mastoparan-x and 1-palmitoyl-2-oleoylphosphatidylcholine

Antimicrobial peptides are known to interact strongly with negatively charged lipid membranes, initially by peripheral insertion of the peptide into the bilayer, which for some antimicrobial peptides will be followed by pore formation, and successive solubilization of the membranes resulting in mixed peptide-lipid micelles. We have investigated the mode of action of the antimicrobial peptide mastoparan-X using isothermal titration calorimetry (ITC) and cryo-transmission electron microscopy (cryo-TEM). The results show that mastoparan-X induces a range of structural transitions of POPC/POPG (3:1) lipid membranes at different peptide/lipid ratios. It has been established that ITC can be used as a fast method for localizing membrane transitions and when combined with DLS and cryo-TEM can elucidate structural changes, including the threshold for pore formation and micellation. Cryo-TEM was employed to confirm the structural changes associated with the thermodynamic transitions found by ITC. The pore-formation process has furthermore been investigated in detail and the thermodynamic parameters of pore formation have been characterized using a system-specific temperature where the enthalpy of peptide partitioning becomes zero (T(zero)). This allows for an exclusive study of the pore-formation process. The use of ITC to find T(zero) allows for characterization of the thermodynamic parameters of secondary processes on lipid membranes.

Topics: Calorimetry; Cryoelectron Microscopy; Intercellular Signaling Peptides and Proteins; Membranes, Artificial; Micelles; Peptides; Phosphatidylcholines; Phosphatidylglycerols; Porosity; Thermodynamics; Titrimetry

Many antimicrobial peptides (AMPs) function by forming various oligomeric structures and/or pores upon binding to bacterial membranes. Because such peptide aggregates are capable of inducing membrane thinning and membrane permeabilization, we expected that AMP binding would also affect the diffusivity or mobility of the lipid molecules in the membrane. Herein, we show that measurements of the diffusion times of individual lipids through a confocal volume via fluorescence correlation spectroscopy (FCS) provide a sensitive means of probing the underlying AMP-membrane interactions. In particular, results obtained with two well-studied AMPs, magainin 2 and mastoparan X, and two model membranes indicate that this method is capable of revealing structural information, especially the heterogeneity of the peptide-membrane system, that is otherwise difficult to obtain using common ensemble methods. Moreover, because of the high sensitivity of FCS, this method allows examination of the effect of AMPs on the membrane structure at very low peptide/lipid ratios.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Animals; Antimicrobial Cationic Peptides; Cell Membrane Permeability; Diffusion; Intercellular Signaling Peptides and Proteins; Lipid Bilayers; Magainins; Molecular Probes; Organotechnetium Compounds; Peptides; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Spectrometry, Fluorescence; Unilamellar Liposomes; Xenopus Proteins

We have investigated the wasp venom peptides mastoparan X and polistes mastoparan regarding their apparent potential to induce pore-like defects in phosphatidylcholine unilamellar vesicles. Based on a fundamental theoretical model, the pore activation and deactivation kinetics have been evaluated from the observed efflux of liposome entrapped carboxyfluorescein in relation to the bound peptide to lipid ratio. We can quantitatively describe our experimental data very well in terms of a specific reaction scheme resulting in only a few short-lived pores. They evidently emerge rapidly from a prepore nucleus being produced by two rate-limiting monomeric states of bound peptide. These peculiar states would be favorably populated in an early stage of bilayer perturbation, but tend to die out in the course of a peptide/lipid restabilization process.

Topics: Amino Acid Sequence; Animals; Dextrans; Fluorescein-5-isothiocyanate; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Kinetics; Liposomes; Peptides; Phosphatidylcholines; Protein Binding; Wasp Venoms