Determining the structure of membrane-active peptides inside lipid bilayers is essential to understand their mechanism of action. Molecular dynamics simulations can easily provide atomistic details, but need experimental validation. We assessed the reliability of self-assembling (or "minimum-bias") and potential of mean force (PMF) approaches, using all-atom (AA) and coarse-grained (CG) force-fields. The LAH4 peptide was selected as a stringent test case, since it is known to attain different orientations depending on the protonation state of its four histidine residues. In all simulations the histidine side-chains inserted in the membrane when neutral, while they interacted with phospholipid headgroups in their charged state. This led to transmembrane orientations for neutral-His LAH4 in all minimum-bias AA simulations and in most CG trajectories. By contrast, the charged-His peptide stabilized membrane defects in AA simulations, whereas it was located at the membrane surface in some CG trajectories, and interacted with both lipid leaflets in others. This behavior is consistent with the higher antimicrobial activity and membrane-permeabilizing behavior of the charged-His LAH4. In addition, good agreement with solid-state NMR orientational data was observed in AA simulations. PMF calculations correctly predicted a higher membrane affinity for the neutral-His peptide. Interestingly, the structures and relative populations of PMF local free-energy minima corresponded to those determined in the less computationally demanding minimum-bias simulations. These data provide an indication about the possible membrane-perturbation mechanism of the charged-His LAH4 peptide: by interacting with lipid headgroups of both leaflets through its cationic side-chains, it could favor membrane defects and facilitate translocation across the bilayer.
LAH4-L1 is a synthetic amphipathic peptide with antimicrobial activity. The sequence of the 23 amino acid peptide was inspired by naturally occurring frog peptides such as PGLa and magainin. LAH4-L1 also facilitates the transport of nucleic acids through the cell membrane. We have investigated the membrane binding properties and energetics of LAH4-L1 at pH 5.5 with physical-chemical methods. CD spectroscopy was employed to quantitate the membrane-induced random coil-to-helix transition of LAH4-L1. Binding isotherms were obtained with CD spectroscopy as a function of the lipid-to-protein ratio for neutral and negatively charged membranes and were analyzed with both the Langmuir multisite adsorption model and the surface partition/Gouy-Chapman model. According to the Langmuir adsorption model each molecule LAH4-L1 binds 4 POPS molecules, independent of the POPS concentration in the membrane. This is supported by the surface partition/Gouy-Chapman model which predicts an electric charge of LAH4-L1 of z = 4. Binding affinity is dominated by electrostatic attraction. The thermodynamics of the binding process was elucidated with isothermal titration calorimetry. The ITC data revealed that the binding process is composed of at least three different reactions, that is, a coil-to-helix transition with an exothermic enthalpy of about -11 kcal/mol and two endothermic processes with enthalpies of ∼4 and ∼8 kcal/mol, respectively, which partly compensate the exothermic enthalpy of the conformational change. The major endothermic reaction is interpreted as a deprotonation reaction following the insertion of a highly charged cationic peptide into a nonpolar environment.
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