Influence of Lipid Saturation, Hydrophobic Length and Cholesterol on Double-Arginine-Containing Helical Peptides in Bilayer Membranes

Abstract

Membrane proteins are essential for many cell processes yet are more difficult to investigate than soluble proteins. Charged residues often contribute significantly to membrane protein function. Model peptides such as GWALP23 (acetyl-GGALW⁵ LAL⁸ LALALAL¹⁶ ALW¹⁹ LAGA-amide) can be used to characterize the influence of specific residues on transmembrane protein domains. We have substituted R8 and R16 in GWALP23 in place of L8 and L16, equidistant from the peptide center, and incorporated specific ² H-labeled alanine residues within the central sequence for detection by solid-state ² H NMR spectroscopy. The resulting pattern of [² H]Ala quadrupolar splitting (Δν_q ) magnitudes indicates the core helix for R^8,16 GWALP23 is significantly tilted to give a similar transmembrane orientation in thinner bilayers with either saturated C12:0 or C14:0 acyl chains (1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)) or unsaturated C16:1 Δ9 cis acyl chains. In bilayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC; C18:1 Δ9 cis) multiple orientations are indicated, whereas in longer, unsaturated 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEiPC; C20:1 Δ11 cis) bilayers, the R^8,16 GWALP23 helix adopts primarily a surface orientation. The inclusion of 10-20 mol % cholesterol in DOPC bilayers drives more of the R^8,16 GWALP23 helix population to the membrane surface, thereby allowing both charged arginines access to the interfacial lipid head groups. The results suggest that hydrophobic thickness and cholesterol content are more important than lipid saturation for the arginine peptide dynamics and helix orientation in lipid membranes.

Keywords: GWALP23; arginine; cholesterol; protein-lipid interactions; solid-state NMR spectroscopy.

Figure 1.

Molecular models of GWALP R^8,16, (A) shown in an arbitrary tilted orientation, with the side chains of A15 and A17 space-filling red and the side chains of W5, R8, R16 and W19 shown as sticks; (B) an end view of the helix to show the relative locations of the larger side chains.

Figure 2.

Solid-state ²H NMR spectra of labeled alanines A15 and A17 of GWALP23-R^8,16 in aligned bilayers of DLPC, DMPC, DPoPC, DOPC. The peaks labeled Cα-D arise from the backbone Cα deuteron of residue A17. Sample orientation β = 90°; temperature 50 °C.

Figure 3.

Solid-state ²H NMR spectra of labeled alanines A15 and A17 GWALP23-R^8,16 in aligned bilayers of DEiPC (C20:1) or of DOPC with varying amounts of cholesterol. Sample orientation β = 90°; temperature 50 °C.

Figure 4.

Quadrupolar wave analysis for the tilted helix of GWALP23-R^8,16 in bilayer membranes. A. Results for aligned bilayers of DLPC, DMPC, DPoPC and DOPC. The major orientation in DOPC (solid black curve) is surface-bound, while the helix orientation in the thinner lipids and the minor orientation in DOPC (dashed curve) are transmembrane. B. Results for aligned bilayers of DEiPC and of DOPC with 20%, 10% or 0% cholesterol. The minor orientation in DOPC (dashed curve) is transmembrane while the other orientations represent surface-bound helices. Labeled alanine positions are numbered in panel B. Residue A7 does not fit the curve for the surface-bound orientation.

Figure 5.

Pymol models of GWALP23 R^8,16: (A) tilted in DMPC, and (B) surface bound in DOPC.