A Molecular View on the Role of Cholesterol upon Membrane Insertion, Aggregation, and Water Accessibility of the Antibiotic Lipopeptide Trichogin GA IV As Revealed by EPR

Abstract

Trichogin GA IV is a membrane-active lipopeptide, the antibiotic activity of which was proposed to be based on its capability to induce leakage due to formation of pores into the bacterial cell membrane. However, less attention has been paid to its biological selectivity, i.e., discrimination between bacterial versus cholesterol containing (mammalian) membranes. This is the reason which motivated us to study the role of cholesterol on penetration of the peptide into the membrane and formation of water channels. The ESEEM technique was used to measure the modulation amplitudes for TOAC spin-labeled trichogin GA IV peptide analogues in hydrated membranes of phosphatidylcholine (PC) lipid in the presence of 50 mol % cholesterol-d7. From the interaction between the nitroxide spin-label and the nearby located deeply membrane inserted deuterons, the N-terminus was found to be positioned at the core of the membrane. Separately, ESEEM measurements for the FTOAC-8 labeled peptide, but in D2O hydrated cholesterol/PC membranes, provide strong evidence for the polar C-terminus situated near the membrane surface. The apparently too high modulation amplitude measured for the buried FTOAC-1 label is likely attributed to the presence of peptide associated water. In cholesterol depleted membrane, however, the long axes of the helical molecules are found oriented parallel to the membrane surface even at high peptide concentration. Continuous wave EPR spectroscopy indicates that, for cholesterol containing membrane, peptide insertion is accompanied by self-aggregation of parallelly aligned transmembrane peptide molecules, while for cholesterol lacking membranes they are monomolecularly distributed. Thus, cholesterol tends to stabilize the transmembrane peptide aggregate.

Trichogin GA IV Alignment and Oligomerization in Phospholipid Bilayers

“…Here, for the first time, the peptide alignment at a P/L value below the threshold for peptide aggregation and insertion (≤1:20) has been investigated in a liquid‐crystalline bilayer by using a noninvasive 15 N and 31 P solid‐state NMR approach (Figure ). The data are indicative of a well‐defined alignment of the trichogin helix parallel to the membrane surface (Figures and S1); this is in good agreement with previous investigations using 2,2,6,6‐tetramethyl‐piperidine‐1‐oxyl‐4‐amino‐4‐carboxylic acid (TOAC) nitroxide‐labeled trichogin and EPR spectroscopy in PC membranes or of fluorophore‐labeled trichogin . The fluorescence spectroscopy investigation also showed a location of the fluorophores at about 1 nm from the bilayer center.…”

Interaction of hydrophobic and amphipathic antimicrobial peptides with lipid bicelles

“…Although the detailed mechanism is still under debate, there is a general consensus that at low P/L ratios, the peptide is monomeric and absorbed parallel to the membrane surface below the polar headgroups. It becomes oligomeric and adopts a transmembrane orientation at high P/L ratios . The insertion depth of trichogin GA IV at low P/L ratios explains the plateau of the order parameter that we observe for P/L ratio up to 1 : 300.…”

Peptide–Membrane Interactions by Spin-Labeling EPR

“…For example, the convolution approach was employed to extract effective distance and distance distribution to characterize the membrane-induced aggregation of alamethicin in bicelles (Bortolus, De Zotti, Formaggio, & Maniero, 2013). Rigid limit CW EPR linewidth broadening as measured for frozen samples (T=78.5 K) was used to estimate spin-spin distances for membrane imbedded TOAC-labeled trichogin GA IV, a lipopeptide with antibiotic activity, assuming formation of a dimer in a model membrane (Syryamina et al, 2012). …”