Fluorophore position of headgroup-labeled Gb3 glycosphingolipids in lipid bilayers

Fluorescent lipid probes are an invaluable tool for investigating lipid membranes. In particular, localizing certain receptor lipids such as glycosphingolipids within phase-separated membranes is of pivotal interest to understanding the influ-ence of protein-receptor lipid binding on membrane organization. However, fluorescent labeling can readily alter the phase behavior of a lipid membrane because of the interaction of the fluorescent moiety with the membrane interface. Here, we inves-tigated Gb3 glycosphingolipids, serving as receptor lipids for the protein Shiga toxin, with a headgroup attached BODIPY fluorophore separated by a polyethylene glycol (PEG) spacer of different lengths. We found that the diffusion coefficients of the fluorescently labeled Gb3 species in 1,2-dioleoyl-sn-glycero-3-phosphocholine/Gb3 (98:2, n/n) supported lipid bilayers are unaltered by the PEG spacer length. However, quenching as well as graphene-induced energy transfer experiments indicated that the length of the PEG spacer (n = 3 and n = 13) alters the position of the BODIPY fluorophore. In particular, the graphene-induced energy transfer technique provided accurate end-to-end distances between the fluorophores in the two leaflets of the bilayer thus enabling us to quantify the distance between the membrane interface and the fluorophore with sub-nanometer resolution. The spacer with three oligo ethylene glycol groups positioned the BODIPY fluorophore directly at the membrane inter-face favoring its interaction with the bilayer and thus may disturb lipid packing. However, the longer PEG spacer (n = 13) separated the BODIPY moiety from the membrane surface by 1.5 nm.SIGNIFICANCE Glycosphingolipids are pivotal for the binding of bacterial toxins to lipid membranes. To investigate their distribution in membranes and the influence of protein binding on receptor lipid reorganization, fluorescently labeled glycosphingolipids are highly desirable. However, attaching a fluorophore to the lipid headgroup can readily alter the membrane's phase behavior owing to the interaction of the fluorescent moiety with the membrane interface. Graphene-induced energy transfer experiments on supported bilayers allowed us to quantify the distance between the fluorophore and the membrane interface with sub-nanometer resolution. These results in conjunction with fluorescence recovery after photobleaching and quenching experiments allowed the unraveling of the interaction of the BODIPY moeity of Gb3 with lipid bilayers as a function of the polyethylene glycol spacer length.