Membrane Insertion and Bilayer Perturbation by Antimicrobial Peptide CM15

Department of Biophysics and National Biomedical Electron Paramagnetic Resonance Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Biophysical Journal (Impact Factor: 3.97). 10/2007; 93(5):1651-60. DOI: 10.1529/biophysj.107.104034
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Antimicrobial peptides (AMPs) are an important component of innate immunity and have generated considerable interest as a potential new class of antibiotic. The biological activity of AMPs is strongly influenced by peptide-membrane interactions; however, for many of these peptides the molecular details of how they disrupt and/or translocate across target membranes are not known. CM15 is a linear, synthetic hybrid AMP composed of the first seven residues of the cecropin A and residues 2-9 of the bee venom peptide mellitin. Previous studies have shown that upon membrane binding CM15 folds into an alpha-helix with its helical axis aligned parallel to the bilayer surface and have implicated the formation of 2.2-3.8 nm pores in its bactericidal activity. Here we report site-directed spin labeling electron paramagnetic resonance studies examining the behavior of CM15 analogs labeled with a methanethiosulfonate spin label (MTSL) and a brominated MTSL as a function of increasing peptide concentration and utilize phospholipid-analog spin labels to assess the effects of CM15 binding and accumulation on the physical properties of membrane lipids. We find that as the concentration of membrane-bound CM15 is increased the N-terminal domain of the peptide becomes more deeply immersed in the lipid bilayer. Only minimal changes are observed in the rotational dynamics of membrane lipids, and changes in lipid dynamics are confined primarily to near the membrane surface. However, the accumulation of membrane-bound CM15 dramatically increases accessibility of lipid-analog spin labels to the polar relaxation agent, nickel (II) ethylenediaminediacetate, suggesting an increased permeability of the membrane to polar solutes. These results are discussed in relation to the molecular mechanism of membrane disruption by CM15.

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    • "The linker by which nitroxide probes conjugate to the protein confers an intrinsic conformational flexibility. To reduce probe flexibility, MTSL has been modified in the 4-position of the pyrrole ring to include bulky groups such as phenyl (Fawzi et al., 2011) or bromo moieties (Altenbach et al., 2001; Pistolesi et al., 2007; Georgieva et al., 2012), and the latter MTSL analog (4-bromo-MTSL) is commercially available. Additional restriction of spin label motion can be incorporated by bifunctional SL that enable cross-linking of two target sites on a peptide or protein (Fleissner et al., 2011). "
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    ABSTRACT: The resurgence of pulsed electron paramagnetic resonance (EPR) in structural biology centers on recent improvements in distance measurements using the double electron-electron resonance (DEER) technique. This unit focuses on EPR-based distance measurements by site-directed spin labeling (SDSL) of engineered cysteine residues in soluble proteins, with HIV-1 protease used as a model. To elucidate conformational changes in proteins, experimental protocols were optimized and existing data analysis programs were employed to derive distance-distribution profiles. Experimental considerations, sample preparation, and error analysis for artifact suppression are also outlined herein. Curr. Protoc. Protein Sci. 74:17.17.1-17.17.29. © 2013 by John Wiley & Sons, Inc.
    Current protocols in protein science / editorial board, John E. Coligan ... [et al.] 01/2013; 74:17.17.1-17.17.29. DOI:10.1002/0471140864.ps1717s74
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    • "u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a m e m detergent-like mechanism [8], leading to two distinct sets of events either below or above a threshold concentration [9] [10] [11] [12]. These peptides accumulate onto the bilayer surface until a threshold concentration is reached, leading to membrane disruption [13] [14] [15]. The threshold concentration may vary with lipid composition of the target membrane and peptide environment. "
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    ABSTRACT: Warnericin RK is an antimicrobial peptide, produced by a Staphyloccocus warneri strain, described to be specifically active against Legionella, the pathogenic bacteria responsible for Legionnaires' disease. Warnericin RK is an amphiphilic alpha-helical peptide, which possesses a detergent-like mode of action. Two others peptides, δ-hemolysin I and II, produced by the same S. warneri strain, are highly similar to S. aureus δ-hemolysin and also display anti-Legionella activity. It has been recently reported that S. aureus δ-hemolysin activity on vesicles is likewise related to phospholipid acyl-chain structure, such as chain length and saturation. As staphylococcal δ-hemolysins were highly similar, we thus hypothesized that fatty acid composition of Legionella's membrane might influence the sensitivity of the bacteria to warnericin RK. Relationship between sensitivity to the peptide and fatty acid composition was then followed in various conditions. Cells in stationary phase, which were already described as less resistant than cells in exponential phase, displayed higher amounts of branched-chain fatty acids (BCFA) and short chain fatty acids. An adapted strain, able to grow at a concentration 33 fold higher than minimal inhibitory concentration of the wild type (i.e. 1μM), was isolated after repeated transfers of L. pneumophila in the presence of increased concentrations of warnericin RK. The amount of BCFA was significantly higher in the adapted strain than in the wild type strain. Also, a transcriptomic analysis of the wild type and adapted strains showed that two genes involved in fatty acid biosynthesis were repressed in the adapted strain. These genes encode enzymes involved in desaturation and elongation of fatty acids respectively. Their repression was in agreement with the decrease of unsaturated fatty acids and fatty acid chain length in the adapted strain. Conclusively, our results indicate that the increase of BCFA and the decrease of fatty acid chain length in membrane were correlated with the increase in resistance to warnericin RK. Therefore, fatty acid profile seems to play a critical role in the sensitivity of L. pneumophila to warnericin RK.
    Biochimica et Biophysica Acta 12/2010; 1808(4):1146-53. DOI:10.1016/j.bbamem.2010.12.011 · 4.66 Impact Factor
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    • "CM15, an artificial hybrid of the N-termini of cecropin A and melittin, is the shortest potent antimicrobial peptide sequence. It disrupts and reorganizes membranes through an unknown mechanism that begins with its binding to the membrane surface.(19) Each of these two peptides is unstructured in solution and helical when bound to lipids. "
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    ABSTRACT: Cyanylated cysteine, or beta-thiocyanatoalanine, is an artificial amino acid that can be introduced into peptides and proteins by post-translational chemical modification of solvent-exposed cysteine side chains, and thus it can be used in any protein with a suitable expression and mutagenesis system. In this study, cyanylated cysteine is introduced at selected sites in two model peptides that have been shown to bind to membrane interfaces: a membrane-binding sequence of the human myelin basic protein and the antimicrobial peptide CM15. Far-UV circular dichroism indicates that the secondary structures of the bound peptides are not influenced by introduction of the artificial side chain. Infrared spectra of both systems in buffer and exposed to dodecylphosphocholine micelles indicate that the CN stretching absorption band of cyanylated cysteine can clearly distinguish between membrane burial and solvent exposure of the artificial side chain. Since infrared spectroscopy can be applied in a wide variety of lipid systems, and since cyanylated cysteine can be introduced into proteins of arbitrary size via mutagenesis and post-translational modification, this new probe could see wide use in characterizing the protein-lipid interactions of membrane proteins.
    Journal of Physical Chemistry Letters 03/2010; 1(5):850-855. DOI:10.1021/jz1000177 · 7.46 Impact Factor
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