Abstract
Alamethicin has been extensively studied as an antimicrobial peptide (AMP) and is widely used as a simple model for ion channel proteins. It has been shown that the antimicrobial activity of AMPs is related to their cell membrane orientation, which may be influenced by the phase of the lipid molecules in the cell membrane. The "healthy" cell membranes contain fluid phase lipids, while gel phase lipids can be found in injured or aged cells or in some phase separated membrane regions. Thus, investigations on how the phase of the lipids influences the membrane orientation of AMPs are important to understand more details regarding the AMP's action on cell membranes. In this study, we determined the orientational changes of alamethicin molecules associated with planar substrate supported single lipid bilayers (serving as model cell membranes) with different phases (fluid or gel) as a function of peptide concentration using sum frequency generation (SFG) vibrational spectroscopy. The phase changes of the lipid bilayers were realized by varying the sample temperature. Our SFG results indicated that alamethicin lies down on the surface of fluid and gel phase 1,2-dimyristoyl(d54)-sn-glycero-3-phosphocholine (d-DMPC) lipid bilayers when the lipid bilayers are in contact with a peptide solution with a low concentration of 0.84 μM. However, at a medium peptide concentration of 10.80 μM, alamethicin inserts into the fluid phase lipid bilayer. Its orientation switches from a transmembrane to an in-plane (or lying down) orientation when the phase of the lipid bilayer changes from a fluid state to a gel state. At a high peptide concentration of 21.60 μM, alamethicin adopts a transmembrane orientation while associated with both fluid and gel phase lipid bilayers. We also studied the structural changes of the fluid and gel phase lipid bilayers upon their interactions with alamethicin molecules at different peptide concentrations.