; Fluorescence; Förster resonance energy transfer (FRET); GLTP; GLTP-fold; Glycosphingolipid; Lipid intermembrane transfer; Membrane interaction; Unilamellar lipid bilayer vesicle nanoparticles.
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