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Membrane fusion is one of the most important processes for the survival of eukaryotic cells and entry of enveloped viruses to the host cells. Lipid composition plays a crucial role in the process by modulating the organization and dynamics of the membrane, as well as the structure and conformation of membrane proteins. Phosphatidylethanolamine (PE), a lipid molecule with intrinsic negative curvature, promotes membrane fusion by stabilizing the non-lamellar intermediate structures in the fusion process. Conversely, oleic acid (OA), with intrinsic positive curvature, inhibits membrane fusion. The current study aimed to investigate polyethylene glycol-mediated lipid mixing, content mixing, content leakage, and depth-dependent membrane organization and dynamics, using arrays of steady-state and time-resolved fluorescence techniques, to determine the causative role of PE and OA in membrane fusion. The results demonstrated that the presence of 30 mol % PE in the membrane promotes membrane fusion through a mechanism that circumvents the classical stalk model. On the contrary, membranes containing OA showed reduced rate and extent of fusion, despite following the same mechanism. Collectively, our findings in terms of membrane organization and dynamics indicated a plausible role of PE and OA in membrane fusion.

Graphic Abstract

Membrane fusion is the primary step in the entry of enveloped viruses into the host cell. Membrane composition modulates the membrane fusion by changing the organization dynamics of the fusion proteins, peptides, and membranes. The asymmetric lipid compositions of the viral envelope and the host cell influence the membrane fusion. Cholesterol is an important constituent of mammalian cells and plays a vital role in the entry of several viruses. In our pursuit of developing peptide-based general fusion inhibitors, we have previously shown that a coronin 1-derived peptide, TG-23, inhibited polyethylene glycol-induced fusion between symmetric membranes without cholesterol. In this work, we have studied the effect of TG-23 on the polyethylene glycol-mediated fusion between 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG) (60/30/10 mol %) and DOPC/DOPE/DOPG/CH (50/30/10/10 mol %) membranes and between DOPC/DOPE/DOPG (60/30/10 mol %) and DOPC/DOPE/DOPG/CH (40/30/10/20 mol %) membranes. Our results demonstrate that the TG-23 peptide inhibited the fusion between membranes containing 0 and 10 mol % cholesterol though the efficacy is less than that of symmetric fusion between membranes devoid of cholesterol, and the inhibitory efficacy becomes negligible in the fusion between membranes containing 0 and 20 mol % cholesterol. Several steady-state and time-resolved fluorescence spectroscopic techniques have been successfully utilized to evaluate the organization, dynamics, and membrane penetration of the TG-23 peptide. Taken together, our results demonstrate that the reduction of the inhibitory effect of TG-23 in asymmetric membrane fusion containing cholesterol of varying concentrations is not due to the altered peptide structure, organization, and dynamics, rather owing to the intrinsic negative curvature-inducing property of cholesterol. Therefore, the membrane composition is an added complexity in the journey of developing peptide-based membrane fusion inhibitors.