Asymmetry in membranes strongly affects their biophysical characteristics and behaviour. It is a very important factor to consider for understanding the functioning of membranes in biological systems, especially in regards to lipid molecules that are known to localize in one leaflet over the other. Phosphatidic acid is one representative of this family of molecules. Reported to play a crucial role in lipid metabolism and suspected to participate in cellular signalling, its specific nature might only be exhibited in asymmetric systems. Here, using giant unilamellar vesicles, we studied two asymmetric phosphocholine-based bilayer systems with 1-palmitoyl-2-oleoyl phosphatidic acid (POPA) localized in either the inner or the outer membrane leaflet. We systematically characterized and compared the investigated systems to their symmetric counterpart. To verify and quantify the presence of POPA in the membrane, α-synuclein-mEGFP was used. We report an increase in the area compressibility and bending rigidity moduli in the asymmetric membranes compared to the symmetric ones. Furthermore, we show area compressibility differences depending on the localization of POPA in the leaflets. The results suggest the asymmetry affects both mechanics and phase state of the membrane.
