We investigated the dependence of membrane binding on amino acid sequence for a series of amphipathic peptides derived from δ-lysin. δ-Lysin is a 26 amino acid, N-terminally formylated, hemolytic peptide that forms an amphipathic α-helix bound at membrane–water interfaces. A shortened peptide, lysette, was derived from δ-lysin by deletion of the four N-terminal amino acid residues. Five variants of lysette were synthesized by altering the amino acid sequence such that the overall hydrophobic moment remained essentially the same for all peptides. Peptide–lipid equilibrium dissociation constants and helicities of peptides bound to zwitterionic lipid vesicles were determined by stopped-flow fluorescence and circular dichroism. We found that binding to phosphatidylcholine bilayers was a function of the helicity of the bound peptide alone and independent of the a priori hydrophobic moment or the ability to form intramolecular salt bridges. Molecular dynamics (MD) simulations on two of the peptides suggest that sequence determines the insertion depth into the bilayer. The location of the two aspartate residues at the C-terminus of lysette-2 leads to a loss of helical content in the simulations, which correlates with faster desorption from the bilayer as compared to lysette. We also found a systematic deviation of the experimentally determined dissociation constant and that predicted by the Wimley–White interfacial hydrophobicity scale. The reason for the discrepancy remains unresolved but appears to correlate with a predominance of isoleucine over leucine residues in the lysette family of peptides.
Daptomycin is a clinically important 13 amino acid lipopeptide antibiotic. Its N-terminus is acylated with n-decanol and its C-terminal 10 amino acids form a ring. It is structurally and size-wise different from other well-known membrane-active antimicrobials. Daptomycin is known to disrupt the cytoplasmic membrane function of Gram-positive bacteria by causing leakage of potassium (and potentially other) ions, leading to the loss of membrane potential and cell death. The critical factor affecting the function of daptomycin is its interaction with negatively charged lipids such as PG in a calcium (Ca þþ) dependent manner. Based on previous research on cell membranes, daptomycin has been assumed to insert and aggregate in the membrane, and then to alter the membrane curvature. However these details have not been demonstrated by biophysical studies. In our aspirated GUV experiments, we found that with a DOPG-containing GUV and a sufficient concentration of Ca þþ , daptomycin can extract lipid molecules, and form lipid-peptide aggregations. The lipid-peptide aggregates did not occur if cardiolipin replaced PG, or if other divalent ions, such as Mg þþ , Ba þþ replaced Ca þþ. Similarly, daptomycin did not bind to a GUV when cardiolipin substituted for DOPG or in the absence of Ca þþ. Daptomycin with Ca þþ did bind to a pure DOPC GUV, but had no other effects. Furthermore, with the presence of daptomycin, DOPG and Ca þþ , we found Ca þþ permeates into the GUV, while a content dye, Texas red dextran, did not leak out. This result suggests that daptomycin and Ca þþ do not form pores on the membrane of DOPG-contained GUV, but cause leakage of ions. Finally, daptomycin with DOPG and Ca þþ produces a negative exciton CD couplet centered at the 225 nm absorption peak of Trptophan1 and Kynurenine13, whereas in all other conditions, the exciton CD couplet is positive.
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