Lysozyme interaction with negatively charged lipid bilayers: protein aggregation and membrane fusion

Abstract

We report on the interaction of hen egg-white lysozyme (HEWL) with lipid vesicles in terms of surface-induced protein conformational variation and subsequent aggregation. In particular, we investigated the variations of the secondary structure of native lysozyme in the presence of liposomes with different surface charge density, resulting from different molar ratios of the zwitterionic POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and the negatively charged POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)). It is well known that the main driving force involved in the interaction between globally anionic liposomes and lysozyme is electrostatic compensation, which, in some cases, produces extended aggregation. Moreover the presence of membranes can induce unfolding in the protein. In order to understand the main determinants of such phenomena, we probed simultaneously lysozyme-induced vesicle fusion events, variations in the secondary structure of the protein and its effect on liposomal membrane fluidity. We found that above a charge-density threshold, the association with vesicles results in modifications of the native structure associated with a decrease of liposomal membrane fluidity. Electron microscopy images revealed that the above described interactions result in mesoscopic structural changes, i.e. liposome clustering and fusion, together with the appearance of elongated structures, reminiscent of fibrillar aggregates. Additionally, a confocal microscopy analysis revealed that upon interaction with giant unilamellar vesicles (GUVs) of the same lipid composition where the above interactions were observed, a prompt insertion of lysozyme in the membrane occurs, leading to vesicle clustering, with the appearance of elongated structures where both the lipid and the protein are present.