Influence of pH and Side-Chain Negative Charge on the Behavior of Designed Transmembrane Peptides in Lipid Bilayers

Abstract

GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA-amide) is a favorable model peptide for investigations of single-residue effects on protein-lipid interactions and the properties of membrane-spanning helices (J. Biol. Chem. 285, 31723). GWALP23 has favorable properties in bilayer membranes because the peptide exhibits only limited dynamic averaging of NMR observables such as the 2H quadrupolar splitting or the 15N-1H dipolar coupling (Biophys. J. 101, 2939). To investigate the potential influence of negatively charged side chains upon system properties, we have substituted a single Leu residue with Glu at different positions and incorporated specific 2H-Ala labels in the core of the single-Trp peptide Y5GWALP23 (see Biochemistry 51, 2044). Solid state 2H NMR experiments were used to examine the peptide orientation and dynamics as functions of the lipid bilayer thickness and pH in hydrated lipid bilayer membranes. We observed well defined 2H quadrupolar splittings for Y5GWALP23-E16 in the pH range from 4.0 to 8.2, suggesting that the peptide helix is well oriented in DOPC lipid bilayers. The glutamic acid residue, though protonated, seemed to confer multi-state behavior at pH 2.5, and the resulting populations exhibited slow exchange on the NMR time scale. The deprotonation of E16 at pH 8.2 did not have any effect on the peptide orientation, perhaps suggesting that the close proximity of E16 to W19 (on the next helical turn) could provide stability to the peptide helix. We are also studying the peptide-lipid behavior when Glu is substituted in position 12 and/or 14, individually or together.