Asymmetric distribution of aminophospholipids stiffens fluid membranes

Cellular envelopes contain a large number of lipid species that distribute asymmetrically between the two leaflets of the bilayer. For example, mammalian plasma membranes are composed of an outer leaflet enriched in cholinephospholipids, while the majority of the aminophospholipids are confined to the inner leaflet. Membrane asymmetry can theoretically lead to distinct effects regarding its elastic behavior, either due to lipid specific properties (e.g. size, shape), or simply lipid over/under-crowding of a given leaflet. Corresponding experimental data are scarce. This prompted us to determine the structure and bending rigidity of minimal mammalian plasma membrane mimics composed of milk sphingomyelin (MSM), palmitoyl oleoyl phosphatidylcholine (POPC), palmitoyl oleoyl phosphatidylethanolamine (POPE), and palmitoyl oleoyl phosphatidylserine (POPS) using small-angle neutron/X-ray scattering and neutron spin echo spectroscopy. Most strikingly, we observed an anomalous stiffening of freely-floating fluid vesicles (size ∼ 100 nm) in case of inner leaflets composed of POPE/POPS mixtures and outer leaflets enriched in POPC. That is, the bending rigidities of POPCout/(POPE/POPS)in vesicles did not only exceed those of their scrambled (symmetric) analogs, but also those of symmetric vesicles mimicking either their inner and outer leaflets. Addition MSM in the outer leaflet reduced but not abolished this effect, possibly due to hydrocarbon chain interdigitation. To reconcile our findings, we speculate that lipid shape and charge asymmetry may increase the weight of short-wavelength undulatory modes in membranes by inducing a coupling at distances smaller than the membrane thickness.