Ionization-Dependent Behavior of Transmembrane Helices that Incorporate Glu or Tyr Residues

Abstract

GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA-amide) is a constructive low-dynamic model peptide for investigations of single-residue influence on protein-lipid interactions and the properties of membrane-spanning helices (J. Biol. Chem. 285, 31723). To investigate the pH dependence, ionization behavior and orientational constraints when potentially negatively charged glutamic acid side chains are present, we have substituted a single Leu residue with Glu at different positions and incorporated specific 2H-Ala labels in the core GWALP23 or Y5GWALP23. Solid state NMR experiments show well defined 2H-Ala quadrupolar splittings for Y5GWALP23-E14 over the pH range of 6 to 10, suggesting that the peptide helix is well oriented in DLPC and DOPC lipid bilayers. Spectral changes are evident above pH 11 (in ether-lipid bilayers), but the titration of the Glu is uncertain because the Tyr residue itself appears to titrate around pH 11.5. GWALP23-E14 shows no spectral changes over the pH range 6.0 to 11.5, yet a change in quadrupolar splittings is observed at pH 13. The combined results suggest that the Glu residue may titrate with a pKa near 12. In bilayers of DLPC and DOPC, GWALP23-E16 shows similar trend and is suggested to have a pKa of around 12. The rather modest shifts in the 2H quadrupolar splittings, nevertheless, suggest that the orientation of the transmembrane helix actually may change rather little at high pH. It is conceivable that the close proximity of either E14 or E16 to W19 could provide stability to the neutral peptide helix and perhaps influence the results. We additionally are investigating the possibilities for helix unwinding near the ends of these peptides.