Membrane fusion, essential to eukaryotic life, is broadly envisioned as a three-step process proceeding from contacting bilayers through two semistable, nonlamellar lipidic intermediate states to a fusion pore. Here, we introduced a new, to our knowledge, experimental approach to gain insight into the nature of the transition states between initial, intermediate, and final states. Recorded time courses of lipid-mixing, content-mixing, and content-leakage associated with fusion of 23 nm vesicles in the presence of poly(ethylene glycol) at multiple temperatures were fitted globally to a three-step sequential model to yield rate constants and thereby activation thermodynamics for each step of the process, as well as probabilities of occurrence of lipid-mixing, content-mixing, or content-leakage in each state. Experiments with membranes containing hexadecane, known to reduce interstice energy in nonlamellar structures, provided additional insight into the nature of fusion intermediates and transition states. The results support a hypothesis for the mechanism of stalk formation (step-1) that involves acyl chain protrusions into the interbilayer contact region, a hypothesis for a step-2 mechanism involving continuous interconversion of semistable nonlamellar intermediates, and a hypothesis for step-3 (pore formation) mechanism involving correlated movement of whole lipid molecules into hydrophobic spaces created by geometry mismatch between intermediate structures.

Viral fusion peptides are short N-terminal regions of type-1 viral fusion proteins that are critical for virus entry. Although the importance of viral fusion peptides in virus-cell membrane fusion is established, little is known about how they function. We report the effects of wild-type (WT) hemagglutinin (HA) fusion peptide and its G1S, G1V, and W14A mutants on the kinetics of poly(ethylene glycol)(PEG)-mediated fusion of small unilamellar vesicles composed of dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, sphingomyelin, and cholesterol (molar ratio of 35:30:15:20). Time courses of lipid mixing, content mixing, and content leakage were obtained using fluorescence assays at multiple temperatures and analyzed globally using either a two-step or three-step sequential ensemble model of the fusion process to obtain the rate constant and activation thermodynamics of each step. We also monitored the influence of peptides on bilayer interfacial order, acyl chain order, bilayer free volume, and water penetration. All these data were considered in terms of a recently published mechanistic model for the thermodynamic transition states for each step of the fusion process. We propose that WT peptide catalyzes Step 1 by occupying bilayer regions vacated by acyl chains that protrude into interbilayer space to form the Step 1 transition state. It also uniquely contributes a positive intrinsic curvature to hemi-fused leaflets to eliminate Step 2 and catalyzes Step 3 by destabilizing the highly stressed edges of the hemi-fused microstructures that dominate the ensemble of the intermediate state directly preceding fusion pore formation. Similar arguments explain the catalytic and inhibitory properties of the mutant peptides and support the hypothesis that the membrane-contacting fusion peptide of HA fusion protein is key to its catalytic activity.

The N-terminal fusion peptide (residues 770−788) of an S2 glycoprotein of the severe acute respiratory syndrome coronavirus (SARS-CoV), exposed upon receptor binding, is crucial for virus entry into the host cell. The fusion peptide alters the membrane organization and dynamics of the host membrane to facilitate membrane fusion. Generally, the effect of the fusion peptide on the membrane is sensitive to the lipid composition of target membranes. In the present work, we have utilized steady-state and time-resolved fluorescence spectroscopy in tandem with circular dichroism spectroscopy to elucidate the binding, oligomeric status, and secondary structure of the fusion peptide and its impact on the depthdependent membrane organization and dynamics. We have used depth-dependent fluorescence probes, 1,6-diphenyl-1,3,5-hexatriene (DPH) and its trimethylammonium derivative (TMA-DPH), to evaluate the effect of the peptide binding along the bilayer normal. We have exploited the energy transfer efficiency of tryptophan between TMA-DPH and DPH to determine the relative location of the solitary tryptophan present in the membrane-bound fusion peptide. We have further evaluated the effect of membrane cholesterol on the binding and organization of the peptide and the impact of peptide binding on the depth-dependent physical properties of the membrane at various cholesterol concentrations. Our results clearly demonstrate that the membrane cholesterol alters the oligomeric status of the membrane-bound peptide and the effect of peptide binding on the depth-dependent membrane organization and dynamics. The role of cholesterol is important, as the eukaryotic host cells contain a good amount of cholesterol that might be important for the entry of pathogenic viruses.

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Membrane fusion, one of the most essential processes in the life of eukaryotes, occurs when two separate lipid bilayers merge into a continuous bilayer and internal contents of two separated membranes mingle. There is a certain class of proteins that assist the binding of the viral envelope to the target host cell and catalyzing fusion. All class I viral fusion proteins contain a highly conserved 20-25 amino-acid amphipathic peptide at the N-terminus, which is essential for fusion activity and is termed as the 'fusion peptide'. It has been shown that insertion of fusion peptides into the host membrane and the perturbation in the membrane generated thereby is crucial for membrane fusion. Significant efforts have been given in the last couple of decades to understand the lipid-dependence of structure and function of the fusion peptide in membranes to understand the role of lipid compositions in membrane fusion. In addition, the lipid compositions further change the membrane physical properties and alter the mechanism and extent of membrane fusion. Therefore, lipid compositions modulate membrane fusion by changing membrane physical properties and altering structure of the fusion peptide.Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.