# Insights into Membrane Damage by \(\alpha\)-Helical and \(\beta\)-Sheet Peptides
## 4 Conclusions  
In summary, this paper compared the lipid membrane damage by four neutral amphipathic peptides. Two of these peptides, named M01 and M02, are 8-residue isomers made using four phenylalanine, two glutamic acids, and two lysines. At the water-lipid interface, peptides M01 and M02 fold into \(\beta\)-strands and \(\alpha\)-helices, respectively. In unbiased all-atom simulations, M01 peptides were found to aggregate promptly into long \(\beta\)-sheets that spanned both leaflets of the bilayer, allowing water to permeate the membrane. In contrast, \(\alpha\)-helical M02 peptides remained dispersed on the membrane surface, without spanning its leaflets or causing water leakage. To provide insights into the potential of \(\alpha\)-helices to damage lipid membranes, two longer (16 residues) versions of the M02 peptide, named M03 and M04, were studied. The M03 peptide is made by repeating the M02 sequence twice. This results in a peptide with \(\mu=0.57\), and N- and C-termini that are nonpolar and charged, respectively. The M04 peptide is made by moving one of the non-polar amino acids in the middle of the M03 sequence to the C-terminal. The latter modification accounts for a sequence in which non-polar and charged amino acids are more sharply segregated at the water-lipid interface (\(\mu=0.87\)), and with both N- and C-terminals exhibiting non-polar residues. In leakage experiments, this modification increased the minimal peptide concentration required to induce water permeation by at least a factor of 5. The peptide concentration was less than 1:50 peptide-lipid for M03 peptides and 1:10 for M04. This difference in behavior in the all-atom simulations is explained by the higher propensity of M03 peptides to insert themselves into the bilayer spanning its leaflets. This starts with peptides parallel to the membrane rotating around charged residues in the C-terminal, which behave as a pivot point. In this process, water molecules are dragged to the interior of the bilayer. In contrast, rotation is mostly inhibited for M04 peptides, as non-polar residues close to the pivot point at the C-terminal become partially exposed to the solvent when M04 helices rotate to span the bilayer. Thus, in the computer simulations and experiments, M04 peptides only induced water permeation at higher concentrations compared to M03. Taken together, our results highlight properties in the amino acid sequence that account for membrane damage by amphipathic peptides. These include partitioning of polar-nonpolar residues at the water-lipid interface, which determines the secondary structure of peptides; the length of these secondary structures compared to the bilayer thickness; and the difference in hydropathy between the N- and C-terminals. Consistent with other studies, we found that peptides that form secondary structures with end-to-end distance of \(\sim\)2 nm or more can induce water permeation [35; 36; 37]. This is the case for the 8-residue \(\beta\)-strand (M01) and 16-residue \(\alpha\)-helical (M03 and M04) peptides studied here, but not for the 8-residue \(\alpha\)-helical M02 peptide. Compared to M03, the M04 peptide could have been expected to be more efficient at inducing water permeation, since its amphiphilicity is enhanced. However, our simulations showed that its C-terminal non-polar residue protrudes from the membrane as its N-terminus inserts into the bilayer, causing it to become hydrated and less prone to forming pores. Thus, although our study confirms that amphiphilicity is important in accounting for secondary structure formation, other aspects of the amino acid sequence, e.g., differences in the hydropathy of N- and C-terminals, can modulate the ability of a peptide to damage the membrane via pore formation. We anticipate that some of the effects of C-terminal modifications reported in the literature [42; 43] may be explained by this difference, which should be further explored as a design principle.