The Effect of Membrane Spanning Peptides on Laurdan and Di-4-ANEPPDHQ Emission Spectra

Abstract

Laurdan and di-4-ANEPPDHQ are membrane order reporting probes whose peak emission wavelengths depend on the lipid environment. The probes report membrane order through different mechanisms, laurdan by sensing changes in the level of water penetration into the lipid bilayer and Di-4-ANEPPDHQ by sensing dipole potential changes in the membrane. Laurdan and di-4-ANEPPDHQ are excited by UV and blue light, respectively, and both show an ∼50 nm blue shift in emission for membranes in liquid-ordered (lo) phase versus membranes in liquid-disordered (ld) phase.Large unilamelar vesicles (LUVs) in lo phase were created by mixing sphingomyelin, DOPC spiked with 5% DPPG and cholesterol in 1:1:2 ratio. LUVs in ld phase were created only using DOPC spiked with 5% DPPG. Transmembrane polypeptides, mastoparan (a 14-residue peptide toxin isolated from wasp venom) or bovine prion protein (N-terminal residues 1-30), were added to 100 nm LUVs stained with 1μM laurdan or di-4-ANEPPDHQ in up to 1:10 protein/total lipid ratio. The laurdan and the di-4-ANEPPDHQ emission spectra were measured for both lo and ld phase LUVs before and after the addition of polypeptides and remained unchanged for all conditions. The integrity and size distribution of the LUVs upon addition of the polypeptides were determined by dynamic laser light scattering and no changes were detected. The insertion efficiency of the polypeptides into LUVs was determined by measuring their 3D polypeptide structure by circular dichroism. Both polypeptides had an alpha helical conformation compatible with them being inserted into the lipid bilayer. Our results suggest that the presence of proteins in biological membranes does not influence the spectra of laurdan and di-4-ANEPPDHQ showing that the dyes report solely on lipid order.