Viral membrane fusion is an orchestrated process triggered by membrane-anchored viral fusion glycoproteins. The S2 subunit of the spike glycoprotein from severe acute respiratory syndrome (SARS) coronavirus (CoV) contains internal domains called fusion peptides (FP) that play essential roles in virus entry. Although membrane fusion has been broadly studied, there are still major gaps in the molecular details of lipid rearrangements in the bilayer during fusion peptide-membrane interactions. Here we employed differential scanning calorimetry (DSC) and electron spin resonance (ESR) to gather information on the membrane fusion mechanism promoted by two putative SARS FPs. DSC data showed the peptides strongly perturb the structural integrity of anionic vesicles and support the hypothesis that the peptides generate opposing curvature stresses on phosphatidylethanolamine membranes. ESR showed that both FPs increase lipid packing and head group ordering as well as reduce the intramembrane water content for anionic membranes. Therefore, bending moment in the bilayer could be generated, promoting negative curvature. The significance of the ordering effect, membrane dehydration, changes in the curvature properties and the possible role of negatively charged phospholipids in helping to overcome the high kinetic barrier involved in the different stages of the SARS-CoV-mediated membrane fusion are discussed.

Primaquine (PQ) is a potent therapeutic agent used in the treatment of malaria and its mechanism of action still lacks a more detailed understanding at a molecular level. In this context, we used differential scanning calorimetry (DSC), pressure perturbation calorimetry (PPC), and electron spin resonance (ESR) to investigate the effects of PQ on the lipid phase transition, acyl chain dynamics, and on volumetric properties of lipid model membranes. DSC thermograms revealed that PQ stabilizes the fluid phase of the lipid model membranes and interacts mainly with the lipid headgroups. This result was revealed by the great effect on the pretransition of phosphatidylcholines and the destabilization of the inverted hexagonal phase of a phosphatidylethanolamine bilayer. Spin probes located at different positions along the lipid chain were used to monitor different membrane regions. ESR results indicated that PQ is effective in changing the acyl chain ordering and dynamics of the whole chain of dimyristoylphosphatidylcholine (DMPC) phospholipid in the rippled gel phase. The combined ESR and PPC results revealed that the slight DMPC volume changes at the main phase transition induced by the presence of PQ is probably due to a less dense lipid gel phase. At physiological pH, the cationic amphiphilic PQ strongly interacts with the lipid headgroup region of the bilayers, causing considerable disorganization in the hydrophobic core. These results shed light on the molecular mechanism of primaquine-lipid interaction, which may be useful in the understanding of the complex mechanism of action and/or the adverse effects of this antimalarial drug.

Antimicrobial peptides (AMPs) isolated from several organisms have been receiving much attention due to some specific features that allow them to interact with, bind to, and disrupt cell membranes. The aim of this paper was to study the interactions between a membrane mimetic and the cationic AMP Ctx(Ile21)-Ha as well as analogues containing the paramagnetic amino acid 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) incorporated at residue positions n = 0, 2, and 13. Circular dichroism studies showed that the peptides, except for [TOAC13]Ctx(Ile21)-Ha, are unstructured in aqueous solution but acquire different amounts of α-helical secondary structure in the presence of trifluorethanol and lysophosphocholine micelles. Fluorescence experiments indicated that all peptides were able to interact with LPC micelles. In addition, Ctx(Ile21)-Ha and [TOAC13]Ctx(Ile21)-Ha peptides presented similar water accessibility for the Trp residue located near the N-terminal sequence. Electron spin resonance experiments showed two spectral components for [TOAC0]Ctx(Ile21)-Ha, which are most likely due to two membrane-bound peptide conformations. In contrast, TOAC2 and TOAC13 derivatives presented a single spectral component corresponding to a strong immobilization of the probe. Thus, our findings allowed the description of the peptide topology in the membrane mimetic, where the N-terminal region is in dynamic equilibrium between an ordered, membrane-bound conformation and a disordered, mobile conformation; position 2 is most likely situated in the lipid polar head group region, and residue 13 is fully inserted into the hydrophobic core of the membrane.

Fusion peptides (FP) are prominent hydrophobic segments of viral fusion proteins that play critical roles in viral entry. FPs interact with and insert into the host lipid membranes, triggering conformational changes in the viral protein that leads to the viral-cell fusion. Multiple membrane-active domains from the severe acute respiratory syndrome (SARS) coronavirus (CoV) spike protein have been reported to act as the functional fusion peptide such as the peptide sequence located between the S1/S2 and S2’ cleavage sites (FP1), the S2’-adjacent fusion peptide domain (FP2), and the internal FP sequence (cIFP). Using a combined biophysical approach, we demonstrated that the α -helical coiled-coil-forming internal cIFP displayed the highest membrane fusion and permeabilizing activities along with membrane ordering effect in phosphatidylcholine (PC)/phosphatidylglycerol (PG) unilamellar vesicles compared to the other two N-proximal fusion peptide counterparts. While the FP1 sequence displayed intermediate membranotropic activities, the well-conserved FP2 peptide was substantially less effective in promoting fusion, leakage, and membrane ordering in PC/PG model membranes. Furthermore, Ca 2+ did not enhance the FP2-induced lipid mixing activity in PC/phosphatidylserine/cholesterol lipid membranes, despite its strong erythrocyte membrane perturbation. Nonetheless, we found that the three putative SARS-CoV membrane-active fusion peptide sequences here studied altered the physical properties of model and erythrocyte membranes to different extents. The importance of the distinct membranotropic and biological activities of all SARS-CoV fusion peptide domains and the pronounced effect of the internal fusion peptide sequence to the whole spike-mediated membrane fusion process are discussed.