(4-24)-ply(a) and dimyristoylphosphatidylglycerol

How can highly charged, cationic antimicrobial peptides (AMPs) translocate across hydrophobic lipid bilayers despite the prohibitive energetic penalty to do so? A common explanation has been the formation of peptide-lined channels. However, for most AMPs, no structures of membrane pores have been found despite clear evidence of membrane leakage and antimicrobial activity. The study here suggests an alternative and simple reason: for the AMP PGLa from Xenopus laevis (charge +5), such pores are not needed to explain both leakage and peptide translocation. Elevated-temperature multimicrosecond equilibrium simulations at all-atomistic level reveal that peptides spontaneously translocate across the membrane individually on a timescale of tens of microseconds, without forming pores. Both surface-bound peptides and lipids assist in the one-by-one translocation of the charged side chains. Single peptides can remain in a transmembrane orientation for many microseconds, snorkeling some charged residues to one interface and some to the opposite, but without inducing a water channel. Instead of stable pores, short-lived water bridges occur when two or three peptides connect at their termini, allowing both ion translocation and lipid flip-flop via a brushlike mechanism usually involving the C terminus of one peptide. The results here suggest that for some specific antimicrobial and other membrane active peptides, pore formation may not have to be invoked at all to explain peptide translocation and membrane permeabilization, which may explain why no channel structures for them have been determined experimentally.

Topics: Animals; Antimicrobial Cationic Peptides; Cations, Monovalent; Dimyristoylphosphatidylcholine; Hydrophobic and Hydrophilic Interactions; Kinetics; Lipid Bilayers; Molecular Dynamics Simulation; Permeability; Phosphatidylglycerols; Sodium; Temperature; Water; Xenopus laevis; Xenopus Proteins

Dynamic Nuclear Polarization (DNP) has been introduced to overcome the sensitivity limitations of nuclear magnetic resonance (NMR) spectroscopy also of supported lipid bilayers. When investigated by solid-state NMR techniques the approach typically involves doping the samples with biradicals and their investigation at cryo-temperatures. Here we investigated the effects of temperature and membrane hydration on the topology of amphipathic and hydrophobic membrane polypeptides. Although the antimicrobial PGLa peptide in dimyristoyl phospholipids is particularly sensitive to topological alterations, the DNP conditions represent well its membrane alignment also found in bacterial lipids at ambient temperature. With a novel membrane-anchored biradical and purpose-built hardware a 17-fold enhancement in NMR signal intensity is obtained by DNP which is one of the best obtained for a truly static matrix-free system. Furthermore, a membrane anchor sequence encompassing 19 hydrophobic amino acid residues was investigated. Although at cryotemperatures the transmembrane domain adjusts it membrane tilt angle by about 10 degrees, the temperature dependence of two-dimensional separated field spectra show that freezing the motions can have beneficial effects for the structural analysis of this sequence.

Topics: Amino Acid Sequence; Anti-Infective Agents; Antimicrobial Cationic Peptides; Cold Temperature; Dimyristoylphosphatidylcholine; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Magnetic Resonance Spectroscopy; Phosphatidylcholines; Phosphatidylglycerols

To understand the molecular mechanisms of amphiphilic membrane-active peptides, one needs to study their interactions with lipid bilayers under ambient conditions. However, it is difficult to control the pH of the sample in biophysical experiments that make use of mechanically aligned multilamellar membrane stacks on solid supports. HPLC-purified peptides tend to be acidic and can change the pH in the sample significantly. Here, we have systematically studied the influence of pH on the lipid interactions of the antimicrobial peptide PGLa embedded in oriented DMPC/DMPG bilayers. Using solid-state NMR (31P, 2H, 19F), both the lipid and peptide components were characterized independently, though in the same oriented samples under typical conditions of maximum hydration. The observed changes in lipid polymorphism were supported by DSC on multilamellar liposome suspensions. On this basis, we can present an optimized sample preparation protocol and discuss the challenges of performing solid-state NMR experiments under controlled pH. DMPC/DMPG bilayers show a significant up-field shift and broadening of the main lipid phase transition temperature when lowering the pH from 10.0 to 2.6. Both, strongly acidic and basic pH, cause a significant degree of lipid hydrolysis, which is exacerbated by the presence of PGLa. The characteristic re-alignment of PGLa from a surface-bound to a tilted state is not affected between pH of 7 to 4 in fluid bilayers. On the other hand, in gel-phase bilayers the peptide remains isotropically mobile under acidic conditions, displays various co-existing orientational states at pH7, and adopts an unknown structural state at basic pH.

