Dynamics and conformational studies of TOAC spin labeled analogues of Ctx(Ile(21))-Ha peptide from Hypsiboas albopunctatus

Abstract

Antimicrobial peptides (AMPs) isolated from several organisms have been receiving much attention due to some specific features that allow them to interact with, bind to, and disrupt cell membranes. The aim of this paper was to study the interactions between a membrane mimetic and the cationic AMP Ctx(Ile(21))-Ha as well as analogues containing the paramagnetic amino acid 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) incorporated at residue positions n = 0, 2, and 13. Circular dichroism studies showed that the peptides, except for [TOAC(13)]Ctx(Ile(21))-Ha, are unstructured in aqueous solution but acquire different amounts of α-helical secondary structure in the presence of trifluorethanol and lysophosphocholine micelles. Fluorescence experiments indicated that all peptides were able to interact with LPC micelles. In addition, Ctx(Ile(21))-Ha and [TOAC(13)]Ctx(Ile(21))-Ha peptides presented similar water accessibility for the Trp residue located near the N-terminal sequence. Electron spin resonance experiments showed two spectral components for [TOAC(0)]Ctx(Ile(21))-Ha, which are most likely due to two membrane-bound peptide conformations. In contrast, TOAC(2) and TOAC(13) derivatives presented a single spectral component corresponding to a strong immobilization of the probe. Thus, our findings allowed the description of the peptide topology in the membrane mimetic, where the N-terminal region is in dynamic equilibrium between an ordered, membrane-bound conformation and a disordered, mobile conformation; position 2 is most likely situated in the lipid polar head group region, and residue 13 is fully inserted into the hydrophobic core of the membrane.

Figure 1. Definition of the principal magnetic axes (x_m, y_m, z_m) oriented relative to the nitroxide molecular frame in a TOAC-labeled α-helical peptide.

The y_m axis is perpendicular to the others, forming a right-handed coordinate system. The rotational diffusion axes (x_R, y_R, z_R) are taken to coincide with the magnetic frame (see text). The local director, z_D, is defined as the average orientation of the z_R axis over the course of its motion. In the rotational diffusion frame, where z_R is fixed, z_D traces a trajectory around z_R. The orienting potential is then expressed as a function of the polar angles of z_D in the rotational diffusion frame. The TOAC spin label was inserted at position 13 according to Ghimire and collaborators using MolMol program .

Figure 2. A Schiffer–Edmundson helical wheel of the peptide Ctx(Ile²¹)-Ha.

The modifications/additions of the paramagnetic amino acid TOAC are in the square highlighted.

Figure 3. CD spectra of synthetic peptides.

The spectra were obtained in aqueous solution (A), in the presence of 60% (v/v) trifluoroethanol (B), and in the presence of LPC 10 mmol L⁻¹ (C). The peptide concentration was 80 µmol L⁻¹.

Figure 4. Fluorescence emission maxima of the synthetic peptides as a function of the concentration of LPC micelles in TRIS buffer pH 7.4 at 25°C.

The concentration of the peptide was 10 µmol L⁻¹.

Figure 5. Variation of the Trp fluorescence for the peptides Ctx(Ile²¹)-Ha, [TOAC⁰]Ctx(Ile²¹)-Ha and [TOAC¹³]Ctx(Ile²¹)-Ha in aqueous solution (A) and in LPC micelles (B), in presence of acrylamide at different concentrations.

Figure 6. ESR spectra (solid lines) of the TOAC-labeled peptides and the best nonlinear least-squares fits (dashed lines) in aqueous solution (A) and LPC micelles (B) acquired at 22°C.

The concentration of the peptide was 80 µmol L⁻¹.

Figure 7. Representative ESR spectra acquired at 10, 15, and 50°C corresponding to a dynamic equilibrium between the membrane-bound and membrane-unbound N-terminal conformations (A).

Note the heightening of the spin population experiencing a more restricted re-orientational motion at low temperatures. [TOAC⁰]Ctx(Ile²¹)-Ha ESR spectrum in LPC micelles (solid lines) acquired at 22°C and the best nonlinear least-squares fits (dashed lines) using one (top) or two (bottom) spectral components (B).

Figure 8. Schematic representation of the Ctx(Ile²¹)-Ha topology determined by ESR and nonlinear least-squares simulations showing the two N-terminal conformations: the immobilized, ordered membrane-bound state and the mobile, disordered form, fully exposed to the water phase.