Detailed insight into the interplay between antimicrobial peptides and biological membranes is fundamental to our understanding of the mechanism of bacterial ion channels and the action of these in biological host-defense systems. To explore this interplay, we have studied the incorporation, membrane-bound structure, and conformation of the antimicrobial peptide alamethicin in lipid bilayers using a combination of 1H liquid-state NMR spectroscopy and molecular dynamics (MD) simulations. On the basis of experimental NMR data, we evaluate simple in-plane and transmembrane incorporation models as well as pore formation for alamethicin in DMPC/DHPC (1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine/1,2-dihexanoyl-sn-glycero-3-phosphatidylcholine) bicelles. Peptide-lipid nuclear Overhauser effect (NOE) and paramagnetic relaxation enhancement (PRE) data support a transmembrane configuration of the peptide in the bilayers, but they also reveal that the system cannot be described by a single simple conformational model because there is a very high degree of dynamics and heterogeneity in the three-component system. To explore the origin of this heterogeneity and dynamics, we have compared the NOE and PRE data with MD simulations of an ensemble of alamethicin peptides in a DMPC bilayer. From all-atom MD simulations, the contacts between peptide, lipid, and water protons are quantified over a time interval up to 95 ns. The MD simulations provide a statistical base that reflects our NMR data and even can explain some initially surprising NMR results concerning specific interactions between alamethicin and the lipids.

Pardaxin is a 33-amino-acid neurotoxin from the Red Sea Moses sole Pardachirus marmoratus, whose mode of action shows remarkable sensitivity to lipid chain length and charge, although the effect of pH is unclear. Here we combine optical spectroscopy and dye release experiments with laser scanning confocal microscopy and natural abundance (13)C solid-state nuclear magnetic resonance to provide a more complete picture of how pardaxin interacts with lipids. The kinetics and efficiency of release of entrapped calcein is highly sensitive to pH. In vesicles containing zwitterionic lipids (PC), release occurs most rapidly at low pH, whereas in vesicles containing 20% anionic lipid (PG), release occurs most rapidly at high pH. Pardaxin forms stable or transient pores in PC vesicles that allow release of contents without loss of vesicle integrity, whereas the inclusion of PG promotes total vesicle collapse. In agreement with this, solid-state nuclear magnetic resonance reveals that pardaxin takes up a trans-membrane orientation in 14-O-PC/6-O-PC bicelles, whereas the inclusion of 14-0-PG restricts it to contacts with lipid headgroups, promoting membrane lysis. Pore formation in zwitterionic vesicles is more efficient than lysis of anionic vesicles, suggesting that electrostatic interactions may trap pardaxin in several suboptimal interconverting conformations on the membrane surface.

We present the use of 2H magic-angle spinning (MAS) NMR on methyl-deuterated alpha-amino isobutyric acid (Aib) as a new method to obtain fast and accurate structural constraints on peptaibols in membrane-bound environments. Using nonoriented vesicle-reconstituted samples we avoid the delicate preparation of oriented samples, and the use of MAS ensures high sensitivity and thereby very fast acquisition of experimental spectra. Furthermore, given the high content ( approximately 40%) of Aib in peptaibols and the fact that the amino acid Aib may be synthesized from cheap starting materials, even in the case of 2H, 13C, or 15N labeling, this method is ideally suited for studies of the membrane-bound conformation of peptaibols.

We demonstrate that a significant improvement in the spectral resolution may be achieved in solidstate NMR experiments of proteins in inhomogeneously disordered oriented lipid bilayers. Using 1 H homonuclear decoupling instead of standard 1 H heteronuclear decoupling, the 15 N linewidths may be reduced by up to seven times for such samples. For large oriented membrane proteins such resolution-enhancements may be crucial for assignment and structural interpretation.Over the past decades solid-state NMR spectroscopy on macroscopically oriented lipidbilayer or magnetically oriented bicelle samples has attracted considerable attention for determination of the structure of membrane-bound proteins. 1,2 So far, most studies have addressed smaller membrane proteins and peptides for which it has been possible to obtain well-resolved peaks in 2D separated-local-field (SLF) spectra correlating the amide 1 H-15 N dipole-dipole couplings and 15 N chemical shifts. In less favorable cases, in particular for larger proteins and high protein:lipid ratios, the resonances are substantially broader and display significant overlap. [2][3][4] Despite large efforts in sample preparation, 3,5 it is believed that a major cause of linebroadening is imperfect sample alignment, e.g., mosaic spread, which in many cases appears to be an intrinsic and unavoidable property of the system. The inevitable consequences are low signal-to-noise ratio and increased risk for spectral overlap.In this Communication, we demonstrate a new method that may significantly improve the resolution in the 15 N dimension of 1D 15 N and 2D 1 H −15 N SLF experiments for oriented Correspondence to: Thomas Vosegaard. Supporting Information Available: Details on the sample preparation, the experimental setup of the solid-state NMR experiments, the numerical simulations, and the MD simulation. This material is available free of charge via the Internet at