ANIONIC PHOSPHOLIPIDS MODULATE PEPTIDE INSERTION INTO MEMBRANES

While the insertion of a hydrophobic peptide or membrane protein segme nt into the bilayer can be spontaneous and driven mainly by the hydrop hobic effect, anionic lipids, which comprise ca. 20% of biological mem branes, provide a source of electrostatic attractions for binding of p roteins/peptides into membranes. To unravel the interplay of hydrophob icity and electrostatics in the binding of peptides into membranes, we designed peptides de novo which possess the typical sequence a-Trp-Al a-Ala-X-Ala-Ala-Ala-Lys-Lys-Lys-Lys-amide, where X residues correspond to ''guest'' residues which encompass a range of hydrophobicity (Leu, Ile, Gly, and Ser). Circular dichroism spectra demonstrated that pept ides were partially (40-90%) random in aqueous buffer but were promote d to form 100% a-helical structures by anionic lipid micelles. In neut ral lipid micelles, only the relatively hydrophobic peptides (X = L an d I) spontaneously adopted the a-helical conformation, but when 25% of negatively charged lipids were mixed in to mimic the content of anion ic lipids in biomembranes, the less hydrophobic (X = S and G) peptides then formed a-helical conformations. Consistent with these findings, fluorescence quenching by the aqueous-phase quencher iodide indicated that in anionic (dimyristoylphosphatidylglycerol) vesicles, the peptid e Trp residue was buried in the lipid vesicle hydrophobic core, while in neutral (dimyristoylphosphatidylcholine) vesicles, only hydrophobic (X = L and I) peptides were shielded from the aqueous solution. Trp e mission spectra of peptides in the presence of phospholipids doxyl-lab eled at the 5-, 7-, 10-, 12-, and 16-fatty acid positions implied not only a transbilayer orientation for inserted peptides but also that mi xed peptide populations (transbilayer + surface-associated) may arise. Overall results suggest that for hydrophobic peptides with segmental threshold hydrophobicity below that which promotes spontaneous membran e insertion, primary electrostatic attractions provided by anionic pho spholipids become essential for peptide binding and insertion to membr anes.