Ordering Effect Induced by SARS-CoV Fusion Peptides on Membranes Containing Negatively Charged Lipids Might be Important to Membrane Fusion

Abstract

vesicles, and peptide translocation across their membranes. First, we found that daptomycin binds to POPC:POPG GUVs in the presence of 0.5, 2.0, and 20 mM Ca 2þ . Second, we placed the vesicles in a solution containing carboxyfluorescein (CF) to test for influx. However, influx was never observed. Third, GUVs containing inner vesicles were observed to test for the translocation of daptomycin across the outer membrane of the GUVs, and onto the membrane of the inner vesicles. Again, translocation was never observed. However, in solutions containing 2 mM Ca 2þ a significant amount (about 50%) of membranes that had bound daptomycin collapsed shortly after binding occurred, resulting in the destruction of the GUVs. Furthermore, at high Ca 2þ concentrations (20 mM), the formation of daptomycin-rich clusters on the GUV membrane was observed. At lower concentrations of Ca 2þ (2 mM), daptomycin was distributed evenly on the GUV membrane. Lysette is a 22 amino acid peptide derived from staphylococcal d-lysin that forms an amphipathic a-helix when bound at membrane-water interfaces. We previously found that the experimentally determined DG of binding for lysette is more favorable than that predicted by the Wimley-White interfacial hydrophobicity scale by about 4 kcal/mol. Closer investigation of the amino acid composition of other peptides that are well described by the Wimley-White interfacial scale, such as melittin, led us to hypothesize that a preponderance of isoleucine (Ile) over leucine residues (Leu), as found in lysette, may be responsible for the deviation from the Wimley-White prediction. To test our hypothesis, we designed lysette variants lysette-I and lysette-L. In lysette-I, all Leu residues were replaced by Ile, and in lysette-L, all Ile were replaced by Leu. We also designed a melittin variant, iso-melittin, in which all Leu residues were replace by Ile. Peptide-lipid equilibrium dissociation constants and helicities of peptides bound to zwitterionic phosphatidylcholine (POPC) vesicles were determined by stopped-flow fluorescence and circular dichroism. If the hypothesis were correct, Lysette-I and iso-melittin should bind significantly better to zwitterionic bilayers than their Leu-rich counterparts, if the helicities of the lipid-bound peptides were not influenced by the Leu-Ile substitution. We found that the lysette-I bound significantly better to POPC vesicles than the lysette-L variant, as predicted. In the case of melittin, however, iso-melittin bound with roughly the same affinity to POPC bilayers as the original Leurich melittin. The results are discussed in terms of peptide structures in solution and when bound at the water-lipid interface, and the location of the lipid-bound states in the membrane.