Topics: Amino Acid Sequence; Antimicrobial Cationic Peptides; Calorimetry, Differential Scanning; Dimyristoylphosphatidylcholine; Hydrogen-Ion Concentration; Lipid Bilayers; Liposomes; Magnetic Resonance Spectroscopy; Membrane Lipids; Molecular Sequence Data; Phosphatidylglycerols; Phospholipids; Protein Binding; Transition Temperature

The antimicrobial activity of amphipathic alpha-helical peptides is usually attributed to the formation of pores in bacterial membranes, but direct structural information about such a membrane-bound state is sparse. Solid state (2)H-NMR has previously shown that the antimicrobial peptide PGLa undergoes a concentration-dependent realignment from a surface-bound S-state to a tilted T-state. The corresponding change in helix tilt angle from 98 to 125 degrees was interpreted as the formation of PGLa/magainin heterodimers residing on the bilayer surface. Under no conditions so far, has an upright membrane-inserted I-state been observed in which a transmembrane helix alignment would be expected. Here, we have demonstrated that PGLa is able to assume such an I-state in a 1:1 mixture with magainin 2 at a peptide-to-lipid ratio as low as 1:100 in dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol model membranes. This (2)H-NMR analysis is based on seven orientational constraints from Ala-3,3,3-d(3) substituted in a non-perturbing manner for four native Ala residues as well as two Ile and one Gly. The observed helix tilt of 158 degrees is rationalized by the formation of heterodimers. This structurally synergistic effect between the two related peptides from the skin of Xenopus laevis correlates very well with their known functional synergistic mode of action. To our knowledge, this example of PGLa is the first case where an alpha-helical antimicrobial peptide is directly shown to assume a transmembrane state that is compatible with the postulated toroidal wormhole pore structure.

Topics: Amino Acid Sequence; Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Dimerization; Dimyristoylphosphatidylcholine; Drug Synergism; Lipid Bilayers; Membrane Lipids; Models, Chemical; Molecular Sequence Data; Nuclear Magnetic Resonance, Biomolecular; Peptides; Phosphatidylglycerols; Sequence Homology, Amino Acid; Skin; Xenopus laevis

The conformational properties of the magainin family of antimicrobial peptides in aqueous solution and in model membranes have been probed by Fourier transform infrared spectroscopy. The magainins were found to be structureless in aqueous solution at neutral pD, confirming other studies by Raman and circular dichroism spectroscopy. Increasing the pD to 10 induced the formation of predominantly alpha-helical secondary structures, with some beta-sheet. In the presence of negatively charged liposomes (dimyristoylphosphatidylglycerol), the peptides folded into alpha-helical secondary structures with some beta-sheet structure evident. On the other hand, in the presence of zwitterionic phospholipids (dimyristoylphosphatidylcholine), the spectra were identical to those in aqueous solution. For some magainins, the interaction with charged liposomes was modulated by the presence of cholesterol; cholesterol was found to promote the formation of beta-sheet structures, as evidenced by the appearance of amide I bands at 1614 and 1637 cm-1. Differences in structure were observed between the amidated and nonamidated forms of some peptides. From the data, a mechanism of antimicrobial action of the magainin family of peptides is proposed.

Topics: Amino Acid Sequence; Animals; Antimicrobial Cationic Peptides; Dimyristoylphosphatidylcholine; Fourier Analysis; Liposomes; Magainins; Molecular Sequence Data; Oligopeptides; Peptide Fragments; Peptides; Phosphatidylglycerols; Protein Conformation; Sequence Homology, Nucleic Acid; Spectrophotometry, Infrared; Xenopus laevis; Xenopus Proteins