Amphipathic antimicrobial piscidin in magnetically aligned lipid bilayers

Abstract

The amphipathic antimicrobial peptide piscidin 1 was studied in magnetically aligned phospholipid bilayers by oriented-sample solid-state NMR spectroscopy. (31)P NMR and double-resonance (1)H/(15)N NMR experiments performed between 25 °C and 61 °C enabled the lipid headgroups as well as the peptide amide sites to be monitored over a range of temperatures. The α-helical peptide dramatically affects the phase behavior and structure of anionic bilayers but not those of zwitterionic bilayers. Piscidin 1 stabilizes anionic bilayers, which remain well aligned up to 61 °C when piscidin 1 is on the membrane surface. Two-dimensional separated-local-field experiments show that the tilt angle of the peptide is 80 ± 5°, in agreement with previous results on mechanically aligned bilayers. The peptide undergoes fast rotational diffusion about the bilayer normal under these conditions, and these studies demonstrate that magnetically aligned bilayers are well suited for structural studies of amphipathic peptides.

Figure 1

One-dimensional ³¹P NMR spectra as a function of temperature of anionic magnetically aligned bilayers with molar composition 2.6:0.6:1.0 14-O-PC/DMPG/6-O-PC in the absence (−p1) and presence (+p1) of piscidin 1 (left and right, respectively). As marked, the sample is equilibrated at 25°C, 30°C, 34°C, 42°C, 48°C, 55°C, and 61°C (bottom to top). The ³¹P NMR isotropic chemical-shift frequency (0 ppm) is marked by the vertical dotted lines.

Figure 2

One-dimensional ¹⁵N NMR (left) and ³¹P NMR spectra (right) of ¹⁵N-(F2I5G8)-p1-NH₂ in anionic magnetically aligned bilayers with molar composition 2.6:0.6:1.0 14-O-PC/DMPG/6-O-PC, equilibrated at 34°C, 42°C, 48°C, 55°C, and 61°C (bottom to top). The ³¹P NMR isotropic chemical-shift frequency (0 ppm) is marked by the vertical dotted line.

Figure 3

Two-dimensional ¹⁵N/¹H SLF spectra obtained with the SAMPI-4 pulse sequence. (A) ¹⁵N-(F2I5G8)-p1-NH₂. (B) ¹⁵N-(V10G13I16)-p1-NH₂. (C) PISA-wheel simulations obtained using the ¹⁵N and ¹H chemical shift anisotropy tensors of an ideal α-helix that has a tilt angle of 76°, 80°, and 84° with respect to the bilayer normal. The standard order parameter of S = 0.85 is incorporated into the simulations. (D) ¹⁵N-(I5F6G8I9V10V12G13I16L19V20)-p1-NH₂ in anionic 2.6:0.6:1.0 14-O-PC/DMPG/6-O-PC perpendicular bicelles at 61°C. Spectra from ¹⁵N-(F2I5G8)-p1-NH₂, ¹⁵N-(V10G13I16)-p1-NH₂, and ¹⁵N-(F6V12)-p1-NH₂ were used to confirm the bicelle assignments of this sample.

Figure 4

One-dimensional ¹⁵N NMR (left) and ³¹P NMR (right) of ¹⁵N-(F2I5G8)-p1 NH₂ in zwitterionic magnetically aligned bilayers with molar composition 3.2:1.0 14-O-PC/6-O-PC, acquired at 42°C and 55°C, as designated.

Figure 5

(A) Two-dimensional ¹⁵N/¹H SLF SAMPI-4 spectra of ¹⁵N-(F2I5G8)-p1-NH₂ in zwitterionic 3.2:1.0 14-O-PC/6-O-PC perpendicular bicelles at 55°C. A third broad, weak resonance with a ¹H-¹⁵N dipolar coupling of ∼500 Hz is visible only at lower contour levels. Slices in the dipolar dimension corresponding to the three observable resonances marked by dotted lines are shown at the top. (B) PISA-wheel simulations of an ideal α-helix with tilt angles of 55° (blue) and 60° (red) with respect to the bilayer normal.

Figure 6

Simulations of the effect of uniaxial motional averaging around the bilayer normal on ¹⁵N/¹H SLF spectra of a uniformly ¹⁵N-labeled in-plane 18-residue α-helix with a tilt of 80° with respect to the bilayer normal in perpendicular bicelles, at rotational diffusion rates of: 3 × 10⁵ s⁻¹, 10 × 10⁵ s⁻¹, and 20 × 10⁵ s⁻¹ (top to bottom). Red crosses show the predicted positions of the resonances.

Figure 7

Comparison between solid-state NMR data of piscidin 1 in 3:1 DMPC/DMPG lipid bilayers mechanically aligned on glass plates at 40°C and piscidin 1 in 2.6:0.6:1.0 14-O-PC/DMPG/6-O-PC magnetically aligned bilayers at 61°C. (A) Simulated bicelle spectra calculated from the ¹⁵N/¹H SLF PISEMA data for piscidin 1 in mechanically aligned bilayers (A. A. De Angelis, C. V. Grant, M. K. Baxter, J. A. McGavin, S. J. Opella, and M. L. Cotten, unpublished results) using Eq. 1 and bicelle order parameter S = 0.85. (B) Experimental results in magnetically aligned bilayers combining data from two samples: ¹⁵N-(I5F6G8I9V10V12G13I16L19V20)-p1-NH₂ and ¹⁵N-(F2I5G8)-p1-NH₂. Phe², which provides a starting point for resonance assignment using PISA wheels, is shown in gray.