title: The effect of variable liposome brightness on quantifying lipid-protein interactions using fluorescence correlation spectroscopy.
abstract: Fluorescence correlation spectroscopy (FCS) has been increasingly used to study the binding of fluorescently-labeled peptides and proteins to phospholipid vesicles. In this work, we present a new method to analyze partition data obtained by this technique based on the assumption that the number of fluorescently-labeled protein molecules bound per liposome follows a Poisson distribution. To not overestimate the recovered partition coefficients, we first show that the variation in liposome brightness caused by this statistical distribution must be considered explicitly in data analysis when the parameter used to establish the partition curves is the fractional instead of the absolute amplitudes associated with the slowest diffusing particles in the system (lipid vesicles), a choice frequently made in FCS partition studies. We further extend the theoretical model describing the membrane partition of a fluorescently-labeled protein by considering the presence of a trace amount of free fluorescent dye (non-binding component) in the system. We show that this situation can account for an apparent maximal binding level lower than 100% in the experimental partitioning curves obtained for Alexa 488 fluorescently-labeled lysozyme and liposomes prepared with variable anionic phospholipid content. The extreme sensitivity of the FCS technique allowed uncoupling lysozyme partition from the protein-induced liposome aggregation, confirming that lysozyme binding to negatively charged liposomes is dominantly driven by electrostatic interactions.
authors.affiliation: Institute of Nanoscience and Nanotechnology, Instituto Superior Técnico, U.T.L, Complexo I, Av. Rovisco Pais, Lisboa, Portugal.
authors.forename: Ana M
authors.initials: AM
authors.lastname: Melo
authors.forename: Manuel
authors.initials: M
authors.lastname: Prieto
authors.forename: Ana
authors.initials: A
authors.lastname: Coutinho
mesh_terms: D008055:Lipids / Q000737:chemistry*; D008081:Liposomes; D011506:Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D008055:Lipids; D008081:Liposomes; D011506:Proteins