Free energy barrier for melittin reorientation from a membrane-bound state to a transmembrane state.

An important step in a phospholipid membrane pore formation by melittin antimicrobial peptide is a reorientation of the peptide from a surface into a transmembrane conformation. Experiments measure the fraction of peptides in the surface state and the transmembrane state, but no computational study exists that quantifies the free energy curve for the reorientation. In this work we perform umbrella sampling simulations to calculate the potential of mean force (PMF) for the reorientation of melittin from a surface-bound state to a transmembrane state and provide a molecular level insight in understanding the peptide-lipid properties that influence the existence of the free energy barrier. The PMFs were calculated for a peptide to lipid (P/L) ratio of 1/128 and 4/128. We observe that the free energy barrier is reduced when the P/L ratio increases. In addition, we study the cooperative effect; specifically we investigate if the reorientation barrier is smaller for a second melittin, given that another neighboring melittin was already in the transmembrane orientation. We observe that indeed the barrier of the PMF curve is reduced in this case, thus confirming the presence of a cooperative effect.

Melittin creates transient pores in a lipid bilayer: results from computer simulations.

To study the interaction between melittin peptides and lipid bilayer, we performed coarse-grained simulations on systems containing melittin interacting with a bilayer containing zwitterionic dipalmitoylphosphatidylcholine (DPPC) and anionic palmitoyloleoylphosphatidylglycerol (POPG) phospholipids in a 7:3 ratio. Eight different systems were considered: four at low and four at high peptide to lipid (P/L) ratios. In case of low P/L ratio we did not observe any pore creation in the bilayer. In two out of four of the simulations with the high P/L ratio, appearance of transient pores in the bilayer was observed. These pores were created due to an assembly of 3-5 melittin peptides. Not all of the peptides in the pores were in a transmembrane conformation; many of them had their termini residues anchored to the same leaflet, and these peptides assumed bent, U-shaped, conformations. We propose that when an assembly of melittin peptides creates pores, such an assembly acts as a "wedge" that splits the bilayer. To get a more detailed description of melittin on the bilayer surface and in transient pores, we performed coarse-grained to united-atom scale transformations and after that performed 50 ns molecular dynamics simulations using the united atom description of the systems. While these simulations did not show much of the change in the pore structure during the 50 ns time interval, they clearly showed the presence of water in the transient pores. The appearance of transient pores together with the translocation of peptides across the membranes is consistent with the mechanism proposed to explain graded dye leakage from large vesicles in the presence of melittin.

Binding and reorientation of melittin in a POPC bilayer: computer simulations.

We performed, using an all-atom force field, molecular dynamics computer simulations to study the binding of melittin to the POPC bilayer and its subsequent reorientation in this bilayer. The binding process involves a simultaneous folding and adsorption of the peptide to the bilayer, followed by the creation of a "U shaped" conformation. The reorientation of melittin from the parallel to the perpendicular conformation requires charged residues to cross the hydrophobic core of the bilayer. This is accomplished by a creation of defects in the bilayer that are filled out with water. The defects are caused by peptide charged residues dragging the lipid headgroup atoms along with them, as they reorient. With increased concentration of melittin water defects form stable pores; this makes it easier for the peptide N-terminus to reorient. Our results complement experimental and computational observations of the melittin/lipid bilayer interaction.

Influence of the arrangement and secondary structure of melittin peptides on the formation and stability of toroidal pores.

Melittin interactions with lipid bilayers and melittin formed pores are extensively studied to understand the mechanism of the toroidal pore formation. Early experimental studies suggested that melittin peptide molecules are anchored by their positively charged residues located next to the C-terminus to only one leaflet of the lipid bilayer (asymmetric arrangement). However, the recent non-linear spectroscopic experiment suggests a symmetric arrangement of the peptides with the C-terminus of the peptides anchored to both bilayers. Therefore, we present here a computational study that compares the effect of symmetric and asymmetric arrangements of melittin peptides in the toroidal pore formation. We also investigate the role of the peptide secondary structure during the pore formation. Two sets of the symmetric and asymmetric pores are prepared, one with a helical peptide from the crystal structure and the other set with a less helical peptide. We observe a stable toroidal pore being formed only in the system with a symmetric arrangement of the less helical peptides. Based on the simulation results we propose that the symmetric arrangement of the peptides might be more favorable than the asymmetric arrangement, and that the helical secondary structure is not a prerequisite for the formation of the toroidal pore.