Unnatural Amino Acids with Abbreviations
2.3. Results and Discussions
2.3.9. Structure Determination and Peptide: Membrane Interaction Studies through MD Simulations
To probe this interaction further, we performed MD simulations with three different N-terminal protonated peptides P4, P5, P6 in presence and absence of SDS micelles.
Figure 2.13. Solvent accessibility of the peptides, P4 and P5 in DPC micelle: Two- dimensional 1H-1H TOCSY NMR spectra of (a) P4 and (b) P5 in perpetuated DPC micelle (black contour) and in the presence of MnCl2 (red contour). The experiment was recorded using a Bruker Avance III 500 MHz NMR spectrometer and at 310 K. Most of the cross-peaks (including Trp) broadened in the presence of 0.1 mM MnCl2 solution. The data conformed that the peptides were mostly solvent exposed in DPC micelle.
Free peptides in aqueous environment showed various random coil conformations (Figure 2.14a), which validated the observation from NMR and CD. In order to understand the binding event, we carried out MD simulations with an initial configuration where peptides were placed 10 Å away from SDS micelle (Figure 2.1). The MD trajectory analysis suggested that peptides started interacting with micelles within ~ 1 ns and a stable bound conformation (Figure 2.14 b) was attained within ~ 15 ns, which remained stable throughout the molecular dynamics trajectory (total ~ 50 ns). SDS bound peptides sampled limited conformational space or attained a specific conformation with respect to the flexible random coil SDS free state (Figure 2.15).
Though the conformation of micelle bound peptide was distinct and stable, it could not be categorized as any classical secondary structure.
Figure 2.14. Structure from molecular dynamics trajectory. (a) Few representative random coil structures of peptide P4 in water, (b) SDS Micelle bound P4 peptide conformation after 50 ns of molecular dynamics run. N-terminal has been colored blue and C-terminal red in the peptide (cartoon); SDS micelle (surface and lines) has sulfates in red and aliphatic side chain in grey.
Hydrogens are not shown for clarity.
The binding of the peptide to the SDS micelle followed a sequential order (Figure 2.16). At first, the positively charged peptide landed on the surface of negatively charged SDS micelles through electrostatic interaction. Subsequently, the SDS bound peptide was further stabilized by hydrophobic interaction between neutral side chains of the peptide and aliphatic chain of micelle, leading to a local deformation on micelle surface.
Figure 2.15. Residue specific Ramachandran Map for a) Free P4, SDS micelle bound b) P4, c) P5 and d) P6, indicating the flexible conformation of P4 in Free State vs the fixed random coil conformations of P4, P5 and P6 upon binding to SDS during the MD trajectory.
Figure 2.16. MD Snapshots of peptide (sticks, NH3+-LKWLKKL-CONH2: Model I) binding to SDS micelle (surface and lines). a) Peptide approaching to micelle at 5 ps, b) anchoring interaction between peptide and micelle is established at 0.07 ns, c) hydrophobic interaction along with the electrostatic anchoring could be seen at 0.2 ns, d) representative snapshot at 50 ns describing stable conformation of peptide bound to the micelle.
Peptide backbone satisfied its hydrogen bond requirement by interacting with waters and/or sulfates of SDS micelle. Micelle-peptide interaction has been shown in Figure 2.17 a-c for P4, P5 and P6 respectively.
Figure 2.17. MD snapshot describing the electrostatic, hydrophobic and hydrogen bonding interactions between peptide and micelle. Water as ball and stick, micelle as surface and lines, peptide as stick. Only water hydrogens are shown. Oxygen is red, Nitrogen is blue and carbon is gray. a) P4 bound micelle, b) P5 bound micelle, c) P6 bound micelle and d) peptide with deprotonated carboxy terminal –COO- (derived from P4) bound to micelle.
Similarly, side chain residues of L1, W3 and L4 in P5 were buried inside the micelle, whereas L7 was present at the membrane water interphase (Figure 2.17b). R2, R5, R6 side chains of P5 formed anchoring interaction by direct and water mediated interactions with sulfates of micelle (Figure 2.17b). In case of peptide P6, only L1 and W3 primarily formed hydrophobic interactions with aliphatic side chains of the micelles (Figure 2.17c). The hydrogen bond requirement of peptide backbone was satisfied by interaction with water molecules and/or sulfate of micelles. Interaction area between the peptides and SDS micelles were ~ 753 (±92)
Å2, 778 (±108) Å2 and 519 (±66) Å2 for P4, P5 and P6, respectively. Lack of positively charged side chains (Neutral His has pka ~ 6.0) in P6 was responsible for lowest interaction area.
In order to understand the role of the C-terminal end of the peptides, in their interaction with SDS micelle, we performed simulations with a peptide containing -COO- terminus (Model I, Figure 2.17d). Model I was similar to P4, differing only at the C-terminal end with a carboxyl group instead of the amide (-CONH2) group. The C-terminal end of Model I lost its interaction with micelles due to electrostatic reasons. The calculated area of interaction for Model I was ~ 670 (±88) Å2. This proved that electrostatic interaction between peptide and the micelle was crucial for initial binding. The removal of positive charge (as in P6) or deprotonation of the carboxyl group at the C-terminal end (as in Model I) led to poor association, which was reflected in the poor anti-microbial activity of P6 and P1-3, respectively (Table 2.2).
To prove our point further, we performed MD simulation with P4 in the presence of DPC micelle, a neutral lipid mimicking the zwitterionic mammalian membrane. It is interesting to note that P4 could not bind with dodecylphosphocholine micelles (DPC) even after 50 ns of dynamics due to electrostatic reasons. This clearly proved that electrostatic interaction was the primary and crucial interaction between the peptide and the membrane-mimic (SDS/DPC). Trp residue W3 in both P4 and P5 was seen to be buried in the hydrophobic core of the SDS micelles from MD simulations and PRE NMR experiments. Significant blue shift in the fluorescence emission of the two peptides (Figure 2.18) in presence of SDS (about 9 nm in case of P4 and 11 nm in the case of P5) supported our claim for the burial of the Trp residue within the hydrophobic core of the SDS micelle. This might be the second additional drive for the interaction of P4 and P5 with the membrane and hence their activity.
Figure 2.18. Trp emission spectra for a) P4 and b) P5 in water (green line) and in the presence of 30 mM SDS (blue line).