Influence of Glutamic Acid Residues on the Properties of Model Membrane-Spanning Helices

Abstract

GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA- amide) is a constructive 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 glutamic acid 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 showed well defined 2H quadrupolar splittings for Y5GWALP23-E16 in the pH range from 4.0 to 9.0, suggesting that the peptide helix is well oriented in DOPC lipid bilayers. The E16-containing peptide seems to exhibit multi-state behavior at pH 10.9, in bilayers formed by ether-linked lipids, suggesting a pKa that is above pH 9 for the E16 side chain. The rather modest shift in the 2H quadrupolar splittings suggests that the orientation of the transmembrane peptide helix changes rather little at high pH. It is conceivable that the close proximity of E16 to W19 could provide stability to the neutral peptide helix and perhaps influence the pKa of E16. The molecular cousin having E14 instead of E16 shows multi-state behavior from pH 4.0 to pH 10.9 rendering pKa determination enigmatic at this time. Some puzzles remain with respect to comparisons between E16 and E14. We are additionally investigating the peptide-lipid behavior when Glu is introduced in position 12, at the peptide center.