# Insights into Membrane Damage by \(\alpha\)-Helical and \(\beta\)-Sheet Peptides
## 3 Results  
### Water Permeation  
All-atom simulations of peptide-membrane systems were performed to assess the potential of our model peptides to disrupt the integrity of lipid bilayers. Figure 2 shows the cumulative number of water permeation events over time, with dashed lines representing control simulations without peptides. Figure 2A presents the results for the 8-residue peptides M01 [Ac-(FKFE)\({}_{2}\)-NH\({}_{2}\)] and M02 [Ac-FFKKFFEE-NH\({}_{2}\)], which preferentially adopted \(\beta\)-sheet and \(\alpha\)-helical conformations, respectively, at the water-lipid interfaces [28]. In these simulations, 10 peptides were added into the solution every 1 us, which allowed for their gradual adsorption and aggregation on the membrane. This corresponds to an insertion of 80 amino acids at each step-Figures S1 and S2. M01 peptides adsorb to the membrane and progressively form \(\beta\)-sheet-rich aggregates. Dimers and trimers appeared in simulations containing 20-30 peptides, and larger assemblies--tetramers and pentamers--emerged after the insertion of an additional 10 peptides (totaling 40)-Figure S1. These clusters continued to grow as the simulation progressed and they eventually penetrated the bilayer. Membrane insertion occurred when \(\beta\)-sheets on opposing leaflets aligned to form a transmembrane water channel. Additional \(\beta\)-sheets were recruited to form a cylindrical structura around the pore, with nonpolar side chains oriented toward the lipid core and polar residues lining the aqueous interior. This structural rearrangement explains the sharp rise in water permeation at 5 us-cyan line in Figure 2A. Panel I shows the resulting pore formed by M01, with the largest \(\beta\)-sheets composed of up to 10 peptides spanning the membrane. In simulations with peptide M02, the cumulative water permeation curve (purple line) showed no sharp increase indicative of pore formation (Figure 2A). The molecular structure of our system at the end of the simulation shows that the 40 adsorbed peptides were dispersed on the membrane leaflets folded into helical structures-panel II. The latter conformation partitions polar and nonpolar residues to the solvent and membrane interior, respectively, and it leaves only a reduced number of dangling peptide hydrogen donors and acceptors-Figure 1. In contrast, M01 adopts extended \(\beta\)-strand conformations, driven by its alternating polar-nonpolar sequence pattern. This arrangement leaves many hydrogen donor and acceptor sites exposed, which is energetically unfavorable unless passivated--explaining the spontaneous \(\beta\)-sheet formation observed in Figure 2B. The picture that emerges from Figure 2A is that M01 peptides are able to create pores due to their ability to form large \(\beta\)-sheet aggregates that can span both membrane leaflets. This contrasts with the tendency of M02 peptides to remain dispersed on the lipid membrane. In addition, individually, M02 peptides are too short to penetrate deeply within the bilayer and cause damage. These insights inspired us to study two longer 16-residue peptides that can span the bilayer when folded into an \(\alpha\)-helix. The first peptide sequence, Ac-(FFKKFFEE)\({}_{2}\)-NH\({}_{2}\) (M03), corresponds to the juxtaposition of two M02 repeats. As shown in Figure 1, this sequence produces \(\alpha\)-helices with one face that is mostly (but not completely) non-polar and the other polar. The sequence of the second studied peptide named M04 is Ac-(FFKKFFEE)(FKKFFEE)-NH\({}_{2}\) and corresponds to the juxtaposition of the M02 sequence with one of its 8-residue permutations. This combination is designed to allow for perfect segregation of polar-nonpolar residues to different faces of an \(\alpha\)-helix--Figure 1. Thus, both peptides contain the same sixteen amino acids and share the same N-terminal sequence, beginning with two nonpolar residues. They differ in their ability to segregate polar and non-polar residues, and in their last C-terminal residue, which is negatively charged in M03 and nonpolar in M04.