Molecular collisions or resonance energy transfer in lipid vesicles? A methodology to tackle this question

Abstract

In this work, molecular interactions in a lipid membrane are discussed through fluorescence spectroscopy data, both experimentally and theoretically. In particular, the fluorescence quenching mechanisms between the fluorescent probe 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan) and the potential drug 2-nitrobenzaldehyde-thiosemicarbazone (2-TSC) were studied, both inserted in a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DMPC) model membrane. The fluorescence intensity and the lifetime of Laurdan decrease dramatically in the presence of 2-TSC, in both gel and fluid phases of the DMPC bilayer. It is shown here how to identify the correct quenching mechanism, by conducting a careful analysis of the fluorescence data. The analysis of the bimolecular constant values obtained through the Stern-Volmer equation, considering the collisional mechanism, made clear the incompatibility of the obtained values with estimated diffusion coefficients for Laurdan and 2-TSC inserted into lipid bilayers. On the other hand, using the Förster’s theory of resonance energy transfer (FRET) we obtained results in good agreement with the already known dynamic characteristics of a DMPC bilayer, at its both gel and fluid phases. Through spectroscopy data and computational calculation, Förster distance, energy transfer efficiency and distance distribution were obtained for the donor/acceptor pair Laurdan/2-TSC, at both gel and fluid phases of the bilayer. The distance distribution reflects the occurrence of FRET involving donor/acceptor pairs in the same leaflet of the lipid bilayer and pairs in opposite leaflet, and these results are in good agreement with our previous proposal about the lateral organization and position of Laurdan and 2-TSC molecules in a DMPC bilayer. All these results lead us to conclude that FRET between the donor Laurdan and the acceptor 2-TSC is the mechanism responsible for non-radiative deexcitation of Laurdan. The methodology used here could be extended to other pairs of donor/acceptor molecules, to contribute to the knowledge about their localizations in lipid membranes.