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Lysylated phospholipids stabilize models of bacterial lipid bilayers and protect against antimicrobial peptides.

Biochimica et biophysica acta (2014-05-02)
Elizabeth Cox, Austen Michalak, Sarah Pagentine, Pamela Seaton, Antje Pokorny
ABSTRACT

Aminoacylated phosphatidylglycerols are common lipids in bacterial cytoplasmic membranes. Their presence in Staphylococcus aureus has been linked to increased resistance to a number of antibacterial agents, including antimicrobial peptides. Most commonly, the phosphatidylglycerol headgroup is esterified to lysine, which converts anionic phosphatidylglycerol into a cationic lipid with a considerably increased headgroup size. In the present work, we investigated the interactions of two well-studied antimicrobial peptides, cecropin A and mastoparan X, with lipid vesicles composed of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), containing varying fractions of an aminoacylated phosphatidylethanolamine, a stable analog of the corresponding phosphatidylglycerol-derivative. To differentiate between the effects of headgroup size and charge on peptide-lipid interactions, we synthesized two different derivatives. In one, the headgroup was modified by the addition of lysine, and in the other, by glutamine. The modification by glutamine results in a phospholipid with a headgroup size comparable to that of the lysylated version. However, whereas lysyl-phosphatidylethanolamine (Lys-PE) is cationic, glutaminyl-phosphatidylethanolamine (Gln-PE) is zwitterionic. We found that binding of mastoparan X and cecropin A was not significantly altered if the content of aminoacylated phosphatidylethanolamines did not exceed 20mol.%, which is the concentration found in bacterial membranes. However, a lysyl-phosphatidylethanolamine content of 20mol% significantly inhibits dye release from lipid vesicles, to a degree that depends on the peptide. In the case of mastoparan X, dye release is essentially abolished at 20mol.% lysyl-phosphatidylethanolamine, whereas cecropin A is less sensitive to the presence of lysyl-phosphatidylethanolamine. These observations are understood through the complex interplay between peptide binding and membrane stabilization as a function of the aminoacylated lipid content. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
L-Lysine, ≥98% (TLC)
Sigma-Aldrich
Ethyl alcohol, Pure, 200 proof, meets USP testing specifications
Sigma-Aldrich
HBTU, ≥98.0% (T)
Sigma-Aldrich
L-Lysine monohydrochloride, reagent grade, ≥98% (HPLC)
Sigma-Aldrich
L-Lysine monohydrochloride, from non-animal source, meets EP, JP, USP testing specifications, suitable for cell culture, 98.5-101.0%
Sigma-Aldrich
L-Lysine, crystallized, ≥98.0% (NT)
Supelco
L-Lysine monohydrochloride, Pharmaceutical Secondary Standard; Certified Reference Material
Supelco
Ethanol solution, certified reference material, 2000 μg/mL in methanol
Supelco
L-Lysine, analytical standard
Sigma-Aldrich
L-Lysine monohydrochloride, BioUltra, ≥99.5% (AT)
Lysine hydrochloride, European Pharmacopoeia (EP) Reference Standard
Supelco
L-Lysine monohydrochloride, certified reference material, TraceCERT®
Sigma-Aldrich
L-Lysine acetate salt, ≥98% (HPLC)
Supelco
L-Lysine Acetate, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
Cecropin A, ≥97% (HPLC), powder
Lysine acetate, European Pharmacopoeia (EP) Reference Standard
Sigma-Aldrich
Ethanol, absolute, denaturated with 0.5-1.5 Vol.% 2-butanone and approx. 0.001% Bitrex (GC), ≥98% (GC)