## 3 Results and Discussions
### Molecular Dynamics Simulations  
Previously, it has been reported that the peptides L-Phes and D-Phes adopt \(\alpha\)-helical conformations in a mixture of acetonitrile and water, using solution NMR technique [18]. Here, we evaluated the structural stability and interactions of the peptides with DMPC membrane with three different types of simulations. The initial structures were shown in Figure A3, and Table A1 displays the contents of systems. First, we simulated the helical structures inside the model membrane. Both peptides exhibited the stable helical conformation during these simulations, and the first and last snapshots of the simulations are shown in Figure A4a. We identified the most populated structures during the last 25 ns of the simulations, which are shown in Figure A4b. We applied the structures as the initial conformations for the next simulations. Second, we placed the peptides in the water region of the membrane system to examine differences between L-Phes and D-Phes in their initial interactions with DMPC membrane. During these simulations, we did not observe stable interactions between the membrane and the peptides. This indicated that the peptides need longer simulation time to show initial insertions into DMPC membrane. Perhaps, the peptides need to establish an initial electrostatic interaction with negatively charged phospholipids, or alternatively, the concentrations of the peptides may need to be increased in the membrane system to stimulate the process. The last snapshots and secondary structure profiles are shown in the Figure 4. The relative positions of the peptides were almost similar with their initial position along the z-axis. Although both peptides partially lost their helical conformations during the last-half of the simulations, L-Phes exhibited the higher helicity than D-Phes. Especially, the second position of L-Phes mostly formed \(\alpha\)-helical secondary structures compared to the random coil structure  
Figure 3: \({}^{31}\)P solid-state NMR spectra of DMPC (grey), DMPC with L-Phes (black), and DMPC with D-Phes (red) under static condition. of D-Phes. These observations were consistent with our CD experiments, which found the slightly higher helicity for L-Phes. Third, we performed the pseudo-transmembrane simulations with three different replicas. We inserted C-terminus of the peptides into DMPC membrane, because the charged residues of the peptides are near to N-terminus. We extensively analyzed these simulations to understand the mechanism of action and membrane interactions of the peptides at the membrane interface. The series of snapshots are displayed in Figure 5. Both peptides took their positions within 50 ns of simulations, which were the same as their last snapshots. Figure 6 displays the secondary structure profiles during the simulations of L-Phes and D-Phes. The second residue of L-Phes mostly formed \(\alpha\)-helical secondary structure during all three runs. In contrast, the second residue of D-Phes presented the random coil conformations in all three runs. These results agreed with the secondary structure results of CD and VCD experiments. This suggested that the membrane interactions of N-terminal side could be different between L-Phes and D-Phes. The pseudo-transmembrane position of L-Phes transferred into transmembrane state in the three different runs. In contrast, only one simulation showed the fully inserted D-Phes. During the simulations, L-Phes peptide tilted around 39\({}^{\circ}\) in all three simulations. When D-Phes transferred to transmembrane state (Run 1), the average tilt angle was equal to around 39\({}^{\circ}\). This indicated that when the peptides get inserted, they would tilt similarly in the model membrane. Second and third runs of D-Phes displayed around 82\({}^{\circ}\) of the tilt angles. The average values of the tilt angles in the last 100 ns simulations are summarized in Table 1. These data possibly indicate the heterogeneity of membrane-bound states of D-Phes at the interface of DMPC membrane.