Molecular Dynamics Simulations.

The package NAMD⁴⁴ was used to carry out all-atom MD simulations. The simulated system consisted of a dipeptide in a planar membrane–water lamellae system, which included 142 POPC lipid molecules and 8019 water molecules. The size of the simulation cell was ∼69.6 × 69.6 × 85.1 Å with periodic boundary conditions applied along x, y, and z directions. The x and y dimensions of the cell were kept fixed to ensure that the surface area per lipid headgroup was equal to the value of 68.3 Ų measured in the recent X-ray scattering experiments.⁴⁵ The pressure was kept at 1 atm along the z-direction, perpendicular to the membrane surface. A constant temperature of 303 K was maintained using the Langevin friction force with the damping coefficient at 5 ps⁻¹. In all simulations, a time step of 2 fs was used. The particle mesh Ewald scheme was applied to calculate long-ranged electrostatic interactions, with the grid 72 × 72 × 90. The cutoff of 13.5 Å was used for van der Waals interactions.

The updated version of CHARMM potentials for phospholipids^46–48 and the TIP3P model⁴⁹ of water were used to describe interatomic interactions in the system. The three selected dipeptides—phenylalanine-leucine, serine-leucine, and serine-serine—are all blocked at the N- and C-terminal with acetyl group and N-methylamide, and are referred to as Ace-Phe-Leu-NMe, Ace-Ser-Leu-NMe, and Ace-Ser-Ser-NMe, respectively. The recently optimized CHARMM force field for proteins⁵⁰ was applied to the dipeptides. The CMAP term⁵¹ for the backbone torsion angle was applied to the N- and C-termini.

To test the potential functions, benchmark MD simulations were carried out for the Ace-Phe-Leu-NMe dipeptide in water and in decane, which forms a nonpolar environment similar to the lipid tail region. The simulations in water were 250 ns in length, with constant pressure of 1 atm and temperature of 298 K in a simulation box of 38.8 × 38.8 × 38.8 Å. The dipeptide backbone was mainly distributed between three states: polyproline II (P_II), β, and α_R (see the Ramachandran map plotted in Figure S1). The extended P_II and β states were populated more frequently than the coiled α_R state: 65% vs 23% for Phe and 63% vs 28% for Leu. In addition, there were small contributions (total ∼10%) from other states. The observed dominance of the P_II and β states is in agreement with the recent NMR and circular dichroism (CD) spectroscopic studies on blocked Ace-X₁-X₂-NH₂ peptides in water.⁵² This reflects the sought-after improvement in the updated CHARMM force field for proteins⁵⁰ compared to the old one,^46,51 which systematically overestimated the α_R state.⁵⁰ The high population of the P_II state shown for a number of short peptides in previous spectroscopic and theoretical studies (see references in a recent review⁵³) is the cornerstone for the P_II hypothesis initially put forward by Tiffany and Krimm^54,55 to understand the structure of unfolded proteins.^53,56

For the dipeptide in decane, a trajectory 200 ns in length was obtained at T = 310 K and constant pressure of 1 atm. The dimensions of the simulation box were 57.9 × 57.9 × 57.9 Å. The conformation of the backbone was found predominantly in the extended β state (the populations of β, P_II, and α_R were 72%, 17%, and 10%, respectively, for Phe and 75%, 14%, and 10% for Leu; see also the Ramachandran map plotted in the upper panel of Figure S2). These preferences are similar to those calculated for alanine dipeptide in the gas phase.⁵⁷ For comparison, population of the β state in aqueous solution is only 16% for Phe and 30% for Leu. A similar dependence of the backbone conformation on solvent polarity was previously observed in experimental studies on different tripeptides containing alanine.⁵⁸ In these studies, it was found that β-sheet conformation dominated in DMSO, whereas conformational distribution in water was markedly broadened. Similar backbone conformations were found for the dipeptide at the POPC membrane center (see the Ramachandran map plotted in the lower panel of Figure S2). The interconversion between the different conformation states was rather fast, occurring on the nanosecond time scale.