# Insights into Membrane Damage by \(\alpha\)-Helical and \(\beta\)-Sheet Peptides
## 3 Results
### Experimental Investigation  
It is important to highlight that all-atom simulations were performed starting with M03 and M04 peptides already folded into \(\alpha\)-helices. This was motivated by simulations of M02 peptides that, starting from random coil conformations, spontaneously folded into \(\alpha\)-helices at the water-lipid interface-Figure 2-II. An \(\alpha\)-helix conformation for M03 and M04 peptides was also expected, based on the segregation of polar and nonpolar residues to different faces of a helical wheel--Figure 1. However, since \(\alpha\)-helix conformations were critical for pore formation in the simulations, the ability of M03 and M04 peptides to form this secondary structure needed to be tested experimentally. In addition, the larger permeation of M03 compared to M04 in the simulations was unexpected, since sequences with higher hydrophobic moments (\(\mu\)) are usually associated with more intensive water leakage [41], whereas M03 (\(\mu\) = 0.59) had a smaller moment than M04 (\(\mu\) = 0.87) [47]. Here, experiments were performed to test this water leakage trend.  
In the following section, we discuss the secondary structure and membrane damage properties of M03 and M04. While modeling shows that M01 is capable of damaging membranes, experimentally the peptide self-assembled into supramolecular \(\beta\)-sheet rich fibers [27; 65]. This happened immediately upon dissolution in aqueous solvent; likely before it was able to interact with the membranes. Therefore, it was difficult to determine the effect of M01 peptides on membranes experimentally, as the leakage observed was likely due to mechanical disruption of the liposomes as the peptide monomers assembled into their final fibril structure. While modeling of M02 suggests that it forms an \(\alpha\)-helix, experimentally, the analysis of its secondary structure was less straightforward. The IR spectra (Figure S3) contain peaks that overlap with both the random coil and \(\alpha\)-helical region, indicating that more complex methods of secondary structure determination are needed to experimentally validate the models. Despite this ambiguity, the leakage assays  
Figure 4: Solvent exposure of the C-terminal. (**A,B**) Schematics of M03 and M04 peptides with their N-termini aligned parallel and inserted into the membrane. (**C,D**) Solvent-accessible surface area (SASA) of the last phenylalanine of the M03 and M04 peptides that are inserted in the membrane. A 15-point moving average (white line) is shown for clarity. Horizontal arrows indicate time intervals when the N-terminal remained embedded in the membrane.  
of M02 were consistent with the modeling and showed minimal leakage, even at high concentrations of peptide (Figure S4).  
Notice that the experiments and simulations were performed under different conditions. The former used room temperature and a buffer salt solution, whereas the simulations were performed at a higher temperature of 350 K and in pure water. The latter temperature was necessary to accelerate the dynamics enabling pores to form within a few microseconds of simulation. The addition of salts to the solution was expected to accelerate peptide aggregation, as well as to affect secondary structure formation, and peptide adsorption onto lipid membranes [20; 66; 67; 68; 69; 25]. Direct comparison between the simulations and experiments is therefore not possible. However, the ranking of peptide sequences based on their ability to form pores is robust across a broad range of conditions/solutions. This allows in silico and in vitro studies to inform each other and will be explored here.