Insights into peptide‐membrane interactions of newly synthesized, nitroxide‐containing analogs of the peptaibiotic trichogin GAIV using EPR

Abstract

Trichogin GA IV is a short-length (10-amino acid long), mostly hydrophobic, peptaibiotic with an N-terminal fatty acyl chain and a C-terminal 1,2-amino alcohol. A cardinal role of the terminal moieties in the cytotoxic activity of trichogin has been recently found. Previously, peptide orientation and dynamics of trichogin analogs in the membrane were studied using methyl ester derivatives. Therefore, in the present work we synthesized several trichogin analogs with naturally occurring terminal groups to verify whether these moieties have any effect on peptide-membrane interaction. These trichogin analogs, both neutral and carrying a positively charged Lys residue, bear the nitroxide-containing α-amino acid TOAC to study them using EPR spectroscopy. Vesicles were used to investigate orientation and penetration depth of the peptide at room temperature. Bicelles were employed to evaluate the order, dynamics, and orientation of the peptide at a near physiological temperature. In addition, the position of the N-terminal 1-octanoyl chain in the membrane was studied by labeling it with a nitroxide. The secondary structure of the peptides in vesicles was studied by CD spectroscopy showing that they adopt a mostly α-helical structure. In vesicles, the analogs insert below the lipid headgroups with the helix axis oriented parallel to the membrane surface at a peptide-to-lipid (P:L) ratio of 1:100. The presence of the single, positively charged Lys residue does not alter the orientation adopted by the peptides. In bicelles at P:L ratios 1:100 and 1:60, the peptide adopts a transmembrane orientation characterized by a very low orientational order, whereas at a 1:15 P:L ratio it severely disrupts the membrane. Our data shows that overall orientation and insertion in model membranes of the native trichogin GA IV are strictly comparable to those of its methyl ester analogs previously examined.

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.…”

“…These data suggest that the inserted, aggregated species is responsible for membrane leakage possibly by defects along the lipid–peptide interface. In view of these correlations, the oligomeric state is of particular interest, and its structure has been studied in further detail here (Figures E, G and ) and in previous EPR and fluorescence spectroscopy investigations …”

“…A correlation was observed between membrane‐perturbing activity and aggregate formation in the membrane and between the fraction of aggregated and inserted peptide. Above a threshold P/L, trichogin has been shown to aggregate and to insert more deeply into the membrane . Notably, the 31 P solid‐state NMR data (Figure B, D, F) are indicative of well‐oriented PC bilayers even in the presence of high trichogin concentrations.…”

“…When a threshold concentration of about 1:50 is reached, dimerization and a deeper penetration of the peptide inside the membrane are observed by EPR and fluorescence spectroscopies . The 15 N solid‐state NMR spectra remain unchanged, thus suggesting that the membrane topology is unaltered between P/L=1:120 and 1:20 (Figure A, C).…”

“…Indeed, previous EPR and fluorescence spectroscopy investigations have identified several possible supramolecular membrane assemblies. At P/L<1:100, an in‐plane alignment of trichogin monomers has been identified in a number of investigations . At P/L≈1:20, the formation of small oligomers/dimers accompanied by a change in membrane topology and trichogin dynamics has been observed .…”