# Insights into Membrane Damage by \(\alpha\)-Helical and \(\beta\)-Sheet Peptides
## 3 Results
### Mechanism of Membrane Damage  
To investigate how peptide-lipid interactions at the bilayer surface lead to pore formation and water leakage, Figure 3A,B track the z-coordinates of the N- and C-termini of each peptide during the simulations. This analysis focuses on the 2-3 us interval, when water permeation begins to occur for both peptides--Figure 2B. The results show that both M03 and M04 peptides inserted into the membrane via their N-termini (upper panels), while their C-termini (lower panels) remained anchored at the membrane surface throughout the simulation. This behavior was initially surprising, given that the first eight residues are identical in both sequences, yet the extent of their water leakage differed substantially. Figure 3 also shows that, in the M03 system, three peptides (black, green, and purple lines) from the upper leaflet and one (pink) from the lower leaflet penetrated the membrane after 2.4 us. In contrast, intermittent leakage in the M04 system arose between 2.2 and 2.4 us, as one peptide from each leaflet (green and brown lines) converged near the center of the bilayer.  
Figure 3C,D illustrate the sequence of events leading to pore formation by M03 and M04 peptides. The deposition of small aggregates on the membrane surface (panels C-I and D-I) indicates that peptides can self-assemble in solution before interacting with lipids. As the simulation progressed, some of these peptide-peptide interactions were exchanged for peptide-lipid interactions. Pore formation starts when water molecules (red spheres) surrounding the N-termini (cyan spheres) of peptides in opposing leaflets converge at the bilayer center-C-II and D-II. This coalescence forms a continuous water column across the membrane, which enables the polar face of the helices to stabilize in the non-polar environment of the bilayer-C-IV and D-III. This pore remained stable until the end of the simulation for M03 peptides. In contrast, the pore was only metastable for M04 peptides and the membrane healed itself within 0.2 us (panel D-IV), expelling peptides to its surface.  
To understand why insertions of M03 peptides are more stable those of M04, Figure 4 examines the interaction of their C-termini with the lipid bilayer. Specifically, we focus on the terminal nonpolar residue, which is positioned closer to the C-terminus in M04 than in M03 (Figure 4A,B). When peptides rotate to insert their N-termini into the membrane, this nonpolar residue can become exposed to the aqueous environment--an energetically unfavorable configuration. We hypothesize that such exposure is more pronounced for M04, potentially explaining its reduced membrane-disrupting ability. To test this, we computed the solvent-accessible surface area (SASA) of the terminal phenylalanine, averaged over peptides that penetrated the membrane. For the M03 peptides, which become inserted into the membrane after 2.4 us, the SASA remained relatively constant throughout the simulation--Figure 4C. In contrast, the SASA of the M04 peptides that inserted between 2.2 and 2.4 us increased significantly, indicating greater exposure of the non-polar residues to water. This unfavorable interaction likely contributed to the reduced pore stability and membrane disruption observed for M04.  
Figure 3: (**A,B**) Positions of N-termini (top panels) and C-termini (bottom panels) for each peptide in simulations from Figure 2B: M03 (black) and M04 (red). Dashed gray lines correspond to the density peaks of phosphate atoms. (**C,D**) Representative molecular structures from simulations with M03 and M04 peptides during membrane disruption. Phosphate atoms are shown as gray spheres; N- and C-termini are marked by cyan and dark-gray spheres, respectively. Red beads indicate water oxygen atoms inside the bilayer.