Raman spectroscopy of synthetic antimicrobial frog peptides magainin 2a and PGLa

Abstract

Magainin and PGLa are 23-and 21-residue peptides isolated from the skin of the African clawed frog Xenopus lueuis. They protect the frog from infection and exhibit a broad-spectrum antimicrobial activity in vitro. The mechanism of this activity involves the interaction of magainin with microbial membranes. We have measured the secondary structure and membrane-perturbing ability of these peptides to obtain information about this mechanism. Our results show that mgn2a forms a helix with an average length of less than 20 A upon binding to liposomes. At high concentrations (50 mg/mL) mgn2a spontaneously solubilizes phosphatidylcholine liposomes at temperatures above the gel-liquid-crystalline phase transition. Mgn2a appears to bind to the surface of liposomes made of negatively charged lipids without spontaneously penetrating the bilayer. Finally, mgn2a and PGLa interact together with liposomes in a synergistic way that enhances the helix content of one or both of the peptides and allows the peptides to more easily penetrate the bilayer. PGLa mixed with a small nonperturbing amount of magainin 2 amide is 25-43 times as potent as PGLa alone at inducing the release of carboxyfluorescein from liposomes. The results suggest that the mechanism of antimicrobial activity does not involve a channel formed by transmembrane helical peptides.M a g a i n i n , also known as PGSI (Giovannini et al., 1987; Terry et al., 1988), is a family of 23-residue peptides that are secreted from the skin of the African clawed frog. They protect the frog from wound infection and exhibit a broadspectrum antimicrobial activity in vitro (Zasloff, 1987;Zasloff et al., 1988). On a molar basis this activity is as potent as tetracycline against some organisms.PGLa, another antimicrobial peptide isolated from frog skin, has an antimicrobial activity that is comparable to that of magainin 2 (Soravia et al., 1988), but these peptides have little sequence homology. The sequences of magainin 1 amide (mgnla), magainin 2 amide (mgn2a), and PGLa are (Giovannini et al., 1987) mgn 1 a: Gly-He-GI y-Lys-Phe-Leu-His-Ser-Ala-Gly-LysPhe-Gly-Lys-Ala-Phe-Val-Gly-Glu-Ile-Met-Lys-Ser-NH2 mgn2a: Gly-Ile-Gly-Lys-Phe-Leu-His-Ser-Ala-Lys-LysPhe-GI y-Lys-Ala-Phe-Val-GI y-Glu-He-Met-Asn-Ser-NH2 PGLa: Gly-Met-Ala-Ser-Lys-Ala-Gly-Ala-Ile-AlaGly-Lys-Ile-Ala-Lys-Val-Ala-Leu-Lys-Ala-Leu-NH2These peptides are positively charged and can form extremely amphiphilic helices about 30 A in length. These features are common to polypeptides that bind to and effect the permeability of lipid bilayers (Sparrow & Goto, 1982;DeGrado et al., 1981;Green et al., 1987), and it has been suggested that magainins might act as detergents or transmembrane helical channel formers (Zasloff, 1987;Guy & Raghunathan, 1988). Indeed, measurements using a single channel conductance apparatus indicate that magainin forms a voltage-dependent channel in artificial membranes (Cruciani et al., 1988). Magainin 2 amide causes membrane depolarization in Escherichia coli cells and cytochrome oxidase liposo...

Magainin Oligomers Reversibly Dissipate .DELTA..mu.H+ in Cytochrome Oxidase Liposomes

“…Only secondarily, at higher concentrations or dependent on additional forces (Williams et al, 1990), might they cause lysis. The mode of action depicted by Christensen etal.…”

“…In 5% trifluoroethanol, magainin 2 takes on an amphipathic a-helical conformation (Marion et al, 1988), suggesting, as confirmed by Raman spectroscopy (Williams et al, 1990), that it may also do so when present in the hydrophobic environment of the membrane. Magainins have been proposed to line an ion channel (Guy & Raghunatan, 1988;Duclohier et al, 1989;Cruciani et al, 1992;Vaz Gomes et al, 1993) or to lie in the plane of the membrane-solution interface (Bechinger et al,199 1) and perturb membrane impermeability (Cruciani et al, 1992;Grant et al, 1992).…”

Mechanisms of Antimicrobial Peptide Action and Resistance

“…For example, the frog skin peptide PGLa, disordered when exposed to membranes composed of the zwitterionic PC and SM membranes, adopts a helical structure in the presence of membranes composed of PG and PE (Latal et al, 1997). Similarly, magainins only undergo a helical transition when interacting with anionic membranes as demonstated by CD, (Matsuzaki et al, 1989(Matsuzaki et al, , 1991, vibrational/ Raman-FTIR (Williams et al, 1990;Hirsh et al, 1996) and NMR (Bechinger et al, 1993;Hirsh et al, 1996). Examination of cecropin analogs revealed that the extent of ␣-helical conformation is proportionately dependent on the amount of negatively charged phospholipid within the model membrane (Wang et al, 1998).…”

Channel Current Analysis for Pore-forming Properties of an Antimicrobial Peptide, Magainin 1, Using the Droplet Contact Method

“…Magainins have 23 amino acid residues and demonstrate broad-spectrum antibacterial and antifungal activities. 5 To apply a clinically useful antimicrobial therapeutic agent, extensive studies, such as NMR, 6,7 Raman spectroscopy, 8 fluorescence, 3,9 and electrophysiological studies, 1,10 have been performed to analyze the antibacterial mechanism.…”