Properties of membrane-incorporated WALP peptides that are anchored on only one end

Abstract

Peptides of the "WALP" family, acetyl-GWW(LA)(n)LWWA-[ethanol]amide, have proven to be opportune models for investigating lipid-peptide interactions. Because the average orientations and motional behavior of the N- and C-terminal Trp (W) residues differ, it is of interest to investigate how the positions of the tryptophans influence the properties of the membrane-incorporated peptides. To address this question, we synthesized acetyl-GGWW(LA)(n)-ethanolamide and acetyl-(AL)(n)WWG-ethanolamide, in which n = 4 or 8, which we designate as "N-anchored" and "C-anchored" peptides, respectively. Selected (2)H or (15)N labels were incorporated for solid-state nuclear magnetic resonance (NMR) spectroscopy. These peptides can be considered "half"-anchored WALP peptides, having only one pair of interfacial Trp residues near either the amino or the carboxyl terminus. The hydrophobic lengths of the (n = 8) peptides are similar to that of WALP23. These longer half-anchored WALP peptides incorporate into lipid bilayers as α-helices, as reflected in their circular dichroism spectra. Solid-state NMR experiments indicate that the longer peptide helices assume defined transmembrane orientations with small non-zero average tilt angles and moderate to high dynamic averaging in bilayer membranes of 1,2-dioleoylphosphatidylcholine, 1,2-dimyristoylphosphatidylcholine, and 1,2-dilauroylphosphatidylcholine. The intrinsically small apparent tilt angles suggest that interactions of aromatic residues with lipid headgroups may play an important role in determining the magnitude of the peptide tilt in the bilayer membrane. The shorter (n = 4) peptides, in stark contrast to the longer peptides, display NMR spectra that are characteristic of greatly reduced motional averaging, probably because of peptide aggregation in the bilayer environment, and CD spectra that are characteristic of β-structure.

Figure 1

Schematic ribbon models for hydrophobic peptides of length 11 residues or 19 residues, with two Trp residues near the N-terminal (lighter gray) or near the C-terminal (darker gray). The longer peptides could span a lipid bilayer membrane as α-helices, whereas the shorter peptides could span only about one leaflet of a bilayer. Nevertheless, the shorter peptides are not helical under such conditions.

Figure 2

Circular dichroism spectra of N-anchored “n=8” (A), C-anchored “n=8” (B), C-anchored “n=4” (C), and N-anchored “n=4” (D) WALP peptides, in vesicles of DLPC (black), DMPC (blue) or DOPC (red). It is evident that the shorter lipids promote a larger extent of α-helix formation for the longer peptides.

Figure 3

²H NMR spectra of labeled alanines in N-anchored “n=8” (A, B), C-anchored “n=8” (C, D) and C-anchored “n=4” (E, F) WALP peptides, in hydrated bilayers of DMPC oriented at β = 0° (A, C, E) or β = 90° (B, D, F); temperature 50 °C. The ²H-labeled alanine residues and % deuteration are A⁶ and A⁸ 70% 100% (A, B); or A¹³ and A¹⁵ 70% 100% (C, D); or A³ and A⁵ 75% 50% and A⁷_100% (E, F).

Figure 4

SAMPI4 spectra for C-anchored “n=8” WALP peptide with ¹⁵N-labels in residues 5-15 (A), or in residues 11-15 (B). Peak assignments for labeled residues are shown in B. The PISA wheel patterns are based on analysis of a combination of ²H and ¹⁵N data, with equal weights for the ²H quadrupolar couplings, ¹⁵N/¹H dipolar couplings and ¹⁵N chemical shifts.

Figure 5

GALA quadrupolar wave plots for “half-anchored” “n=8” WALP peptides, with the paired Trp anchor residues that are N-proximal (A), or C-proximal (B). The curves represent the fits using Gaussian dynamics in the lipid environments of DLPC (black), DMPC (blue) or DOPC (red). For comparison, the semi-static fits are shown as green curves in DLPC.

Figure 6

Gaussian dynamics. RMSD (στ, σρ) graphs for the Gaussian dynamics analysis of the “n=8” N-anchored (A) and C-anchored (B) WALP peptides in mechanically oriented bilayers of DLPC (black), or in magnetically oriented bicelles (blue). The contour levels are 1.0 and 2.0 kHz.

Figure 7

Orientations of half-anchored peptides. RMSD plots for the apparent tilt of the “n=8” N-anchored (A) and C-anchored (B) WALP peptides in oriented bilayers of DLPC (black), DMPC (blue) or DOPC (red). RMSD is plotted as a function of τ and ρ values for the optimum values of σ_τ and σ_ρ in Gaussian calculations (see Table 4 and Figure 5). Contours are drawn at 1.2 kHz and 2.0 kHz.