Magainins and many other 23 to 37 residue long peptides are newly discovered antimicrobial peptides that appear to be widely distributed in the animal kingdom as a universal means for defense against bacterial infections. What is unusual about these peptide antibiotics is that their target is the cellular lipid bilayer membrane. This is unlike other membrane-active proteins which exert their activity through specific receptors on the cell membrane. Without protein receptors, one might presume the peptide-membrane interactions are stoichiometric and nonspecific. But in fact there are critical concentrations for the peptide activity and these critical concentrations are membrane specific. Various magainin peptides are tumoricidal at concentrations 5-10 times greater than those required for antibiotic effects but 10-20 times less than those toxic to normally differentiated cells. We have recently discovered a new phenomenon of peptide-membrane interactions that can explain such self- catalytic, membrane-specific activity of the magainins. Many of these peptides, including magainins, form an amphipathic helical structure upon association with a membrane. We discovered that amphipathic helical peptides associate with a bilayer membrane in two ways; at low peptide- lipid molar ratios (P/L) the majority of peptide molecules adsorb on the membrane surface; but at high P/L the majority of peptide change into a different state; and most strikingly the majority's transition between the low-concentration state and the high-concentration (HC) state occurs over a narrow range of P/L, indicating that the peptide transition is a cooperative phenomenon; furthermore, the critical P/L value for the cooperative transition is different for different lipid compositions. We believe that the interaction of the HC state of the peptides with the cell membrane is the mechanism of cytolysis. With the experimental techniques we have recently developed, rigorous structural studies of these peptide- membrane systems are now possible. We will perform three types of experiments to try to understand why the peptide transition to the HC state is self-catalytic and why it is membrane specific. (1) Study the chemical variables that influence the cooperative peptide transition, including peptide variables, lipid variables, effect of cholesterol, effect of electric field and effect of ions. (2) Use x-ray and neutron lamellar diffraction to determine the z-coordinate (normal to the plane of membrane) of peptide and use in-plane scattering to determine the state of peptide aggregation in the xy plane. (3) Use the same techniques of (2) to determine the roles of water and ions in the peptide transition. Because of their small size and antimicrobial potency, the magainin family of peptides have therapeutic potential. Our specific goal is to understand the mechanism underlying their activity. In general, this study will also help us to understand the functional role of amphipathic helix motif which is often found in membrane-active proteins and peptides.
