Conclusions

There are two issues we attempt to understand by performing simulations described here. The experimental papers dealing with the AMPs action often refer to the importance of stress caused by the presence of peptides in the system. Therefore, the first issue we consider is related to the somewhat paradoxical situation, that when NPT simulations with equal pressure in three directions are performed, the total stress experienced by the bilayer is zero, even in the presence of AMPs adsorbed on the bilayer surface. The second issue is related to the difference in action of AMPs such as melittin and magainin-h2, and its connection to the difference in stress profiles produced by these AMPs.

Our simulations of the bilayers containing AMPs such as melittin and magainin-h2 confirmed, as expected, that the total stress on the lipid bilayer is zero, when NPT simulations with equal pressure in all three directions are performed. Since most of the simulations of bilayers containing AMPs are done in the NPT ensemble, the total stress on the bilayer in those simulations was zero. Nevertheless, there are still stresses acting on each bilayer leaflet. These stresses are acting in opposite directions and are equal in their absolute values. To remove these stresses pores may be created in membranes, and also because there is a pair of forces acting in

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opposite directions on upper and lower leaflets, membranes may bend. In our simulations we observed that the values of the stresses on monolayers we obtained from the NPT simulations of bilayers with melittin and magainin-h2 are similar; this makes it difficult to explain the

difference in the mode of magainin versus melittin action as due to difference in stress.

Free lipid bilayers in equilibrium experience no stress. When AMPs are adsorbed on the surface of one of the leaflets, the system may find itself initially in a nonequilibrium state with a total stress not equal to zero. To remove the stress the bilayer will rearrange through creating pores and bending, thus moving to a new equilibrium state with the total stress again equal to zero. The NPzAT simulations are better in mimicking the initial stage when the total stress is nonzero. In these simulations the stresses on each monolayer, although still different in the sign, are not equal in their absolute values. The action of a pair of forces acting in different directions and of unequal value should result in a creation of a bilayer with a larger curvature. Figure 2.5 shows the shape of the bilayers with the 18 AMPs adsorbed on the upper leaflet when

simulations were done in NPzAT ensemble. For comparison, the shape of the pure bilayer obtained from the NPT simulation is also shown. While there is little curving present in the simulation of pure lipid bilayer, the curving of the bilayers with AMP is clearly seen. Nevertheless, some words of caution are required to be said here: the geometry of our simulations that are performed using periodic boundary conditions may suppress the curving tendency, or produce a wrong curvature.

When AMPs are adsorbed on the bilayer surface they induce stress. To remove this stress the AMPs may either permeate the membrane and initially create transient pores, or just create pores due to large stress on a membrane. Experiments indicate that melittin chooses the first route, while magainin the second. Our simulations on systems with a total stress experienced by

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the bilayer (i.e., simulations performed in NPzAT ensemble) show that magainin-h2 at larger P/L produces a larger stress on the bilayer compared to stress produced by melittin. This observation is consistent with the suggestions about the mechanism of magainin antimicrobial activity made by Tamba et al. based on their experimental work25_.

Since our simulations were performed at the same value of area and since both peptides are α helical and are almost of the same length, the major difference between our systems with peptides is in the peptide sequence. Different peptide side chains (amino acids) in these two peptides interact in a different way with the membrane producing the difference in modes of AMPs action. Still, in general, further experimental and computational research on the detailed nature of AMP activity is required to shed more light on this complicated but important problem.

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Figure 2.1: Lateral pressure profiles from simulations in the NPT ensemble. Red and green curves depict the pressure profiles obtained from the simulation when 12 peptides were inserted into the top leaflet and no peptides placed into the lower leaflet. Red curve is when peptide is melittin and green curve when it is magainin-h2. For comparison we also present the pressure profile in the peptide free membrane, obtained from the simulation in the NPT ensemble (black curve).

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Figure 2.2: Lateral pressure profiles from simulations in the NPzAT ensemble. Red and green curves depict the pressure profiles obtained from the simulation when 12 peptides were inserted into the top leaflet and no peptides placed into the lower leaflet. Red curve is when peptide is melittin and green curve when it is magainin-h2. For comparison we also present the pressure profile in the peptide free membrane, obtained from the simulation in the NPT ensemble (black curve).

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Figure 2.3: Lateral pressure profiles from simulations in the NPT ensemble. Red and green curves depict the pressure profiles obtained from the simulation when 18 peptides were inserted into the top leaflet and no peptides placed into the lower leaflet. Red curve is when peptide is melittin and green curve when it is magainin-h2. For comparison we also present the pressure profile in the peptide free membrane, obtained from the simulation in the NPT ensemble (black curve).

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Figure 2.4: Lateral pressure profiles from simulations in the NPzAT ensemble. Red and green curves depict the pressure profiles obtained from the simulation when 18 peptides were inserted into the top leaflet and no peptides placed into the lower leaflet. Red curve is when peptide is melittin and green curve when it is magainin-h2. For comparison we also present the pressure profile in the peptide free membrane, obtained from the simulation in the NPT ensemble (black curve).

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Figure 2.5: Bilayer structures obtained from some of the simulations performed in this study. To see better the curvature in the membrane, carbon chains of the lipids and water molecules are deleted. Only phosphate headgroups (PO4 particles) are represented here in red. Panel A is the membrane without any protein, simulated under the NPT ensemble. Panel B shows a bilayer with 18 melittins (green) in the top leaflet, simulated in the NPzAT ensemble. Panel C represents a bilayer with 18 magainin-h2 (yellow) molecules in the top monolayer, also simulated in the NPzAT ensemble.

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Chapter 3: Mechanism of Membrane Poration by Shock Wave Induced Nanobubble